sirolimus and Carcinoma--Hepatocellular

Liver transplantation (LT) is an effective treatment option for end-stage liver disease. Mammalian target of rapamycin (mTOR) inhibitors, such as rapamycin, are widely used post LT.. In this review, we focused on the anti-cancer activities and metabolic side effects of rapamycin after LT. The literature available on PubMed for the period of January 1999-September 2022 was reviewed. The key words were rapamycin, sirolimus, liver transplantation, hepatocellular carcinoma, diabetes, and lipid metabolism disorder.. Rapamycin has shown excellent effects and is safer than other immunosuppressive regimens. It has exhibited excellent anti-cancer activity and has the potential in preventing hepatocellular carcinoma (HCC) recurrence post LT. Rapamycin is closely related to two long-term complications after LT, diabetes and lipid metabolism disorders.. Rapamycin prevents HCC recurrence post LT in some patients, but it also induces metabolic disorders. Reasonable use of rapamycin benefits the liver recipients.

Topics: Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Liver Transplantation; Neoplasm Recurrence, Local; Sirolimus

The effects of mammalian target of rapamycin (mTOR) inhibitors (sirolimus [SRL] and everolimus [EVL]) on survival in liver transplantation (LT) recipients with hepatocellular carcinoma (HCC) remain the subject of intense research. Therefore, we performed this systematic review and meta-analysis to investigate the potential survival benefits of mTOR inhibitors (mTORis). Embase, PubMed, and Cochrane Central Register of Controlled Trials (CENTRAL) were searched for all randomized controlled trials (RCTs) and cohort studies investigating effects of SRL or EVL on LT recipients for HCC. The primary outcomes were 1-, 2-, 3-, and 5-year overall survival (OS), and the secondary outcomes were 1-, 2-, and 3-year recurrence-free survival (RFS) and adverse effects. Pooled relative risks (RRs) with 95% confidence interval (CI) were calculated by a fixed or random effects model with Mantel-Haenszel weighting. Subgroup analyses were performed according to crucial clinical characteristics. We also conducted sensitivity analyses to assess the reliability of our findings. A total of 17 studies were included. OS was improved in both RCTs (1 year: RR, 1.04; 95% CI, 1.00-1.08; 2 years: RR, 1.09; 95% CI, 1.02-1.16; 3 years: RR, 1.13; 95% CI, 1.04-1.24; 5 years: RR, 1.13; 95% CI, 1.02-1.26) and cohort studies (1 year: RR, 1.13; 95% CI, 1.06-1.20; 2 years: RR, 1.24; 95% CI, 1.16-1.32; 3 years: RR, 1.24; 95% CI, 1.15-1.34; 5 years: RR, 1.17; 95% CI, 1.10-1.24), with a lower risk of renal toxicity (RR, 0.75; 95% CI, 0.60 to 0.93). The 1-, 2-, and 3-year RFS were also improved. Current evidence indicates that SRL- or EVL-based immunosuppression improves OS and RFS with a lower risk of renal toxicity compared with mTORi-free immunosuppression. Nevertheless, results must be interpreted with caution.

Topics: Carcinoma, Hepatocellular; Everolimus; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Sirolimus

Sirolimus and everolimus are mammalian target of rapamycin inhibitors (mTORi) that can reduce relapse rates following liver transplantation (LT) for hepatocellular carcinoma (HCC). Herein, we performed a systematic review and meta-analysis to investigate the efficacy of mTORi and calcineurin inhibitors (CNI) in reducing HCC recurrence and survival adverse effects (AEs) in HCC patients after LT. Systematic literature searches were conducted using MEDLINE, EMBASE, and Cochrane Library databases up to October 2021. The primary outcomes of interest were tumor recurrence rates and overall survival. The secondary outcomes were the characterization and incidence of AEs. A total of 38 trials involving 10,607 participants was included in the analysis. The incidence of recurrence and overall mortality was significantly lower in the mTORi than in the CNI group (relative ratio [RR]: .78, 95% confidence interval [CI]: .68-.89 and RR: .76, 95% CI: .67-.86, respectively). The incidence of some AEs and complications such as acne, anemia, abnormal healing, dyslipidemia, depression, diarrhea, edema, headache/migraine, hypercholesterolemia, incisional hernia, infection, leukopenia, mouth ulceration, pyrexia, proteinuria, pruritis, rash, and thrombocytopenia were higher in the mTORi than in the CNI group. mTORi reduced the recurrence incidence and overall 5-year mortality rate but increased many other incidences of AEs compared with that by CNI. Therefore, clinicians should be aware of the risks and benefits of mTORi use when managing patients undergoing LT for HCC.

Topics: Calcineurin Inhibitors; Carcinoma, Hepatocellular; Humans; Immunosuppression Therapy; Immunosuppressive Agents; Liver Neoplasms; Neoplasm Recurrence, Local; Sirolimus

Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer and occurs mainly in patients with liver cirrhosis. The mammalian target of rapamycin (mTOR) signaling pathway is involved in many hallmarks of cancer including cell growth, metabolism re-programming, proliferation and inhibition of apoptosis. The mTOR pathway is upregulated in HCC tissue samples as compared with the surrounding liver cirrhotic tissue. In addition, the activation of mTOR is more intense in the tumor edge, thus reinforcing its role in HCC proliferation and spreading. The inhibition of the mTOR pathway by currently available pharmacological compounds (i.e., sirolimus or everolimus) is able to hamper tumor progression both in vitro and in animal models. The use of mTOR inhibitors alone or in combination with other therapies is a very attractive approach, which has been extensively investigated in humans. However, results are contradictory and there is no solid evidence suggesting a true benefit in clinical practice. As a result, neither sirolimus nor everolimus are currently approved to treat HCC or to prevent tumor recurrence after curative surgery. In the present comprehensive review, we analyzed the most recent scientific evidence while providing some insights to understand the gap between experimental and clinical studies.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Proliferation; Everolimus; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Neoplasm Recurrence, Local; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Previous studies have indicated that sirolimus (SRL) may be effective for HCC patients undergoing liver transplantation (LT). However, the following results are still contradictory and do not have a clear conclusion. Therefore, we conducted an updated meta-analysis by retrieving published data in EMBASE, PubMed, and the Cochrane Library up to October 2017. Both efficiency and safety of SRL were analyzed using pooled odds ratio (ORs) with 95% confidence interval (CIs). A total of 11 studies involving 7,695 HCC patients were included. Compared with control group, SRL prolonged 1-year (OR = 2.44; CI = 1.66-3.59), 3 year (OR = 1.67; CI = 1.08-2.58) and 5-year (OR = 1.68; CI = 1.21-2.33) overall survival, as well as 1-year (OR = 2.13; CI = 1.19-3.81) disease-free survival. Pooled results found that SRL-treated patients had lower recurrence (OR = 0.60; CI = 0.37-0.98), lower recurrence-related mortality (OR = 0.58; CI = 0.42-0.81) and lower overall mortality (OR = 0.62; CI = 0.44-0.89). Moreover, fewer SRL-treated patients suffered from portal vein thrombosis (OR = 0.29; CI, 0.09-0.91) and diabetes (OR = 0.23; CI = 0.12-0.47), while SRL-treated patients were more vulnerable to acne compared with the control group (OR = 4.44; CI = 1.56-12.60). No significant differences in other adverse effects were found between two groups. Taken together, SRL-based immunosuppression is safe and effective in improving survival, as well as reducing recurrence and mortality for HCC patients following LT.

Topics: Carcinoma, Hepatocellular; Diabetes Mellitus; Disease-Free Survival; Graft Rejection; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Neoplasm Recurrence, Local; Portal Vein; Postoperative Complications; Sirolimus; Venous Thrombosis

Calcineurin-inhibitor immunosuppressants (tacrolimus and ciclosporin) have been associated with an exposure-related increase in tumour recurrence following liver transplantation for hepatocellular carcinoma (HCC). Conversely, mechanistic target of rapamycin (mTOR) inhibitors (sirolimus and everolimus) have been suggested to reduce recurrence rates and improve survival in this patient group.. To clarify the potential benefit of mTOR-inhibitors in HCC transplant patients by comparing recurrence and survival outcomes with calcineurin-inhibitor-based immunosuppression.. A systematic review and meta-analysis was performed. The inclusion criteria were observational or interventional studies reporting the effect of early-initiated (<6 months post-transplant) mTOR-inhibitor-based immunosuppression on survival or tumour recurrence in patients transplanted with HCC, compared to a control of calcineurin-inhibitor-based therapy.. Meta-analysis demonstrated that compared with calcineurin-inhibitor controls, recurrence-free-survival was significantly increased with mTOR-inhibitor-based therapy at 1-year (Risk-Ratio (RR): 1.09, 95% CI: 1.01-1.18) and 3-years (RR: 1.1, 95% CI: 1.01-1.21) post-transplant, with a nonsignificant increase at 5-years (RR: 1.15, 95% CI: 0.99-1.35). Overall survival was improved at 1-year (RR: 1.07, 95% CI: 1.02-1.12), 3-years (RR: 1.1, 95% CI: 1.02-1.19), and 5-years (RR: 1.18, 95% CI: 1.08-1.29). Recurrence-rate was lower in the mTOR-inhibitor arm (RR: 0.67, 95% CI: 0.56-0.82), with no significant increase in acute rejection (RR: 1.1, 95% CI: 0.94-1.28).. mTOR-inhibitor-based immunosuppression may be a preferable option in patients transplanted with HCC. It improves recurrence-free-survival over at least three years and reduces the recurrence rate compared with standard calcineurin-inhibitor-based therapy, with no significant increase in the rate of acute rejection. Future research should clarify the effect in higher vs lower risk cohorts.

Topics: Calcineurin Inhibitors; Carcinoma, Hepatocellular; Cyclosporine; Everolimus; Humans; Immunosuppression Therapy; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Neoplasm Recurrence, Local; Sirolimus; Tacrolimus; TOR Serine-Threonine Kinases

Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase in the downstream of the phosphatidylinositol 3-kinases (PI3K) family. This kinase plays an important role in the development and progression of hepatocellular carcinoma (HCC). Preclinical data demonstrate that 40%-50% of HCC patients have dysregulated expression of the effectors of the mTOR signaling pathway, and the activation of the mTOR pathway is associated with poorly differentiated tumors, early tumor recurrence, and poor survival/prognosis. This article reviews the research advances in the potential role of the mTOR signaling pathway and its inhibitors in the treatment of HCC.. 哺乳动物雷帕霉素靶蛋白（mTOR）是磷酸肌醇-3-激酶（PI3K）家族下游的丝氨酸/苏氨酸蛋白激酶。该激酶在肝细胞癌发生和发展中起着重要作用。临床前数据表明mTOR信号通路效应物的失调表达存在于40％～50％的肝细胞癌中，并且mTOR通路的激活与较低分化的肿瘤、较早的肿瘤复发和较差的生存预后相关。现对mTOR信号通路及其抑制剂在肝细胞癌中的研究进展进行综述。.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Humans; Liver Neoplasms; Neoplasm Recurrence, Local; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Sirolimus; TOR Serine-Threonine Kinases

Middle East Respiratory Syndrome (MERS) is a novel respiratory illness firstly reported in Saudi Arabia in 2012. It is caused by a new corona virus, called MERS corona virus (MERS-CoV). Most people who have MERS-CoV infection developed severe acute respiratory illness.. This work is done to determine the clinical characteristics and the outcome of intensive care unit (ICU) admitted patients with confirmed MERS-CoV infection.. This study included 32 laboratory confirmed MERS corona virus infected patients who were admitted into ICU. It included 20 (62.50%) males and 12 (37.50%) females. The mean age was 43.99 ± 13.03 years. Diagnosis was done by real-time reverse transcription polymerase chain reaction (rRT-PCR) test for corona virus on throat swab, sputum, tracheal aspirate, or bronchoalveolar lavage specimens. Clinical characteristics, co-morbidities and outcome were reported for all subjects.. Most MERS corona patients present with fever, cough, dyspnea, sore throat, runny nose and sputum. The presence of abdominal symptoms may indicate bad prognosis. Prolonged duration of symptoms before patients' hospitalization, prolonged duration of mechanical ventilation and hospital stay, bilateral radiological pulmonary infiltrates, and hypoxemic respiratory failure were found to be strong predictors of mortality in such patients. Also, old age, current smoking, smoking severity, presence of associated co-morbidities like obesity, diabetes mellitus, chronic heart diseases, COPD, malignancy, renal failure, renal transplantation and liver cirrhosis are associated with a poor outcome of ICU admitted MERS corona virus infected patients.. Plasma HO-1, ferritin, p21, and NQO1 were all elevated at baseline in CKD participants. Plasma HO-1 and urine NQO1 levels each inversely correlated with eGFR (. SnPP can be safely administered and, after its injection, the resulting changes in plasma HO-1, NQO1, ferritin, and p21 concentrations can provide information as to antioxidant gene responsiveness/reserves in subjects with and without kidney disease.. A Study with RBT-1, in Healthy Volunteers and Subjects with Stage 3-4 Chronic Kidney Disease, NCT0363002 and NCT03893799.. HFNC did not significantly modify work of breathing in healthy subjects. However, a significant reduction in the minute volume was achieved, capillary [Formula: see text] remaining constant, which suggests a reduction in dead-space ventilation with flows > 20 L/min. (ClinicalTrials.gov registration NCT02495675).. 3 组患者手术时间、术中显性失血量及术后 1 周血红蛋白下降量比较差异均无统计学意义（. 对于肥胖和超重的膝关节单间室骨关节炎患者，采用 UKA 术后可获满意短中期疗效，远期疗效尚需进一步随访观察。.. Decreased muscle strength was identified at both time points in patients with hEDS/HSD. The evolution of most muscle strength parameters over time did not significantly differ between groups. Future studies should focus on the effectiveness of different types of muscle training strategies in hEDS/HSD patients.. These findings support previous adverse findings of e-cigarette exposure on neurodevelopment in a mouse model and provide substantial evidence of persistent adverse behavioral and neuroimmunological consequences to adult offspring following maternal e-cigarette exposure during pregnancy. https://doi.org/10.1289/EHP6067.. This RCT directly compares a neoadjuvant chemotherapy regimen with a standard CROSS regimen in terms of overall survival for patients with locally advanced ESCC. The results of this RCT will provide an answer for the controversy regarding the survival benefits between the two treatment strategies.. NCT04138212, date of registration: October 24, 2019.. Results of current investigation indicated that milk type and post fermentation cooling patterns had a pronounced effect on antioxidant characteristics, fatty acid profile, lipid oxidation and textural characteristics of yoghurt. Buffalo milk based yoghurt had more fat, protein, higher antioxidant capacity and vitamin content. Antioxidant and sensory characteristics of T. If milk is exposed to excessive amounts of light, Vitamins B. The two concentration of ZnO nanoparticles in the ambient air produced two different outcomes. The lower concentration resulted in significant increases in Zn content of the liver while the higher concentration significantly increased Zn in the lungs (p < 0.05). Additionally, at the lower concentration, Zn content was found to be lower in brain tissue (p < 0.05). Using TEM/EDX we detected ZnO nanoparticles inside the cells in the lungs, kidney and liver. Inhaling ZnO NP at the higher concentration increased the levels of mRNA of the following genes in the lungs: Mt2 (2.56 fold), Slc30a1 (1.52 fold) and Slc30a5 (2.34 fold). At the lower ZnO nanoparticle concentration, only Slc30a7 mRNA levels in the lungs were up (1.74 fold). Thus the two air concentrations of ZnO nanoparticles produced distinct effects on the expression of the Zn-homeostasis related genes.. Until adverse health effects of ZnO nanoparticles deposited in organs such as lungs are further investigated and/or ruled out, the exposure to ZnO nanoparticles in aerosols should be avoided or minimised.

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The mammalian target of rapamycin (mTOR) inhibitor everolimus is approved for rejection prophylaxis after liver transplantation. The current article pools the experience of French liver transplant surgeons and physicians in use of everolimus and, particularly, practical guidance on dosing, appropriate concomitant immunosuppression and management of adverse events. In terms of indication, introduction of everolimus from week 4 after liver transplantation, with or without concomitant calcineurin inhibitor (CNI) therapy, offers a significant renal benefit without loss of immunosuppressive efficacy. De novo treatment with everolimus, either selectively or systematically, may play a role in the prevention and treatment of recurrence of hepatocellular cancer and de novo malignancies. For maintenance patients, the most frequent indications for introducing everolimus are in response to renal dysfunction, recurrent hepatocellular cancer, diabetes, hypertension, or neurotoxicity, or as a preventative approach to avoid malignancies. Of these, the strongest evidence exists for a renoprotective effect. However, the low rate of acute rejection following switch of maintenance patients from CNI-based to everolimus-based therapy means that this can be considered even where robust data are not yet available. Most adverse events associated with mTOR inhibitors can usually be managed successfully, often with concentration-controlled dose reductions. Dosing algorithms are provided, with suggestions for target ranges in specific settings, and treatment strategies for the most common side effects are proposed. Although further research is required, everolimus has become an established part of the immunosuppressive arsenal for liver transplant recipients over the last decade. Sharing experience from units which have embraced its use may help other centers develop their own protocols.

Topics: Carcinoma, Hepatocellular; Everolimus; France; Graft Rejection; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Neoplasm Recurrence, Local; Sirolimus

Prevention of hepatocellular carcinoma (HCC) recurrence after liver transplantation is a clinical priority. The importance of the mammalian target of rapamycin (mTOR) pathway in cell growth and survival makes it a logical target for antitumor strategies, as borne out by clinical data in various types of malignancy. A number of studies have indicated that the mTOR inhibitors everolimus and sirolimus suppress cell proliferation and tumor growth in animal models of HCC. Coadministration of an mTOR inhibitor could permit lower dosing of chemotherapeutic agents in HCC management, and trials in non-transplant HCC population are exploring combined used with various agents including sorafenib, the vascular endothelial growth factor inhibitor bevacizumab and conventional agents. In terms of a preventive effect after liver transplantation for HCC, data from retrospective studies and non-randomized prospective analyses in which patients received an mTOR inhibitor with concomitant calcineurin inhibitor therapy have indicated that HCC recurrence rates and overall survival may be improved compared to a standard calcineurin inhibitor regimen. Meta-analyses have supported these findings, but controlled trials are required before any firm conclusions can be drawn. In two of the three randomized trials which have assessed de novo mTOR inhibitor therapy after liver transplantation, there was a numerically lower rate of HCC recurrence by one year post-transplant in patients given an mTOR inhibitor versus the control arm, but absolute numbers were low. Overall, based on the available data from retrospective studies, meta-analyses, and post-hoc assessments of randomized trials, it appears advisable to consider mTOR inhibition-based immunosuppression after transplantation for HCC, particularly in patients who exceed the Milan criteria. Prospective data are awaited.

Topics: Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Hepatocellular; Everolimus; Female; Humans; Liver Neoplasms; Liver Transplantation; Male; Neoplasm Recurrence, Local; Prognosis; Randomized Controlled Trials as Topic; Risk Assessment; Sirolimus; TOR Serine-Threonine Kinases; Treatment Outcome

Mechanistic target of rapamycin (mTOR) regulates cell growth, metabolism and aging in response to nutrients, cellular energy stage and growth factors. mTOR is frequently up-regulated in cancer including hepatocellular carcinoma (HCC) and is associated with bad prognosis, poorly differentiated tumors, and earlier recurrence. Blocking mTOR with rapamycin and first generation mTOR inhibitors, called rapalogs, has shown promising reduction of HCC tumor growth in preclinical models. Currently, rapamycin/rapalogs are used in several clinical trials for the treatment of advanced HCC, and as adjuvant therapy in HCC patients after liver transplantation and TACE. A second generation of mTOR pathway inhibitors has been developed recently and is being tested in various clinical trials of solid cancers, and has been used in preclinical HCC models. The results of series of clinical trials using mTOR inhibitors in HCC treatment will emerge in the near future.

Topics: Animals; Carcinoma, Hepatocellular; Chemoembolization, Therapeutic; Clinical Trials as Topic; Humans; Liver Neoplasms; Liver Neoplasms, Experimental; Liver Transplantation; Models, Biological; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Calcineurin inhibitors (CNIs) have been associated in a dose-dependent fashion with an increased risk of post-transplant hepatocellular carcinoma (HCC) recurrence. The mammalian target of rapamycin inhibitors (mTORi) (sirolimus/everolimus) might represent an alternative immunosuppressive regimen with antineoplastic effect. In the present systematic review, the association between mTORi and HCC recurrence after liver transplantation (LT) was evaluated and compared against that of CNIs-treated patients. In total, 3666 HCC liver transplant recipients from 42 studies met the inclusion criteria. Patients under CNIs developed HCC recurrence significantly more frequently, compared with patients under mTORi (448/3227 or 13.8% vs. 35/439 or 8%, P < 0.001), although patients treated with CNIs had a higher proportion of HCC within Milan criteria (74% vs. 69%) and lower rates of microvascular invasion, compared with mTORi-treated patients (22% vs. 44%) (P < 0.05). Patients on everolimus had significantly lower recurrence rates of HCC, compared with those on sirolimus or CNIs (4.1% vs. 10.5% vs. 13.8%, respectively, P < 0.05), but everolimus-treated recipients had shorter follow-up period (13 vs. 30 vs. 43.2 months, respectively) and more frequently been transplanted for HCC within Milan criteria (84% vs. 60.5% vs. 74%, respectively, P < 0.05). Our findings favor the use of mTORi instead of CNIs to control HCC recurrence after LT, but comparative studies with longer follow-up are needed for final conclusions.

Topics: Calcineurin Inhibitors; Carcinoma, Hepatocellular; Cohort Studies; Drug Delivery Systems; Everolimus; Female; Graft Rejection; Graft Survival; Humans; Immunosuppressive Agents; Incidence; Liver Neoplasms; Liver Transplantation; Male; Neoplasm Recurrence, Local; Prognosis; Randomized Controlled Trials as Topic; Sirolimus; Treatment Outcome

The development of immunosuppression has significantly affected the development of liver transplantation and has helped to switch from the experimental method to a standard treatment of life threatening liver conditions. Tacrolimus is the basic immunosuppressant for patients after a liver transplant and thanks to its prolonged-release dosage form, which due to its simplicity and reliability of use, replaces tacrolimus twice daily early after the transplant and in the longterm administration, will apparently, for a while, defend its position. Other widely used medicines include mycophenolic acid and mTOR inhibitors, sirolimus and everolimus. The induction with antilymphocyte antibodies is used in less than 10% of liver recipients. Only a few new immunosuppresants in this century have passed later stages of clinical studies; the last 2 medicines registered for patients after liver transplantation include Advagraf (Astellas) and Certican (Novartis). Personalised immunosuppression should respect at least the following basic clinical situations: recipients renal function, hepatitis C virus infection, and hepatocellular carcinoma as the liver transplant indication. The results of immunotolerance bio-marker research are necessary for a more successful conduct of protocols minimising immunosuppression and leading to immunotolerance, especially under the efforts of complete withdrawal of immunosupression.

Topics: Antilymphocyte Serum; Carcinoma, Hepatocellular; Everolimus; Graft Rejection; Humans; Immunosuppressive Agents; Liver Diseases; Liver Failure; Liver Neoplasms; Liver Transplantation; Mycophenolic Acid; Reproducibility of Results; Sirolimus; Tacrolimus

Everolimus is an orally administrated mammalian target of rapamycin (mTOR) inhibitor. Several large-scale randomized controlled trials (RCTs) have demonstrated the survival benefits of everolimus at the dose of 10 mg/day for solid cancers. Furthermore, mTOR-inhibitor-based immunosuppression is associated with survival benefits for patients with hepatocellular carcinoma (HCC) who have received liver transplantation. However, a low rate of tumor reduction and some adverse events have been pointed out. This review summarizes the antitumor effects and adverse events of everolimus and evaluates its possible application in advanced HCC. For the meta-analysis of adverse events, we used the RCTs for solid cancers. The odds ratios of adverse events were calculated using the Peto method. Manypreclinical studies demonstrated that everolimus had antitumor effects such as antiproliferation and antiangiogenesis. However, some differences in the effects were observed among in vivo animal studies for HCC treatment. Meanwhile, clinical studies demonstrated that the response rate of single-agent everolimus was low, though survival benefits could be expected. The meta-analysis revealed the odds ratios (95% confidence interval [CI]) of stomatitis: 5.42 [4.31-6.73], hyperglycemia: 3.22 [2.37-4.39], anemia: 3.34 [2.37-4.67], pneumonitis: 6.02 [3.95-9.16], aspartate aminotransferase levels: 2.22 [1.37-3.62], and serum alanine aminotransferase levels: 2.94 [1.72-5.02], respectively. Everolimus at the dose of 10 mg/day significantly increased the risk of the adverse events. In order to enable its application to the standard conventional therapies of HCC, further studies are required to enhance the antitumor effects and manage the adverse events of everolimus.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Everolimus; Humans; Liver Neoplasms; Sirolimus; TOR Serine-Threonine Kinases

The aim of this study was to assess the safety and efficacy of sorafenib, with or without everolimus, in the treatment of recurrent hepatocellular carcinoma (HCC) after an orthotopic liver transplantation (OLT).. We reviewed the outcome of our consecutive cohort series of patients. Eleven patients (nine men) with recurrent HCC after OLT were treated. Four patients received cyclosporine plus sorafenib at a starting dose of 400 mg twice daily; seven received the combination of sorafenib (same dosage) and everolimus. Sorafenib was reduced or stopped according to the drug label.. The median time to recurrence was 12 months (range 2-66). The mean age at the start of treatment was 57 ± 9 years. Sorafenib was withdrawn because of intolerance or side-effects in four (36%) patients. Dose reduction because of adverse events or intolerance was required in 91% of patients after 26 ± 11 days from the start of treatment. The average length of treatment was 68 days (range 15-444). One patient died because of a massive gastrointestinal bleeding while receiving sorafenib and everolimus. The most frequent adverse events were fatigue (54%), skin toxicity (45%), and hypophosphatemia (36%). Two patients (18%) showed a radiological partial response, one (9%) had a stable disease, and six (54%) showed a progressive disease. None of the patients achieved a complete response. Treatment response could not be assessed in two (18%) patients. The overall median survival since the start of treatment was 5 months. One-year survival was 18%.. Sorafenib, with or without mammalian target of rapamycin inhibitors, is poorly tolerated and rarely effective in the treatment of recurrent HCC after OLT.

Topics: Aged; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Hepatocellular; Drug Evaluation; Everolimus; Female; Humans; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Niacinamide; Phenylurea Compounds; Recurrence; Retrospective Studies; Sirolimus; Sorafenib; Survival Analysis; Treatment Outcome

The use of sirolimus (SRL)-based immunosuppression protocols have been reported to reduce recurrence rates following liver transplantation (LT) for hepatocellular carcinoma (HCC), although this is still a matter for debate.. To undertake a systematic review and meta-analysis of available literature on the usage of SRL as an immunosuppressive agent following LT for HCC, with a view to comparing cancer outcomes with the commonly used calcineurin inhibitors (CNIs).. Systematic review and meta-analysis carried out in line with the Meta-analysis Of Observational Studies in Epidemiology (MOOSE) guidelines. Primary outcomes of interest were tumour recurrence rate and recurrence-free survival (RFS). Secondary outcomes were recurrence-related mortality and overall survival (OS).. In all, 5 studies met the inclusion criteria (n = 474). The recurrence rate was lower in SRL group (4.9-12.9%) in comparison with CNIs (17.3-38.7%). The 1-, 3- and 5-year RFS was 93-96%, 82-86% and 79-80% for SRL group, which was much better in comparison with the CNIs 70-78%, 64-65% and 54-60% respectively. Similarly, 1-, 3- and 5-year OS was much better for SRL group (94-95%, 85% and 80%) in comparison with CNIs (79-83%, 66% and 59-62%) respectively. Meta-analysis demonstrated lower recurrence (OR = 0.30, 95% CI = 0.16-0.55, P < 0.001), lower recurrence-related mortality (OR = 0.29, 95% CI = 0.12-0.70, P = 0.005) and lower overall mortality (OR = 0.35, 95% CI = 0.20-0.61, P < 0.001) for SRL group.. The review showed lower recurrence rate, longer recurrence-free survival and overall survival and lower recurrence-related mortality in sirolimus-treated patients in comparison with the calcineurin inhibitor-treated patients following liver transplantation for hepatocellular carcinoma.

Topics: Carcinoma, Hepatocellular; Graft Survival; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Neoplasm Recurrence, Local; Sirolimus; Survival Rate

Sirolimus (SRL) is a novel immunosuppressant with antitumor properties. We performed a meta-analysis to determine whether SRL can improve patient survival and decrease the risks of tumor recurrence in patients with a pretransplant diagnosis of hepatocellular carcinoma (HCC). We searched databases for controlled clinical trials assessing the survival and oncological benefits of SRL for liver transplant recipients with pretransplant HCC. Five studies with a total of 2950 participants were included in this study. In comparison with SRL-free regimens, SRL-based regimens improved overall survival at 1 [odds ratio (OR) = 4.53, 95% confidence interval (95% CI) = 2.31-8.89], 3 (OR = 1.97, 95% CI = 1.29-3.00), and 5 years (OR = 2.47, 95% CI = 1.72-3.55). The pooled results showed that in comparison with SRL-free regimens, SRL-based regimens decreased tumor recurrence (OR = 0.42, 95% CI = 0.21-0.83). No significant differences in the frequencies of episodes of major posttransplant complications were observed between the groups. In conclusion, SRL is generally safe and prolongs patient survival in liver transplant recipients with pretransplant HCC.

Topics: Carcinoma, Hepatocellular; Female; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Male; Neoplasm Recurrence, Local; Risk Factors; Sirolimus; Survival Rate

Topics: alpha-Fetoproteins; Antineoplastic Agents; Benzenesulfonates; Biomarkers, Tumor; Carcinoma, Hepatocellular; Chemoembolization, Therapeutic; Diagnostic Imaging; Gene Expression Profiling; Humans; Immunosuppressive Agents; Liver Function Tests; Liver Neoplasms; Liver Transplantation; Niacinamide; Phenylurea Compounds; Prognosis; Pyridines; Sirolimus; Sorafenib; Survival Rate; Treatment Outcome

The development of calcineurin inhibitors (CNIs) led to marked improvements in patient and graft survival after liver transplantation (LTx). We have been left, however, with a dependence on immunosuppressive agents with nephrotoxicity, neurotoxicity, adverse impacts on cardiac risk profile, and risk for malignancy. These challenges need to be met against a dominance of hepatitis C virus (HCV) and hepatocellular carcinoma (HCC) as indications for liver transplant. Unmet needs for immunosuppression (IS) in LTx include: (1) Effective drugs that avoid CNIs toxicities. (2) Agents without adverse impact on HCV recurrence. (3) Compounds that minimize risk of HCC recurrence. New immunosuppressives will need to address the above needs while supporting patient and graft survival equivalent to those achievable with CNIs, ideally without important new toxicities. Two new classes of agents are currently in advanced clinical development: belatacept, and the mammalian target of rapamycin inhibitors (m-TORi). This manuscript will review evidence for a role for m-TORi in LTx in a range of clinical scenarios including patients with CNI nephrotoxicity or neurotoxicity, patients at risk of (or with) HCV recurrence, and patients at risk of HCC recurrence.

Topics: Adaptive Immunity; Calcineurin Inhibitors; Carcinoma, Hepatocellular; Clinical Trials as Topic; Everolimus; Hepatitis C, Chronic; Humans; Immunity, Innate; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Hepatocellular carcinoma (HCC) has an aggressive clinical course with frequent recurrence and metastasis. Orthotopic liver transplantation has been the only curative tool for unresectable HCC; therefore, recent advances in molecular targeted therapy may improve the prognosis of HCC. The multiple kinase inhibitor sorafenib and the macrolide antibiotic rapamycin are currently the most promising agents for treating unresectable HCC. A large population-based clinical trial revealed that sorafenib significantly prolonged the overall survival of HCC patients. However, subsequent clinical studies showed that sorafenib rarely reduced tumor volume and inadequately prolonged survival of patients with severe liver damage. To improve its therapeutic effect, the development of a predictive biomarker and a sorafenib-based combination is awaited. Another molecular targeting agent, rapamycin, has now been considered as a putative agent for preventing tumor recurrence in post-liver transplantation HCC patients, because it not only has immunosuppressive activity but also exerts an anti-tumor effect. In the near future, a combination of molecular targeting agents, such as sorafenib and rapamycin, may become a standard protocol for treating unresectable HCC. For specifying cases with more effective and less harmful modalities, further investigation in clinical and basic research to identify unexpected effects are needed.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Benzenesulfonates; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Molecular Targeted Therapy; Neoplasm Recurrence, Local; Niacinamide; Phenylurea Compounds; Proto-Oncogene Proteins c-raf; Pyridines; Randomized Controlled Trials as Topic; Signal Transduction; Sirolimus; Sorafenib; TOR Serine-Threonine Kinases

Hepatocellular carcinoma (HCC) is a major health problem worldwide. Several molecular pathways involved in HCC growth and progression have recently been identified. Rapamycin analogs are able to inhibit one of the most active oncogenic molecular pathways in HCC cells: the mammalian target of rapamycin (mTOR) pathway.. In this review, the authors analyze the principal molecular features of the mTOR pathway and the use of rapamycin analogs in the treatment of hepatocarcinoma. The article also looks at the reoccurrence of HCC following liver transplantation as well as after the treatment of de novo neoplasms. Finally, the authors discuss the advantage of using a combined HCC pharmacological therapy to obtain a synergistic effect on tumor mass.. Among the available options for the treatment of advanced-stage HCC, mTOR pathway inhibitors show great promise. Once these agents have their safety and efficacy confirmed, in the treatment of liver disease, their use should be considered in patients affected by HCC. This should especially be the case for those who have had liver transplants or suffered with de novo tumors. Moreover, the authors believe that mTOR inhibitors could be used in a combined pharmacological approach to improve HCC molecular-targeted therapy by producing a multiple-level block of tumor intracellular signaling.

Topics: Animals; Carcinoma, Hepatocellular; Clinical Trials as Topic; Drug Evaluation, Preclinical; Enzyme Inhibitors; Humans; Liver Neoplasms; Liver Transplantation; Molecular Targeted Therapy; Neoplasm Staging; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Hepatocellular carcinoma (HCC) has a poor prognosis. Approximately 85% of patients are not candidates for curative treatments at the time of diagnosis; hence palliative modalities (transcatheter arterial chemoembolisation, radiofrequency ablation, systemic chemotherapy) are used. Systemic chemotherapies have disappointing results. The increasing knowledge in the molecular biology of HCC will increase the possibilities of targeted therapy. The multi-tyrosine kinase inhibitor sorafenib is the only drug which has approved. The VEGF-inhibitors (bevacizumab, sunitinib), EGFR-blocker agents (erlotinib), as well as the inhibition of mTOR (rapamycin) are promising. Combination of sorafenib or other anti-angiogenic agents with local ablative procedure (transcatheter arterial chemoembolisation, radiofrequency ablation), or with curative hepatectomy also can be favorable. Alteration of Wnt pathway, retinoid compounds, inhibition of the cell cycle as well as the proteosome, and epigenetic therapy can be other potential promising targets in HCC.

Topics: Angiogenesis Inhibitors; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Benzenesulfonates; Bevacizumab; Biomarkers, Tumor; Carcinoma, Hepatocellular; Catheter Ablation; Cell Cycle; Chemoembolization, Therapeutic; Chemotherapy, Adjuvant; Epigenesis, Genetic; ErbB Receptors; Erlotinib Hydrochloride; Gene Expression Regulation, Neoplastic; Hepatectomy; Humans; Indoles; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Niacinamide; Phenylurea Compounds; Proteasome Endopeptidase Complex; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Pyridines; Pyrroles; Quinazolines; Signal Transduction; Sirolimus; Sorafenib; Sunitinib; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor A; Wnt Proteins

Topics: Angiogenesis Inhibitors; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antimetabolites, Antineoplastic; Antineoplastic Agents; Benzamides; Benzenesulfonates; Bevacizumab; Carcinoma, Hepatocellular; Carcinoma, Renal Cell; Deoxycytidine; Drug Delivery Systems; Gastrointestinal Stromal Tumors; Gemcitabine; Humans; Imatinib Mesylate; Indoles; Neoplasms; Niacinamide; Pancreatic Neoplasms; Phenylurea Compounds; Piperazines; Pyridines; Pyrimidines; Pyrroles; Sirolimus; Sorafenib; Sunitinib

Sirolimus (SRL) is a macrolide antibiotic that has potent antifungal and immunosuppressive properties; preclinical studies suggest that SRL may possess a significant antiproliferative influence in vitro. Recently, several studies have documented a negative effect by SRL on both primary tumor growth and the proliferation of metastatic foci in various rodent models of hepatocellular carcinoma (HCC). Orthotopic liver transplantation (OLT) is increasingly becoming a viable treatment option for patients with end stage liver disease and concomitant HCC. As such, an immunosuppressive agent with antineoplastic activity is inherently attractive in the setting of OLT for malignancy. Regrettably, the cumulative experience with SRL-based immunosuppression in this patient population is limited. Herein, the authors review the experience to date with SRL as a primary immunosuppressive agent following OLT, and discuss the clinical implications of SRL-based therapy in OLT recipients with cirrhosis and cancer.

Topics: Carcinoma, Hepatocellular; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Sirolimus

As newer immunosuppressive regimens have steadily reduced the incidence of acute rejection and have extended the life expectancy of allograft recipients, posttransplant malignancy has become an important cause of mortality. In fact, it is expected that cancer will surpass cardiovascular complications as the leading cause of death in transplant patients within the next 2 decades. An understanding of the underlying pathobiology and how to minimize cancer risks in transplant recipients are essential. The etiology of posttransplant malignancy is believed to be multifactorial and likely involves impaired immunosurveillance of neoplastic cells as well as depressed antiviral immune activity with a number of common posttransplant malignancies being viral-related. Although calcineurin inhibitors and azathioprine have been linked with posttransplant malignancies, newer agents such as mycophenolate mofetil and sirolimus have not and indeed may have antitumor properties. Long-term data are needed to determine if the use of these agents will ultimately lower the mortality due to malignancy for transplant recipients.

Topics: Azathioprine; Calcineurin Inhibitors; Carcinoma, Hepatocellular; Cyclosporine; Hepatitis, Viral, Human; Humans; Immunosuppressive Agents; Incidence; Kidney Transplantation; Lymphoproliferative Disorders; Mycophenolic Acid; Neoplasms; Sirolimus; Skin Neoplasms; Tacrolimus; Transplantation Immunology; Transplantation, Homologous

Mammalian target of rapamycin (mTOR) inhibitor as an attractive drug target with promising antitumor effects has been widely investigated. High quality clinical trial has been conducted in liver transplant (LT) recipients in Western countries. However, the pertinent studies in Eastern world are paucity. Therefore, we designed a clinical trial to test whether sirolimus can improve recurrence-free survival (RFS) in hepatocellular carcinoma (HCC) patients beyond the Milan criteria after LT. This is an open-labeled, single-arm, prospective, multicenter, and real-world study aiming to evaluate the clinical outcomes of early switch to sirolimus-based regimens in HCC patients after LT. Patients with a histologically proven HCC and beyond the Milan criteria will be enrolled. The initial immunosuppressant regimens are center-specific for the first 4-6 weeks. The following regimens integrated sirolimus into the regimens as a combination therapy with reduced calcineurin inhibitors based on the condition of patients and centers. The study is planned for 4 years in total with a 2-year enrollment period and a 2-year follow-up. We predict that sirolimus conversion regimen will provide survival benefits for patients particular in the key indicator RFS as well as better quality of life. If the trial is conducted successfully, we will have a continued monitoring over a longer follow-up time to estimate indicator of overall survival. We hope that the outcome will provide better evidence for clinical decision-making and revising treatment guidelines based on Chinese population data. Trial register: Trial registered at http://www.chictr.org.cn: ChiCTR2100042869.

Topics: Carcinoma, Hepatocellular; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Multicenter Studies as Topic; Neoplasm Recurrence, Local; Prospective Studies; Quality of Life; Sirolimus; Treatment Outcome

We investigate whether low-dose rapamycin is effective in preventing hepatocellular carcinoma (HCC) growth and treating HCC after tumor development in transgenic mice.. We established transgenic mice with HCC induced by activated HrasG12V and p53 suppression. Transgenic mice were randomly assigned to five experimental groups: negative control, positive control, tacrolimus only, rapamycin only, and tacrolimus plus rapamycin. The mice were further divided into two groups according to time to commencement of immunosuppressant treatment: de novo treatment and post-tumor development.. In the de novo treatment group, marked suppression of tumor growth was observed in the rapamycin only group. In the post-tumor development group, the rapamycin only group displayed no significant suppression of tumor growth, compared to the positive control group. In T lymphocyte subset analysis, the numbers of CD4. Low-dose rapamycin might be effective to prevent HCC growth, but may be ineffective as a treatment option after HCC development.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Liver Neoplasms; Mice; Mice, Transgenic; Sirolimus; Tacrolimus

The aim of this study was to evaluate the survival benefit of sirolimus in patients undergoing liver transplantation (LT) for hepatocellular carcinoma (HCC) (exploratory analysis of the SiLVER-trial).. Patients receiving LT) for HCC are at a high risk for tumor recurrence. Calcineurin inhibitors have shown evidence to promote cancer growth, whereas mammalian target of rapamycin (mTOR) inhibitors like sirolimus have anticancer effects. In the SiLVER-trial (Clinicaltrials.gov: NCT00355862), the effect of sirolimus on the recurrence of HCC after LT was investigated in a prospective randomized trial. Although the primary endpoint of improved disease-free survival (DFS) with sirolimus was not met, outcomes were improved for patients in the sirolimus-treatment arm in the first 3 to 5 years. To learn more about the key variables, a multivariate analysis was performed on the SiLVER-trial data.. Data from 508 patients of the intention-to-treat analysis were included in exploratory univariate and multivariate models for overall survival (OS), DFS and a competing risk analysis for HCC recurrence.. Sirolimus use for ≥3 months after LT for HCC independently reduced the hazard for death in the multivariate analysis [hazard ratio (HR): 0.7 (95% confidence interval, CI: 0.52-0.96, P = 0.02). Most strikingly, patients with an alpha-fetoprotein (AFP) ≥10 ng/mL and having used sirolimus for ≥3 months, benefited most with regard to OS, DFS, and HCC-recurrence (HR: 0.49-0.59, P = 0.0079-0.0245).. mTOR-inhibitor treatment with sirolimus for ≥3 months improves outcomes in LT for HCC, especially in patients with AFP-evidence of higher tumor activity, advocating particularly for mTOR inhibitor use in this subgroup of patients.. EudraCT: 2005-005362-36 CLINICALTRIALS.GOV:: NCT00355862.

Topics: Aged; Carcinoma, Hepatocellular; Female; Humans; Immunosuppressive Agents; Intention to Treat Analysis; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Neoplasm Recurrence, Local; Sirolimus; Survival Rate

Middle East Respiratory Syndrome (MERS) is a novel respiratory illness firstly reported in Saudi Arabia in 2012. It is caused by a new corona virus, called MERS corona virus (MERS-CoV). Most people who have MERS-CoV infection developed severe acute respiratory illness.. This work is done to determine the clinical characteristics and the outcome of intensive care unit (ICU) admitted patients with confirmed MERS-CoV infection.. This study included 32 laboratory confirmed MERS corona virus infected patients who were admitted into ICU. It included 20 (62.50%) males and 12 (37.50%) females. The mean age was 43.99 ± 13.03 years. Diagnosis was done by real-time reverse transcription polymerase chain reaction (rRT-PCR) test for corona virus on throat swab, sputum, tracheal aspirate, or bronchoalveolar lavage specimens. Clinical characteristics, co-morbidities and outcome were reported for all subjects.. Most MERS corona patients present with fever, cough, dyspnea, sore throat, runny nose and sputum. The presence of abdominal symptoms may indicate bad prognosis. Prolonged duration of symptoms before patients' hospitalization, prolonged duration of mechanical ventilation and hospital stay, bilateral radiological pulmonary infiltrates, and hypoxemic respiratory failure were found to be strong predictors of mortality in such patients. Also, old age, current smoking, smoking severity, presence of associated co-morbidities like obesity, diabetes mellitus, chronic heart diseases, COPD, malignancy, renal failure, renal transplantation and liver cirrhosis are associated with a poor outcome of ICU admitted MERS corona virus infected patients.. Plasma HO-1, ferritin, p21, and NQO1 were all elevated at baseline in CKD participants. Plasma HO-1 and urine NQO1 levels each inversely correlated with eGFR (. SnPP can be safely administered and, after its injection, the resulting changes in plasma HO-1, NQO1, ferritin, and p21 concentrations can provide information as to antioxidant gene responsiveness/reserves in subjects with and without kidney disease.. A Study with RBT-1, in Healthy Volunteers and Subjects with Stage 3-4 Chronic Kidney Disease, NCT0363002 and NCT03893799.. HFNC did not significantly modify work of breathing in healthy subjects. However, a significant reduction in the minute volume was achieved, capillary [Formula: see text] remaining constant, which suggests a reduction in dead-space ventilation with flows > 20 L/min. (ClinicalTrials.gov registration NCT02495675).. 3 组患者手术时间、术中显性失血量及术后 1 周血红蛋白下降量比较差异均无统计学意义（. 对于肥胖和超重的膝关节单间室骨关节炎患者，采用 UKA 术后可获满意短中期疗效，远期疗效尚需进一步随访观察。.. Decreased muscle strength was identified at both time points in patients with hEDS/HSD. The evolution of most muscle strength parameters over time did not significantly differ between groups. Future studies should focus on the effectiveness of different types of muscle training strategies in hEDS/HSD patients.. These findings support previous adverse findings of e-cigarette exposure on neurodevelopment in a mouse model and provide substantial evidence of persistent adverse behavioral and neuroimmunological consequences to adult offspring following maternal e-cigarette exposure during pregnancy. https://doi.org/10.1289/EHP6067.. This RCT directly compares a neoadjuvant chemotherapy regimen with a standard CROSS regimen in terms of overall survival for patients with locally advanced ESCC. The results of this RCT will provide an answer for the controversy regarding the survival benefits between the two treatment strategies.. NCT04138212, date of registration: October 24, 2019.. Results of current investigation indicated that milk type and post fermentation cooling patterns had a pronounced effect on antioxidant characteristics, fatty acid profile, lipid oxidation and textural characteristics of yoghurt. Buffalo milk based yoghurt had more fat, protein, higher antioxidant capacity and vitamin content. Antioxidant and sensory characteristics of T. If milk is exposed to excessive amounts of light, Vitamins B. The two concentration of ZnO nanoparticles in the ambient air produced two different outcomes. The lower concentration resulted in significant increases in Zn content of the liver while the higher concentration significantly increased Zn in the lungs (p < 0.05). Additionally, at the lower concentration, Zn content was found to be lower in brain tissue (p < 0.05). Using TEM/EDX we detected ZnO nanoparticles inside the cells in the lungs, kidney and liver. Inhaling ZnO NP at the higher concentration increased the levels of mRNA of the following genes in the lungs: Mt2 (2.56 fold), Slc30a1 (1.52 fold) and Slc30a5 (2.34 fold). At the lower ZnO nanoparticle concentration, only Slc30a7 mRNA levels in the lungs were up (1.74 fold). Thus the two air concentrations of ZnO nanoparticles produced distinct effects on the expression of the Zn-homeostasis related genes.. Until adverse health effects of ZnO nanoparticles deposited in organs such as lungs are further investigated and/or ruled out, the exposure to ZnO nanoparticles in aerosols should be avoided or minimised.

Topics: A549 Cells; Acetylmuramyl-Alanyl-Isoglutamine; Acinetobacter baumannii; Acute Lung Injury; Adaptor Proteins, Signal Transducing; Adenine; Adenocarcinoma; Adipogenesis; Administration, Cutaneous; Administration, Ophthalmic; Adolescent; Adsorption; Adult; Aeromonas hydrophila; Aerosols; Aged; Aged, 80 and over; Aging; Agriculture; Air Pollutants; Air Pollution; Airway Remodeling; Alanine Transaminase; Albuminuria; Aldehyde Dehydrogenase 1 Family; Algorithms; AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase; Alzheimer Disease; Amino Acid Sequence; Ammonia; Ammonium Compounds; Anaerobiosis; Anesthetics, Dissociative; Anesthetics, Inhalation; Animals; Anti-Bacterial Agents; Anti-HIV Agents; Anti-Infective Agents; Anti-Inflammatory Agents; Antibiotics, Antineoplastic; Antibodies, Antineutrophil Cytoplasmic; Antibodies, Monoclonal, Humanized; Antifungal Agents; Antigens, Bacterial; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Antimetabolites, Antineoplastic; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Antioxidants; Antitubercular Agents; Antiviral Agents; Apolipoproteins E; Apoptosis; Arabidopsis; Arabidopsis Proteins; Arsenic; Arthritis, Rheumatoid; Asthma; Atherosclerosis; ATP-Dependent Proteases; Attitude of Health Personnel; Australia; Austria; Autophagy; Axitinib; Bacteria; Bacterial Outer Membrane Proteins; Bacterial Proteins; Bacterial Toxins; Bacterial Typing Techniques; Bariatric Surgery; Base Composition; Bayes Theorem; Benzoxazoles; Benzylamines; beta Catenin; Betacoronavirus; Betula; Binding Sites; Biological Availability; Biological Oxygen Demand Analysis; Biomarkers; Biomarkers, Tumor; Biopsy; Bioreactors; Biosensing Techniques; Birth Weight; Blindness; Blood Chemical Analysis; Blood Gas Analysis; Blood Glucose; Blood Pressure; Blood Pressure Monitoring, Ambulatory; Blood-Brain Barrier; Blotting, Western; Body Mass Index; Body Weight; Bone and Bones; Bone Density; Bone Resorption; Borates; Brain; Brain Infarction; Brain Injuries, Traumatic; Brain Neoplasms; Breakfast; Breast Milk Expression; Breast Neoplasms; Bronchi; Bronchoalveolar Lavage Fluid; Buffaloes; Cadherins; Calcification, Physiologic; Calcium Compounds; Calcium, Dietary; Cannula; Caprolactam; Carbon; Carbon Dioxide; Carboplatin; Carcinogenesis; Carcinoma, Ductal; Carcinoma, Ehrlich Tumor; Carcinoma, Hepatocellular; Carcinoma, Non-Small-Cell Lung; Carcinoma, Pancreatic Ductal; Carcinoma, Renal Cell; Cardiovascular Diseases; Carps; Carrageenan; Case-Control Studies; Catalysis; Catalytic Domain; Cattle; CD8-Positive T-Lymphocytes; Cell Adhesion; Cell Cycle Proteins; Cell Death; Cell Differentiation; Cell Line; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cell Phone Use; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Cellulose; Chemical Phenomena; Chemoradiotherapy; Child; Child Development; Child, Preschool; China; Chitosan; Chlorocebus aethiops; Cholecalciferol; Chromatography, Liquid; Circadian Clocks; Circadian Rhythm; Circular Dichroism; Cisplatin; Citric Acid; Clinical Competence; Clinical Laboratory Techniques; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Clostridioides difficile; Clostridium Infections; Coculture Techniques; Cohort Studies; Cold Temperature; Colitis; Collagen Type I; Collagen Type I, alpha 1 Chain; Collagen Type XI; Color; Connective Tissue Diseases; Copper; Coronary Angiography; Coronavirus 3C Proteases; Coronavirus Infections; Cost of Illness; Counselors; COVID-19; COVID-19 Testing; Creatine Kinase; Creatinine; Cross-Over Studies; Cross-Sectional Studies; Cryoelectron Microscopy; Cryosurgery; Crystallography, X-Ray; Cues; Cultural Competency; Cultural Diversity; Curriculum; Cyclic AMP Response Element-Binding Protein; Cyclin-Dependent Kinase Inhibitor p21; Cycloparaffins; Cysteine Endopeptidases; Cytokines; Cytoplasm; Cytoprotection; Databases, Factual; Denitrification; Deoxycytidine; Diabetes Complications; Diabetes Mellitus; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diagnosis, Differential; Diatoms; Diet; Diet, High-Fat; Dietary Exposure; Diffusion Magnetic Resonance Imaging; Diketopiperazines; Dipeptidyl Peptidase 4; Dipeptidyl-Peptidase IV Inhibitors; Disease Models, Animal; Disease Progression; Disease-Free Survival; DNA; DNA Damage; DNA Glycosylases; DNA Repair; DNA-Binding Proteins; DNA, Bacterial; DNA, Viral; Docetaxel; Dose Fractionation, Radiation; Dose-Response Relationship, Drug; Down-Regulation; Doxorubicin; Drosophila; Drosophila melanogaster; Drug Carriers; Drug Delivery Systems; Drug Liberation; Drug Repositioning; Drug Resistance, Bacterial; Drug Resistance, Multiple, Bacterial; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Drug Therapy, Combination; Edema; Edible Grain; Education, Graduate; Education, Medical, Graduate; Education, Pharmacy; Ehlers-Danlos Syndrome; Electron Transport Complex III; Electron Transport Complex IV; Electronic Nicotine Delivery Systems; 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Forced Expiratory Volume; Forests; Fractures, Bone; Fruit and Vegetable Juices; Fusobacteria; G1 Phase Cell Cycle Checkpoints; G2 Phase Cell Cycle Checkpoints; Gamma Rays; Gastrectomy; Gastrointestinal Microbiome; Gastrointestinal Stromal Tumors; Gefitinib; Gels; Gemcitabine; Gene Amplification; Gene Expression; Gene Expression Regulation; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Neoplastic; Gene Expression Regulation, Plant; Gene Knockdown Techniques; Gene-Environment Interaction; Genotype; Germany; Glioma; Glomerular Filtration Rate; Glucagon; Glucocorticoids; Glycemic Control; Glycerol; Glycogen Synthase Kinase 3 beta; Glycolipids; Glycolysis; Goblet Cells; Gram-Negative Bacterial Infections; Granulocyte Colony-Stimulating Factor; Graphite; Greenhouse Effect; Guanidines; Haemophilus influenzae; HCT116 Cells; Health Knowledge, Attitudes, Practice; Health Personnel; Health Services Accessibility; Health Services Needs and Demand; Health Status Disparities; Healthy Volunteers; Heart Failure; Heart Rate; Heart Transplantation; Heart-Assist Devices; HEK293 Cells; Heme; Heme Oxygenase-1; Hemolysis; Hemorrhage; Hepatitis B; Hepatitis B e Antigens; Hepatitis B Surface Antigens; Hepatitis B virus; Hepatitis B, Chronic; Hepatocytes; Hexoses; High-Throughput Nucleotide Sequencing; Hippo Signaling Pathway; Histamine; Histamine Agonists; Histidine; Histone Deacetylase 2; HIV Infections; HIV Reverse Transcriptase; HIV-1; Homebound Persons; Homeodomain Proteins; Homosexuality, Male; Hospice and Palliative Care Nursing; HSP70 Heat-Shock Proteins; Humans; Hyaluronan Receptors; Hydrogen; Hydrogen Peroxide; Hydrogen-Ion Concentration; Hydrolysis; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypoglycemia; Hypoglycemic Agents; Hypoxia; Idiopathic Interstitial Pneumonias; Imaging, Three-Dimensional; Imatinib Mesylate; Immunotherapy; Implementation Science; Incidence; INDEL Mutation; Induced Pluripotent Stem Cells; Industrial Waste; Infant; Infant, Newborn; Inflammation; Inflammation Mediators; Infliximab; Infusions, Intravenous; Inhibitory Concentration 50; Injections; Insecticides; Insulin-Like Growth Factor Binding Protein 5; Insulin-Secreting Cells; Interleukin-1; Interleukin-17; Interleukin-8; Internship and Residency; Intestines; Intracellular Signaling Peptides and Proteins; Ion Transport; Iridaceae; Iridoid Glucosides; Islets of Langerhans Transplantation; Isodon; Isoflurane; Isotopes; Italy; Joint Instability; Ketamine; Kidney; Kidney Failure, Chronic; Kidney Function Tests; Kidney Neoplasms; Kinetics; Klebsiella pneumoniae; Knee Joint; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Lactate Dehydrogenase 5; Laparoscopy; Laser Therapy; Lasers, Semiconductor; Lasers, Solid-State; Laurates; Lead; Leukocyte L1 Antigen Complex; Leukocytes, Mononuclear; Light; Lipid Peroxidation; Lipopolysaccharides; Liposomes; Liver; Liver Cirrhosis; Liver Neoplasms; Liver Transplantation; Locomotion; Longitudinal Studies; Lopinavir; Lower Urinary Tract Symptoms; Lubricants; Lung; Lung Diseases, Interstitial; Lung Neoplasms; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Lymphoma, Mantle-Cell; Lysosomes; Macrophages; Male; Manganese Compounds; MAP Kinase Kinase 4; Mass Screening; Maternal Health; Medicine, Chinese Traditional; Melanoma, Experimental; Memantine; Membrane Glycoproteins; Membrane Proteins; Mesenchymal Stem Cell Transplantation; Metal Nanoparticles; Metalloendopeptidases; Metalloporphyrins; Methadone; Methane; Methicillin-Resistant Staphylococcus aureus; Mexico; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred ICR; Mice, Knockout; Mice, Nude; Mice, SCID; Mice, Transgenic; Microarray Analysis; Microbial Sensitivity Tests; Microbiota; Micronutrients; MicroRNAs; Microscopy, Confocal; Microsomes, Liver; Middle Aged; Milk; Milk, Human; Minority Groups; Mitochondria; Mitochondrial Membranes; Mitochondrial Proteins; Models, Animal; Models, Molecular; Molecular Conformation; Molecular Docking Simulation; Molecular Dynamics Simulation; Molecular Epidemiology; 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Proto-Oncogene Proteins c-ret; Proto-Oncogene Proteins p21(ras); Proton Pumps; Protons; Protoporphyrins; Pseudomonas aeruginosa; Pseudomonas fluorescens; Pulmonary Artery; Pulmonary Disease, Chronic Obstructive; Pulmonary Gas Exchange; Pulmonary Veins; Pyrazoles; Pyridines; Pyrimidines; Qualitative Research; Quinoxalines; Rabbits; Random Allocation; Rats; Rats, Sprague-Dawley; Rats, Wistar; Receptors, Histamine H3; Receptors, Immunologic; Receptors, Transferrin; Recombinant Proteins; Recurrence; Reference Values; Referral and Consultation; Regional Blood Flow; Registries; Regulon; Renal Insufficiency, Chronic; Reperfusion Injury; Repressor Proteins; Reproducibility of Results; Republic of Korea; Research Design; Resistance Training; Respiration, Artificial; Respiratory Distress Syndrome; Respiratory Insufficiency; Resuscitation; Retinal Dehydrogenase; Retreatment; Retrospective Studies; Reverse Transcriptase Inhibitors; Rhinitis, Allergic; Ribosomal Proteins; Ribosomes; Risk Assessment; 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STAT3 Transcription Factor; Streptomyces coelicolor; Stress, Psychological; Stroke; Stroke Volume; Structure-Activity Relationship; Students, Medical; Students, Pharmacy; Substance Abuse Treatment Centers; Sulfur Dioxide; Surface Properties; Surface-Active Agents; Surveys and Questionnaires; Survival Analysis; Survival Rate; Survivin; Sweden; Swine; Swine, Miniature; Sympathetic Nervous System; T-Lymphocytes, Regulatory; Talaromyces; Tandem Mass Spectrometry; tau Proteins; Telemedicine; Telomerase; Telomere; Telomere Homeostasis; Temperature; Terminally Ill; Th1 Cells; Thiamethoxam; Thiazoles; Thiophenes; Thioredoxin Reductase 1; Thrombosis; Thulium; Thyroid Cancer, Papillary; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms; Time Factors; Titanium; Tomography, Emission-Computed, Single-Photon; Tomography, X-Ray Computed; TOR Serine-Threonine Kinases; Transcription Factor AP-1; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transcriptome; Transforming Growth Factor beta1; 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YAP-Signaling Proteins; Yogurt; Young Adult; Zebrafish; Zebrafish Proteins; Ziziphus

We investigated whether sirolimus-based immunosuppression improves outcomes in liver transplantation (LTx) candidates with hepatocellular carcinoma (HCC).. In a prospective-randomized open-label international trial, 525 LTx recipients with HCC initially receiving mammalian target of rapamycin inhibitor-free immunosuppression were randomized 4 to 6 weeks after transplantation into a group on mammalian target of rapamycin inhibitor-free immunosuppression (group A: 264 patients) or a group incorporating sirolimus (group B: 261). The primary endpoint was recurrence-free survival (RFS); intention-to-treat (ITT) analysis was conducted after 8 years. Overall survival (OS) was a secondary endpoint.. Recurrence-free survival was 64.5% in group A and 70.2% in group B at study end, this difference was not significant (P = 0.28; hazard ratio [HR], 0.84; 95% confidence interval [95% CI], 0.62; 1.15). In a planned analysis of RFS rates at yearly intervals, group B showed better outcomes 3 years after transplantation (HR, 0.7; 95% CI, 0.48-1.00). Similarly, OS (P = 0.21; HR, 0.81; 95% CI, 0.58-1.13) was not statistically better in group B at study end, but yearly analyses showed improvement out to 5 years (HR, 0.7; 95% CI, 0.49-1.00). Interestingly, subgroup (Milan Criteria-based) analyses revealed that low-risk, rather than high-risk, patients benefited most from sirolimus; furthermore, younger recipients (age ≤60) also benefited, as well sirolimus monotherapy patients. Serious adverse event numbers were alike in groups A (860) and B (874).. Sirolimus in LTx recipients with HCC does not improve long-term RFS beyond 5 years. However, a RFS and OS benefit is evident in the first 3 to 5 years, especially in low-risk patients. This trial provides the first high-level evidence base for selecting immunosuppression in LTx recipients with HCC.

Topics: Adult; Age Factors; Aged; Australia; Canada; Carcinoma, Hepatocellular; Disease Progression; Disease-Free Survival; Drug Therapy, Combination; Europe; Female; Humans; Immunosuppressive Agents; Intention to Treat Analysis; Kaplan-Meier Estimate; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Neoplasm Recurrence, Local; Prospective Studies; Risk Assessment; Risk Factors; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Treatment Outcome; Young Adult

There is strong rationale to combine temsirolimus (TEM) with Bevacizumab (BEV) for patients with advanced HCC.. A modified two-stage Simon phase II trial was performed with plans to advance to stage 2 if more than 2 patients had confirmed PR or >18 patients were progression free at 6 months out of 25 in stage 1. Toxicity, PFS and overall survival were secondary endpoints. Eligible pts had advanced HCC, Child Pugh A liver status and no prior systemic therapy involving the VEGF or m-TOR targeted agents. Patients were treated with temsirolimus 25 mg IV on Days 1, 8, 15, and 22 of a 28 day cycle and bevacizumab 10 mg/kg IV on Days 1 and 15 of the cycle.. Twenty-eight eligible patients were enrolled, 26 evaluable receiving a median of 6.5 cycles (range 1-18). Drug related toxicities were common including cytopenias, fatigue, mucositis, diarrhea and mild bleeds. Dose reductions or discontinuation of TEM were common. Accrual closed for presumed futility after interim analysis of the first 25 evaluable patients showed only one PR and 16/25 were progression-free at 6 months. However, the final data update in March 2013 demonstrated 4 confirmed PRs, a 5th unconfirmed PR and 16 /26 progression-free at 6 months. Median PFS and OS were 7 and 14 months respectively.. This first-line HCC trial evaluating the BEV/TEM doublet reports an ORR of 19 % and OS of 14 months which is favorable but requires further study at a more optimized dose and schedule.

Topics: Adult; Aged; Angiogenesis Inhibitors; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Bevacizumab; Carcinoma, Hepatocellular; Disease-Free Survival; Female; Humans; Liver Neoplasms; Male; Middle Aged; Protein Kinase Inhibitors; Sirolimus; Treatment Outcome

The oncogenic PI3K/Akt/mTOR pathway is frequently activated in HCC. Data on the mTOR inhibitor, temsirolimus, is limited in HCC patients with concomitant chronic liver disease. The objectives of this study were: (1) In phase I, to determine DLTs and MTD of temsirolimus in HCC patients with chronic liver disease; (2) In phase II, to assess activity of temsirolimus in HCC, and (3) to explore potential biomarkers for response.. Major eligibility criteria included histologically confirmed advanced HCC and adequate organ function. In Phase I part of the study, temsirolimus was given weekly in 3-weekly cycle; dose levels were 20 mg (level 1), 25 mg (level 2) and 30 mg (level 3). The MTD was used in the subsequent phase II part; the primary endpoint was PFS and secondary endpoints were response and OS. In addition, exploratory analysis was conducted on pre-treatment tumour tissues to determine stathmin, pS6, pMTOR or p-AKT expressions as potential biomarkers for response. Overall survival and PFS were calculated using the Kaplan-Meier method. Reassessment CT scans were done every 6 weeks. All adverse events were reported using CTCAE v3.. The Phase I part consisted of 19 patients, 2 of 6 patients at level 3 experienced DLT; dose level 2 was determined to be the MTD. The phase II part consisted of 36 patients. Amongst 35 assessable patients, there were 1 PR, 20 SD and 14 PD. Overall, the median PFS was 2.83 months (95% C.I. 1.63-5.24). The median OS was 8.89 months (95% C.I. 5.89-13.30). Grade ≥ 3 that occurred in > 10% of patients included thrombocytopenia (4) and hyponatraemia (4). Exploratory analysis revealed that disease stabilization (defined as CR + PR + SD > 12 weeks) in tumours having high and low pMTOR H-scores to be 70% and 29% respectively (OR 5.667, 95% CI 1.129-28.454, p = 0.035).. In HCC patients with chronic liver disease, the MTD of temsirolimus was 25 mg weekly in a 3-week cycle. The targeted PFS endpoint was not reached. However, further studies to identify appropriate patient subgroup are warranted.. This study has been registered in ClinicalTrials.gov (Id: NCT00321594) on 1 December 2010.

Topics: Adult; Aged; Antineoplastic Agents; Carcinoma, Hepatocellular; Disease-Free Survival; Female; Humans; Kaplan-Meier Estimate; Liver Neoplasms; Male; Maximum Tolerated Dose; Middle Aged; Sirolimus; Treatment Outcome

Aside from the multikinase inhibitor sorafenib, there are no effective systemic therapies for the treatment of advanced hepatocellular carcinoma.. To assess the efficacy of everolimus in patients with advanced hepatocellular carcinoma for whom sorafenib treatment failed.. EVOLVE-1 was a randomized, double-blind, phase 3 study conducted among 546 adults with Barcelona Clinic Liver Cancer stage B or C hepatocellular carcinoma and Child-Pugh A liver function whose disease progressed during or after sorafenib or who were intolerant of sorafenib. Patients were enrolled from 17 countries between May 2010 and March 2012. Randomization was stratified by region (Asia vs rest of world) and macrovascular invasion (present vs absent).. Everolimus, 7.5 mg/d, or matching placebo, both given in combination with best supportive care and continued until disease progression or intolerable toxicity. Per the 2:1 randomization scheme, 362 patients were randomized to the everolimus group and 184 patients to the placebo group.. The primary end point was overall survival. Secondary end points included time to progression and the disease control rate (the percentage of patients with a best overall response of complete or partial response or stable disease).. No significant difference in overall survival was seen between treatment groups, with 303 deaths (83.7%) in the everolimus group and 151 deaths (82.1%) in the placebo group (hazard ratio [HR], 1.05; 95% CI, 0.86-1.27; P = .68; median overall survival, 7.6 months with everolimus, 7.3 months with placebo). Median time to progression with everolimus and placebo was 3.0 months and 2.6 months, respectively (HR, 0.93; 95% CI, 0.75-1.15), and disease control rate was 56.1% and 45.1%, respectively (P = .01). The most common grade 3/4 adverse events for everolimus vs placebo were anemia (7.8% vs 3.3%, respectively), asthenia (7.8% vs 5.5%, respectively), and decreased appetite (6.1% vs 0.5%, respectively). No patients experienced hepatitis C viral flare. Based on central laboratory results, hepatitis B viral reactivation was experienced by 39 patients (29 everolimus, 10 placebo); all cases were asymptomatic, but 3 everolimus recipients discontinued therapy.. Everolimus did not improve overall survival in patients with advanced hepatocellular carcinoma whose disease progressed during or after receiving sorafenib or who were intolerant of sorafenib.. clinicaltrials.gov Identifier: NCT01035229.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Carcinoma, Hepatocellular; Disease Progression; Double-Blind Method; Everolimus; Female; Humans; Liver; Liver Neoplasms; Male; Middle Aged; Niacinamide; Phenylurea Compounds; Sirolimus; Sorafenib; Survival Analysis; Young Adult

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Everolimus; Female; Humans; Liver Neoplasms; Male; Niacinamide; Phenylurea Compounds; Prognosis; Risk Assessment; Sirolimus; Sorafenib; Survival Analysis; Treatment Failure

Based upon preclinical evidence for improved antitumor activity in combination, this phase I study investigated the maximum-tolerated dose (MTD), safety, activity, pharmacokinetics (PK), and biomarkers of the mammalian target of rapamycin inhibitor, temsirolimus, combined with sorafenib in hepatocellular carcinoma (HCC).. Patients with incurable HCC and Child Pugh score ≤B7 were treated with sorafenib plus temsirolimus by 3 + 3 design. The dose-limiting toxicity (DLT) interval was 28 days. The response was assessed every two cycles. PK of temsirolimus was measured in a cohort at MTD.. Twenty-five patients were enrolled. The MTD was temsirolimus 10 mg weekly plus sorafenib 200 mg twice daily. Among 18 patients at MTD, DLT included grade 3 hand-foot skin reaction (HFSR) and grade 3 thrombocytopenia. Grade 3 or 4 related adverse events at MTD included hypophosphatemia (33%), infection (22%), thrombocytopenia (17%), HFSR (11%), and fatigue (11%). With sorafenib, temsirolimus clearance was more rapid (P < 0.05). Two patients (8%) had a confirmed partial response (PR); 15 (60%) had stable disease (SD). Alpha-fetoprotein (AFP) declined ≥50% in 60% assessable patients.. The MTD of sorafenib plus temsirolimus in HCC was lower than in other tumor types. HCC-specific phase I studies are necessary. The observed efficacy warrants further study.

Topics: Aged; alpha-Fetoproteins; Antineoplastic Combined Chemotherapy Protocols; Biomarkers; Biomarkers, Tumor; Carcinoma, Hepatocellular; Disease-Free Survival; Drug Administration Schedule; Female; Humans; Liver Neoplasms; Male; Maximum Tolerated Dose; Middle Aged; Neoplastic Cells, Circulating; Niacinamide; Phenylurea Compounds; Protein Precursors; Prothrombin; Sirolimus; Sorafenib; Treatment Outcome

Sorafenib is the only therapy shown to improve overall survival in advanced hepatocellular carcinoma (HCC). Combination therapy targeting multiple signaling pathways may improve outcomes. This phase I study was designed to determine the maximum tolerated dose (MTD) of everolimus given with sorafenib 400mg twice daily in patients with advanced HCC of Child-Pugh class A liver function who were naive to systemic therapy.. Everolimus was initiated at 2.5mg once daily and increased per a Bayesian sequential dose-escalation scheme based on the dose-limiting toxicities experienced within the first 28 days of treatment. Adverse events were assessed continuously. Efficacy was evaluated using the best overall response rate per RECIST.. Thirty patients were enrolled; 25 were evaluable for MTD determination. One out of 12 patients treated with everolimus 2.5mg once daily and 6 out of 13 patients treated with everolimus 5.0mg once daily experienced a dose-limiting toxicity, most commonly thrombocytopenia (n=5). All patients experienced 1 adverse event, most commonly diarrhea (66.7%), hand-foot skin reaction (66.7%), and thrombocytopenia (50.0%). Best overall response was stable disease (62.5% and 42.9% in the 2.5-mg and 5.0-mg cohorts, respectively). Median time to progression and overall survival in the 2.5-mg cohort were 4.5 months and 7.4 months, respectively, and 1.8 months and 11.7 months, respectively, in the 5.0-mg cohort.. In patients with advanced HCC, the everolimus MTD in combination with standard-dose sorafenib was 2.5mg once daily. The inability to achieve a biologically effective everolimus concentration at the MTD precluded phase II study of this combination.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Hepatocellular; Everolimus; Female; Humans; Liver Neoplasms; Male; Maximum Tolerated Dose; Middle Aged; Niacinamide; Phenylurea Compounds; Sirolimus; Sorafenib

Deregulation of mammalian target of rapamycin (mTOR) signalling is common in human hepatocellular carcinoma (HCC).. To determine the maximum tolerated dose (MTD) of the oral mTOR inhibitor everolimus in advanced HCC patients.. Patients with locally advanced or metastatic HCC (Child-Pugh class A or B) were enrolled in an open-label phase 1 study and randomly assigned to daily (2.5-10 mg) or weekly (20-70 mg) everolimus in a standard 3 + 3 dose-escalation design. MTD was based on the rate of dose-limiting toxicities (DLTs). Secondary endpoints included safety, pharmacokinetics and tumour response. In a post hoc analysis, serum hepatitis B virus (HBV) DNA levels were quantified.. Thirty-nine patients were enrolled. DLTs occurred in five of 21 patients in the daily and two of 19 patients in the weekly cohort. Daily and weekly MTDs were 7.5 mg and 70 mg respectively. Grade 3/4 adverse events with a ≥10% incidence were thrombocytopenia, hypophosphataemia and alanine transaminase (ALT) elevation. In four hepatitis B surface antigen (HBsAg)-seropositive patients, grade 3/4 ALT elevations were accompanied by significant (>1 log) increases in serum HBV levels. The incidence of hepatitis flare (defined as ALT increase >100 IU/mL from baseline) in HBsAg-seropositive patients with and without detectable serum HBV DNA before treatment was 46.2% and 7.1% respectively (P < 0.01, Fisher exact test). Disease control rates in the daily and weekly cohorts were 71.4% and 44.4% respectively.. The recommended everolimus dosing schedule for future hepatocellular carcinoma studies is 7.5 mg daily. Prophylactic anti-viral therapy should be mandatory for HBsAg-seropositive patients (ClinicalTrials.gov NCT00390195).

Topics: Adult; Aged; Carcinoma, Hepatocellular; DNA, Viral; Dose-Response Relationship, Drug; Everolimus; Female; Hepatitis B; Hepatitis B Surface Antigens; Hepatitis B virus; Humans; Immunosuppressive Agents; Liver Cirrhosis; Liver Neoplasms; Male; Maximum Tolerated Dose; Middle Aged; Sirolimus; Young Adult

Preclinical studies have demonstrated the additive effect of rapamycin with bevacizumab for hepatocellular carcinoma treatment. We conducted a Phase 1 study to evaluate the safety and pharmacokinetics of the combination in patients with hepatocellular carcinoma.. Adult participants with advanced hepatocellular carcinoma received intravenous bevacizumab (5mg/kg every 14 days) and oral rapamycin (1-6 mg/day; 3+3 dose escalation design). Computed tomography assessed tumour response and treatment safety. Pharmacokinetics assessment established rapamycin blood concentrations pre- and post-dose. Dynamic contrast-enhanced computed tomography analysed the tumour region for blood flow, permeability surface area product, fractional intravascular blood volume and extracellular-extravascular volume.. Twenty-four participants were treated. There were two dose limiting toxicities with rapamycin 5mg: grade 3 thrombocytopenia and grade 3 mucositis. The maximally tolerated dose of rapamycin was 4 mg. Adverse events (grade 1-2) included hyperglycaemia (83%), thrombocytopenia (75%), fatigue (46%), mucositis (46%), anorexia (42%), diarrhoea (33%) and proteinuria (12.5%). Of 20 evaluable participants, one reached complete response that lasted 4.5 months, two reached partial response, 14 reached stable disease and three had progressive disease. Median overall survival was 9.4 months; progression-free survival was 5.5 months. Dose level and steady state area under the concentration time curve for hour zero to infinity of rapamycin correlated inversely with blood flow rate and change in permeability-surface area. After 22 days of treatment, there were significant reductions from baseline in blood flow rate, permeability-surface area and fractional intracellular blood volume.. The recommended Phase 2 dose of rapamycin is 4 mg in combination with bevacizumab. Evidence of anti-vascular activity was observed together with promising clinical activity.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Bevacizumab; Carcinoma, Hepatocellular; Dose-Response Relationship, Drug; Female; Hepatectomy; Humans; Liver Neoplasms; Male; Maximum Tolerated Dose; Middle Aged; Sirolimus; Treatment Outcome

There is currently no consensus on the most suitable treatment for the recurrence of hepatocellular carcinoma (HCC) after liver transplantation. This open, multicenter, retrospective, uncontrolled cohort study was designed to evaluate the safety and preliminary efficacy of the combined use of a mammalian target of rapamycin (mTOR) inhibitor and sorafenib in this setting. In 31 patients who suffered from HCC recurrence after liver transplantation, the immunosuppressive therapy was changed to mTOR inhibitors, and systemic treatment with sorafenib was initiated. This combination was maintained until symptomatic tumor progression, death, hepatic decompensation, or unacceptable toxicity occurred. Primary treatment efficacy was determined by overall survival and progression-free survival, and secondary efficacy was determined by the overall response rate. Toxicity parameters associated with the use of sorafenib and mTOR inhibitors were also analyzed. The overall response rate according to the Response Evaluation Criteria in Solid Tumors was 3.8% (1/26), and there was sustained stabilization of the disease in 13 additional cases (50.0%). The median overall survival was 19.3 months [95% confidence interval (CI) = 13.4-25.1 months], and the median time to progression was 6.77 months (95% CI = 2.3-11.1 months). Only 2 grade 3/4 cases of hyperglycemia and 1 case of grade 3/4 mucositis were reported, and they were possibly related to mTOR inhibitors. The most common severe adverse event probably related to sorafenib was diarrhea (12.9%). In conclusion, the coadministration of sorafenib and an mTOR inhibitor could be effective despite notable toxicity in patients with post-liver transplant HCC recurrence not suitable for radical therapy. The toxicity and efficacy need to be further evaluated in randomized controlled studies for this combination to be considered a valid option.

Topics: Adult; Aged; Antineoplastic Agents; Benzenesulfonates; Carcinoma, Hepatocellular; Cohort Studies; Diarrhea; Disease Progression; Drug Therapy, Combination; Everolimus; Female; Humans; Immunosuppressive Agents; Incidence; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Neoplasm Recurrence, Local; Niacinamide; Phenylurea Compounds; Pyridines; Retrospective Studies; Sirolimus; Sorafenib; Survival Rate; TOR Serine-Threonine Kinases; Treatment Outcome

Rapalogs are emerging as promising targeted anticancer drugs. Activation of the PI3K/Akt/mTOR pathway has been observed in 15-50% of hepatocellular carcinomas.. In this phase II study, patients with advanced hepatocellular carcinoma and underlying cirrhosis received sirolimus (20 mg/week for 1 month then 30 mg/week). Tumour response was assessed every 8 weeks. The primary endpoint was the objective tumour response rate according to the Response Evaluation Criteria in Solid Tumours criteria. Secondary endpoints included the objective response according to the modified Response Evaluation Criteria in Solid Tumours criteria, safety, and pharmacokinetic parameters.. Twenty-five patients received sirolimus for a median of 20.6 weeks. Two patients had an objective response (8%, 95CI: 0.98-26.03), including one complete response, and 8 patients had stable disease. There were 2 cases of grade 5 toxicity (infections) and 5 cases of grade 3 toxicity. The main grade 1/2 toxicity was mild transient fatigue (76%). Median time to radiological progression and overall survival were 15.3 weeks (range: 8.2-173.9) and 26.4 weeks (range: 8.2-173.9) respectively. Use of the modified Response Evaluation Criteria in Solid Tumours criteria did not identify any further responders.. These data suggest that first-line sirolimus shows antitumoural efficacy in advanced hepatocellular carcinoma. Larger trials with Child A patients are needed.

Topics: Acne Vulgaris; Adult; Aged; Carcinoma, Hepatocellular; Disease Progression; Epistaxis; Fatigue; Female; Humans; Immunosuppressive Agents; Infections; Kaplan-Meier Estimate; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Mucositis; Multimodal Imaging; Positron-Emission Tomography; Sirolimus; Tomography, X-Ray Computed; TOR Serine-Threonine Kinases; Treatment Outcome

The phosphoinositide 3-kinase/Akt/mammalian target of rapamycin pathway plays a critical role in the pathogenesis of hepatocellular carcinoma (HCC). We performed a single-arm, phase 1/2 study of everolimus in patients with advanced HCC.. Patients with histologically confirmed measurable advanced HCC, 0-2 prior regimens, and adequate hematologic, hepatic, and renal functions received everolimus at 5 mg/day or 10 mg/day orally (6 weeks/cycle). The primary end points were determination of a safe dosage of everolimus (phase 1) and progression-free survival (PFS) at 24 weeks (phase 2).. Twenty-eight patients were enrolled and evaluable for efficacy and toxicity. No dose-limiting toxicities were observed at the 5 mg/day (n = 3) or 10 mg/day (n = 6) dosage level in phase 1. Twenty-five patients received everolimus at 10 mg/day. Grade 3-4 adverse events included lymphopenia (n = 3), aspartate transaminase (n = 3), hyponatremia (n = 2), and 1 patient each with anemia, alanine transaminase, hyperglycemia, proteinuria, rash, and hypoxia. One patient (4%) had partial response (95% confidence interval [CI], 0.9%-19.6%). The median PFS and overall survival were 3.8 months (95% CI, 2.1-4.6) and 8.4 months (95% CI, 3.9-21.1), respectively. The estimated PFS rate at 24 weeks was 28.6% (95% CI, 7.9%-49.3%).. Everolimus was well tolerated in patients with advanced HCC, and 10 mg/day was defined as the phase 2 dosage. Although the study did not proceed to the second stage of phase 2, preliminary antitumor activity was observed with everolimus in patients with advanced HCC, most of whom had prior systemic treatment.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Carcinoma, Hepatocellular; Everolimus; Female; Humans; Liver Neoplasms; Male; Middle Aged; Sirolimus

The potential anti-cancer effects of mammalian target of rapamycin (mTOR) inhibitors are being intensively studied. To date, however, few randomised clinical trials (RCT) have been performed to demonstrate anti-neoplastic effects in the pure oncology setting, and at present, no oncology endpoint-directed RCT has been reported in the high-malignancy risk population of immunosuppressed transplant recipients. Interestingly, since mTOR inhibitors have both immunosuppressive and anti-cancer effects, they have the potential to simultaneously protect against immunologic graft loss and tumour development. Therefore, we designed a prospective RCT to determine if the mTOR inhibitor sirolimus can improve hepatocellular carcinoma (HCC)-free patient survival in liver transplant (LT) recipients with a pre-transplant diagnosis of HCC.. The study is an open-labelled, randomised, RCT comparing sirolimus-containing versus mTOR-inhibitor-free immunosuppression in patients undergoing LT for HCC. Patients with a histologically confirmed HCC diagnosis are randomised into 2 groups within 4-6 weeks after LT; one arm is maintained on a centre-specific mTOR-inhibitor-free immunosuppressive protocol and the second arm is maintained on a centre-specific mTOR-inhibitor-free immunosuppressive protocol for the first 4-6 weeks, at which time sirolimus is initiated. A 21/2 -year recruitment phase is planned with a 5-year follow-up, testing HCC-free survival as the primary endpoint. Our hypothesis is that sirolimus use in the second arm of the study will improve HCC-free survival. The study is a non-commercial investigator-initiated trial (IIT) sponsored by the University Hospital Regensburg and is endorsed by the European Liver and Intestine Transplant Association; 13 countries within Europe, Canada and Australia are participating.. If our hypothesis is correct that mTOR inhibition can reduce HCC tumour growth while simultaneously providing immunosuppression to protect the liver allograft from rejection, patients should experience less post-transplant problems with HCC recurrence, and therefore could expect a longer and better quality of life. A positive outcome will likely change the standard of posttransplant immunosuppressive care for LT patients with HCC.. Trial registered at http://www.clinicaltrials.gov: NCT00355862(EudraCT Number: 2005-005362-36).

Topics: Australia; Canada; Carcinoma, Hepatocellular; Disease-Free Survival; Europe; Humans; Immunosuppressive Agents; Intracellular Signaling Peptides and Proteins; Kaplan-Meier Estimate; Liver Neoplasms; Liver Transplantation; Prospective Studies; Protein Serine-Threonine Kinases; Recurrence; Risk Factors; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Transplantation, Homologous; Treatment Outcome

Hepatocellular cancer (HCC), as well as cholangiocellular cancer (CCC), has an extremely poor prognosis due to the extent of tumor at diagnosis and the underlying liver disease. Sirolimus is used in the transplantation setting as an immunosuppressive agent, but it also possesses antiproliferative and antiangiogenic properties. The objective of the study was to evaluate the effect of sirolimus on HCC and CCC.. In a prospective single-arm protocol, the tumor response to sirolimus as the primary endpoint was studied in 21 patients with advanced HCC and nine with CCC. Sirolimus was administered once daily by mouth, with the dose adjusted to a serum trough level between 4 and 15 mug/ml. Tumor response was evaluated by computed tomography (CT) or magnetic resonance imaging (MRI), according to the Response Evaluation Criteria in Solid Tumors (RECIST), every third month. Secondary measures were overall survival, time to tumor progression, tumor markers, and side effects.. Of the patients with HCC, one had partial remission (PR) and fi ve patients had stable disease (SD) at 3 months. Of the patients with CCC, three had SD. The median survival for patients with HCC was 6.5 months (range, 0.2-36 months) and that for patients with CCC was 7 months (range, 2.6-35 months).. Treatment of HCC and CCC with sirolimus can induce temporary PD or SD. This pilot study indicates that sirolimus might be a promising drug for this treatment, but further clinical studies elucidating the biological effects are advocated.

Topics: Adult; Aged; Antibiotics, Antineoplastic; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Carcinoma, Hepatocellular; Cholangiocarcinoma; Female; Humans; Liver Neoplasms; Male; Middle Aged; Phosphoinositide-3 Kinase Inhibitors; Protein Kinases; Sirolimus; TOR Serine-Threonine Kinases

Long-term survival of transplant recipients is significantly impacted by malignancy. We aimed to determine whether calcineurin inhibitor (CNI)-treated recipients converted to and weaned off molecular target of rapamycin inhibitor (mTOR-I) therapy have favorable changes in their molecular profiles in regard to malignancy risk. We performed gene expression profiling from liver biopsy and blood (PBMC) specimens followed by network analysis of key dysregulated genes, associated diseases and disorders, molecular and cellular functions using IPA software. Twenty non-immune, non-viremic patients were included, and 8 of them achieved tolerance. Two comparisons were performed: (1) tolerance time point vs tacrolimus monotherapy and (2) tolerance time point vs sirolimus monotherapy. Upon achieving tolerance, IPA predicted significant activation of DNA damage response (p = 5.40e-04) and inhibition of DNA replication (p = 7.56e-03). Conversion from sirolimus to tolerance showed decrease in HCC (p = 1.30e-02), hepatic steatosis (p = 5.60e-02) and liver fibrosis (p = 2.91e-02) associated genes. In conclusion, this longitudinal study of patients eventually achieving tolerance reveals an evolving molecular profile associated with decreased cancer risk and improved hepatic steatosis and liver fibrosis. This provides a biological rationale for attempting conversion to mTOR-I therapy and tolerance following liver transplantation particularly in patients at higher risk of cancer incidence and progression post-transplant.

Topics: Carcinoma, Hepatocellular; Graft Rejection; Humans; Immunosuppressive Agents; Leukocytes, Mononuclear; Liver Cirrhosis; Liver Neoplasms; Liver Transplantation; Longitudinal Studies; Sirolimus; TOR Serine-Threonine Kinases; Transplant Recipients

Tumor recurrence after liver transplantation (LT) for selective patients diagnosed with hepatocellular carcinoma (HCC) in the setting of cirrhosis is the greatest challenge effecting the prognosis of these patients. The aim of this study was to evaluate the efficacy of sirolimus on the prognosis for these recipients.. The data from 193 consecutive HCC patients who had undergone LT from January 2015 to December 2019 were retrospectively analyzed. These patients were divided into the sirolimus group [patients took sirolimus combined with calcineurin inhibitors (CNIs) (n = 125)] and non-sirolimus group [patients took CNI-based therapy without sirolimus (n = 68)]. Recurrence-free survival (RFS) and overall survival (OS) were compared between the two groups. The prognostic factors and independent risk factors for RFS and OS were further evaluated.. Non-sirolimus was an independent risk factor for RFS (HR = 2.990; 95% CI: 1.050-8.470; P = 0.040) and OS (HR = 3.100; 95% CI: 1.190-8.000; P = 0.020). A higher proportion of patients beyond Hangzhou criteria was divided into the sirolimus group (69.6% vs. 80.9%, P = 0.030). Compared with the non-sirolimus group, the sirolimus group had significantly better RFS (P < 0.001) and OS (P < 0.001). Further subgroup analysis showed similar results.. This study demonstrated that sirolimus significantly decreased HCC recurrence and prolonged RFS and OS in LT patients with different stage of HCC.

Topics: Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Neoplasm Recurrence, Local; Prognosis; Retrospective Studies; Sirolimus

hsa_circ_0001727 (circZKSCAN1) has been reported to be a tumor-associated circRNA by sponging microRNAs. Intriguingly, we found that circZKSCAN1 encoded a secretory peptide (circZKSaa) in the liver. The present study aims to elucidate the potential role and molecular mechanism of circZKSaa in the regulation of hepatocellular carcinoma (HCC) progression.. The circRNA profiling datasets (RNA-seq data GSE143233 and GSE140202) were reanalyzed and circZKSCAN1 was selected for further study. Mass spectrometry, polysome fractionation assay, dual-luciferase reporter, and a series of experiments showed that circZKSCAN1 encodes circZKSaa. Cell proliferation, apoptosis, and tumorigenesis in nude mice were examined to investigate the functions of circZKSaa. Mechanistically, the relationship between the circZKSaa and mTOR in HCC was verified by immunoprecipitation analyses, mass spectrometry, and immunofluorescence staining analyses.. Receiver operating characteristic (ROC) analysis demonstrated that the secretory peptide circZKSaa encoded by circZKSCAN1 might be the potential biomarker for HCC tissues. Through a series of experiments, we found that circZKSaa inhibited HCC progression and sensitize HCC cells to sorafenib. Mechanistically, we found that the sponge function of circZKSCAN1 to microRNA is weak in HCC, while overexpression of circZKSaa promoted the interaction of FBXW7 with the mammalian target of rapamycin (mTOR) to promote the ubiquitination of mTOR, thereby inhibiting the PI3K/AKT/mTOR pathway. Furthermore, we found that the high expression of cicZKSCAN1 in sorafenib-treated HCC cells was regulated by QKI-5.. These results reveal that a novel circZKSCAN1-encoded peptide acts as a tumor suppressor on PI3K/AKT/mTOR pathway, and sensitizes HCC cells to sorafenib via ubiquitination of mTOR. These findings demonstrated that circZKSaa has the potential to serve as a therapeutic target and biomarker for HCC treatment.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Mammals; Mice; Mice, Nude; MicroRNAs; Peptides; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RNA, Circular; Sirolimus; Sorafenib; TOR Serine-Threonine Kinases

Helicase-like transcription factor (HLTF) has been found to be involved in the maintenance of genome stability and tumour suppression, but whether its downregulation in cancers is associated with posttranslational regulation remains unclear. Here, we observed that HLTF was significantly downregulated in hepatocellular carcinoma (HCC) tissues and positively associated with the survival of HCC patients. Mechanistically, the decreased expression of HLTF in HCC was attributed to elevated β-TrCP-mediated ubiquitination and degradation. Knockdown of HLTF enhanced p62 transcriptional activity and mammalian target of rapamycin (mTOR) activation, leading to HCC tumourigenesis. Inhibition of mTOR effectively blocked β-TrCP overexpression- or HLTF knockdown-mediated HCC tumourigenesis and metastasis. Furthermore, in clinical tissues, decreased HLTF expression was positively correlated with elevated expression of β-TrCP, p62, or p-mTOR in HCC patients. Overall, our data not only uncover new roles of HLTF in HCC cell proliferation and metastasis, but also reveal a novel posttranslational modification of HLTF by β-TrCP, indicating that the β-TrCP/HLTF/p62/mTOR axis may be a new oncogenic driver involved in HCC development. This finding provides a potential therapeutic strategy for HCC patients by targeting the β-TrCP/HLTF/p62/mTOR axis.

Topics: beta-Transducin Repeat-Containing Proteins; Carcinogenesis; Carcinoma, Hepatocellular; Cell Line, Tumor; DNA-Binding Proteins; Humans; Liver Neoplasms; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors

Hepatic forkhead box protein A2 (FOXA2) is a crucial transcription factor for liver development and metabolic homeostasis. However, its role in hepatocellular carcinoma (HCC) progression and lenvatinib-related drug resistance remains unknown. In this study, the level of FOXA2 expression was found to be lower in HCC tissues than in paired adjacent tumor tissues. A low level of FOXA2 expression was associated with aggressive tumor characteristics (vascular invasion and poor differentiation). A low level of FOXA2 expression was found to be an independent risk factor for tumor recurrence (hazard ratio (HR): 1.899, P < 0.001) and long-term survival (HR: 2.011, P = 0.003) in HCC patients after hepatectomy. In xenograft animal models, FOXA2 overexpression significantly inhibited tumor growth. Moreover, FOXA2 overexpression was found to enhance the inhibitory effect of lenvatinib on HCC cells by upregulating the adenosine monophosphate-activated protein kinase-mechanistic target of rapamycin (AMPK-mTOR) pathway. Conversely, inhibition of adenosine monophosphate-activated protein kinase (AMPK) or stimulation of mechanistic target of rapamycin (mTOR) attenuated the sensitization of cells overexpressing FOXA2 to lenvatinib. Similarly, FOXA2 overexpression augmented the antitumor effect of lenvatinib in animal models with xenograft tumors. FOXA2 overexpression increased autophagy in HCC cells treated with lenvatinib. Lenvatinib treatment activated the platelet-derived growth factor receptor-extracellular regulated protein kinase (PDGFR-ERK) pathway in HCC. FOXA2 overexpression further downregulated the PDGFR-ERK pathway through the activation of the AMPK-mTOR axis. In conclusion, FOXA2 was identified as an independent risk factor for HCC after hepatectomy. FOXA2 was found to be closely associated with the biological progression of HCC. By modulating the AMPK-mTOR-autophagy signaling pathway, FOX2 significantly augmented antitumor effect of lenvatinib in HCC.

Topics: AMP-Activated Protein Kinases; Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Drug Resistance, Neoplasm; Hepatocyte Nuclear Factor 3-beta; Humans; Liver Neoplasms; Neoplasm Recurrence, Local; Sirolimus; TOR Serine-Threonine Kinases

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies, whose initiation and development are driven by alterations in driver genes. In this study, we identified four driver genes (TP53, PTEN, CTNNB1, and KRAS) that show a high frequency of somatic mutations or copy number variations (CNVs) in patients with HCC. Four different spontaneous HCC mouse models were constructed to screen for changes in various kinase signaling pathways. The sgTrp53 + sgPten tumor upregulated mTOR and noncanonical nuclear factor-κB signaling, which was shown to be strongly inhibited by rapamycin (an mTOR inhibitor) in vitro and in vivo. The JAK-signal transducer and activator of transcription (STAT) signaling was activated in Ctnnb1

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; DNA Copy Number Variations; Liver Neoplasms; Mice; Proto-Oncogene Proteins p21(ras); Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Both dysregulation of mechanistic target of rapamycin (mTOR) signalling and DNA methylation patterns have been shown to be closely associated with tumor progression and serve as promising targets for hepatocellular carcinoma (HCC) therapy. Although their respective roles in HCC have been extensively revealed, the existence of molecular interactions between them remains largely unknown.. The association of DNA methylation and mTOR signalling in HCC tissues and cell lines was assessed. A Kaplan‒Meier analysis was applied to estimate the overall survival (OS) and recurrence-free survival (RFS) of HCC patients. The modulation of DNMT1 by mTOR in HCC cell lines was determined. The effect of the drug combination in cell lines and mouse models was examined.. The results showed that the DNA methylation level was positively associated with the activation of mTOR signalling in HCC tissues and cell lines. Moreover, HCC patients with higher DNA methylation levels and enhanced activation of mTOR signalling exhibited the worst prognosis. Then, we screened methylation-related enzymes and found that the activation of mTOR signalling increased DNMT1 expression and activity. In addition, mTOR enhanced the translational efficiency of DNMT1 in a 4E-BP1-dependent manner, which is based on the pyrimidine rich translational element (PRTE)-containing 5'UTR of DNMT1. Moreover, we demonstrated that the combined inhibition of mTOR and DNMT synergistically inhibited HCC growth in vitro and in vivo.. In addition to some already identified pro-cancer downstream molecules, the activation of mTOR signalling was found to promote DNA methylation by increasing the translation of DNMT1. Furthermore, combined targeting of mTOR and DNMT1 has been demonstrated to have a more effective tumor suppressive function in HCC.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; DNA (Cytosine-5-)-Methyltransferase 1; DNA Methylation; Liver Neoplasms; Mice; Sirolimus; TOR Serine-Threonine Kinases

Long-term treatment with immunosuppressants is necessary to attenuate allograft rejection following organ transplantation (OT). Consequently, the overall survival of OT recipients with malignancies has been substantially compromised by tumour recurrence. Rapamycin (RAPA) is a clinically approved immunosuppressive agent with antitumour activity that is considered beneficial in preventing posttransplant tumour recurrence. However, the clinical outcome of RAPA is impeded by acquired drug resistance and its poor oral bioavailability.. A nanotherapeutic strategy was developed by supramolecular assembly of RAPA into a polymer cytotoxic 7-ethyl-10-hydroxycamptothecin (SN38) prodrug nanoparticle (termed SRNP) for simultaneous codelivery of cytotoxic/immunosuppressive agents. Cell-based experiments were used to evaluate the cytotoxicity of SRNPs against hepatocellular carcinoma (HCC). The therapeutic efficacy of SRNPs was evaluated in multiple preclinical models including an orthotopic HCC mouse model, an orthotopic liver transplantation (OLT) rat model and a clinically relevant cancer-transplant model to examine its antitumour and immunosuppressive activity.. The combination of SN38 with RAPA resulted in synergetic effects against HCC cells and alleviated RAPA resistance by abrogating Akt/mTOR signalling activation. SRNPs exhibited potent antitumour efficiency in the orthotopic HCC model while substantially prolonging the survival of allografts in the OLT model. In the cancer-transplant model that simultaneously bears tumour xenografts and skin allografts, SRNPs not only effectively inhibited tumour growth but also attenuated allograft damage.. The nanotherapy presented here had enhanced efficacy against tumours and maintained satisfactory immunosuppressive activity and thus has great potential to improve the survival outcomes of patients with a high risk of tumour recurrence following OT.. This work was supported by the National Natural Science Foundation of China (32171368 and 31671019), the Zhejiang Provincial Natural Science Foundation of China (LZ21H180001), the Zhejiang Province Preeminence Youth Fund (LR19H160002), and the Jinan Provincial Laboratory Research Project of Microecological Biomedicine (JNL-2022039c).

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Mice; Neoplasm Recurrence, Local; Rats; Sirolimus

Liver transplantation is one of the most effective treatments for hepatocellular carcinoma (HCC). The balance between inhibiting immune rejection and preventing tumor recurrence after liver transplantation is the key to determining the long-term prognosis of patients with HCC after liver transplantation. In our previous study, we found that capecitabine (CAP), an effective drug for the treatment of HCC, could exert an immunosuppressive effect after liver transplantation by inducing T cell ferroptosis. Recent studies have shown that ferroptosis is highly associated with autophagy. In this study, we confirmed that the autophagy inducer rapamycin (RAPA) combined with metronomic capecitabine (mCAP) inhibits glutathione peroxidase 4 (GPX4) and promotes ferroptosis in CD4

Topics: Animals; Capecitabine; Carcinoma, Hepatocellular; CD4-Positive T-Lymphocytes; Ferroptosis; Humans; Liver Neoplasms; Liver Transplantation; Neoplasm Recurrence, Local; Rats; Sirolimus; T-Lymphocytes

Cabozantinib, mainly targeting cMet and vascular endothelial growth factor receptor 2, is the second-line treatment for patients with advanced hepatocellular carcinoma (HCC). However, the lower response rate and resistance limit its enduring clinical benefit. In this study, we found that cMet-low HCC cells showed primary resistance to cMet inhibitors, and the combination of cabozantinib and mammalian target of rapamycin (mTOR) inhibitor, rapamycin, exhibited a synergistic inhibitory effect on the in vitro cell proliferation and in vivo tumor growth of these cells. Mechanically, the combination of rapamycin with cabozantinib resulted in the remarkable inhibition of AKT, extracellular signal-regulated protein kinases, mTOR, and common downstream signal molecules of receptor tyrosine kinases; decreased cyclin D1 expression; and induced cell cycle arrest. Meanwhile, rapamycin enhanced the inhibitory effects of cabozantinib on the migration and tubule formation of human umbilical vascular endothelial cells and human growth factor-induced invasion of cMet inhibitor-resistant HCC cells under hypoxia condition. These effects were further validated in xenograft models. In conclusion, our findings uncover a potential combination therapy of cabozantinib and rapamycin to combat cabozantinib-resistant HCC.

Topics: Anilides; Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Endothelial Cells; Humans; Liver Neoplasms; Pyridines; Sirolimus; Xenograft Model Antitumor Assays

Rapamycin is widely recognized as an inhibitor of mTOR, and has been approved for clinical use as an immunosuppressant. Its potencies in anti-cancer, anti-aging, and neurodegenerative diseases are emergingly established. The exploration of other targets of rapamycin will further elucidate its underlying mechanisms of action. In this study, we use a chemical proteomics strategy that has identified STAT3, a transcription factor considered to be undruggable, as a direct functional protein target of rapamycin. Together with other multi-dimensional proteomics data, we show that rapamycin treatment in cell culture significantly inhibits c-Myc-regulated gene expression. Furthermore, we show that rapamycin suppresses tumor growth along with a decreased expression of STAT3 and c-Myc in an in vivo xenograft mouse model for hepatocellular carcinoma. Our data suggest that rapamycin acts directly on STAT3 to decrease its transcription activity, providing important information for the pharmacological and pharmaceutical development of STAT3 inhibitors for cancer therapy.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Gene Expression Regulation; Humans; Liver Neoplasms; Mice; Proto-Oncogene Proteins c-myc; Sirolimus; STAT3 Transcription Factor

The MTOR signal is known to be activated in various cancer cells including hepatocellular carcinoma (HCC) cells. Rapamycin, a specific inhibitor of MTOR, has been widely used as an immunosuppressant in organ transplant patients, and its clinical application has been recently expanded to cancer therapy. In this study, the anti-proliferative effect of rapamycin was investigated in four different HCC cell lines. Rapamycin effectively inhibited the proliferation of Huh7 or Hep3B, but not that of HepG2 or SNU3160 cells. Interestingly, rapamycin increased Prospero-related homeobox 1 (PROX1) expression at the protein level, but did not affect its transcript in Huh7 as well as Hep3B cells. Moreover, immunoprecipitation assays showed that PROX1 ubiquitination was downregulated by rapamycin. Furthermore, PROX1 over-expression or siRNA knock-down in Huh7 and Hep3B cells reduced or increased proliferation, respectively. The effect of PROX1 over-expression on the sensitivity to rapamycin was not synergistic, but the effect of MTOR inhibition on cell proliferation was diminished by PROX1 siRNA. Finally, Huh7 cells were inoculated into the flanks of nude mice and rapamycin was injected daily for 14 days. The xenograft volume was decreased and PROX1 expression was increased by rapamycin. These results indicate that PROX1 plays a key role in the anti-proliferative effect of rapamycin and suggest that the increased PROX1 by MTOR inhibition can be used as a useful marker for predicting whether HCC cells can be affected by rapamycin.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Homeodomain Proteins; Humans; Liver Neoplasms; Mice; Mice, Nude; RNA, Small Interfering; Sirolimus; TOR Serine-Threonine Kinases; Tumor Suppressor Proteins

Topics: Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Liver Transplantation; Neoplasm Recurrence, Local; Sirolimus; TOR Serine-Threonine Kinases

Topics: Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Liver Transplantation; Sirolimus; TOR Serine-Threonine Kinases

Dendritic cells (DCs), as the most important antigen-presenting cells, play a crucial role in T cell activation. The latest research showed that inhibition of the mammalian target of rapamycin (mTOR) could enhance DCs maturation, promoting antigen presentation. Matrine has been identified as one of the key alkaloids isolated from the roots of

Topics: Alkaloids; Carcinoma, Hepatocellular; Cytokines; Dendritic Cells; Humans; Lipopolysaccharides; Liver Neoplasms; Matrines; Quinolizines; Sirolimus; TOR Serine-Threonine Kinases

The mammalian target of rapamycin (mTOR) pathway is frequently deregulated and has critical roles in cancer progression. mTOR inhibitor has been widely used in several kinds of cancers and is strongly recommended in patients with hepatocellular carcinoma (HCC) after liver transplantation (LT). However, the poor response to mTOR inhibitors due to resistance remains a challenge. Hypoxia-associated resistance limits the therapeutic efficacy of targeted drugs. The present study established models of HCC clinical samples and cell lines resistance to mTOR inhibitor sirolimus and screened out E2F7 as a candidate gene induced by hypoxia and promoting sirolimus resistance. E2F7 suppressed mTOR complex 1 via directly binding to the promoter of the TSC1 gene and stabilizes hypoxia-inducible factor-1α activating its downstream genes, which are responsible for E2F7-dependent mTOR inhibitor resistance. Clinically, low E2F7 expression could be an effective biomarker for recommending patients with HCC for anti-mTOR-based therapies after LT. Targeting E2F7 synergistically inhibited HCC growth with sirolimus in vivo. E2F7 is a promising target to reverse mTOR inhibition resistance. Collectively, our study points to a role for E2F7 in promoting mTOR inhibitor resistance in HCC and emphasizes its potential clinical significance in patients with HCC after LT.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; E2F7 Transcription Factor; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Liver Neoplasms; Liver Transplantation; MTOR Inhibitors; Sirolimus; TOR Serine-Threonine Kinases

Calcineurin inhibitors (CNIs), which are potent immunosuppressants (ISs), increase the risk for hepatocellular carcinoma (HCC) recurrence after liver transplantation (LTx). Epithelial-mesenchymal transition (EMT) is a key process in which epithelial cancer cells lose their polarity, resulting in cancer progression and metastasis. The aim of this study was to evaluate the effect of sirolimus (SRL) individually and in combination with other ISs to reduce EMT.. HCC SK-Hep1 cells were used and various ISs (SRL, tacrolimus, cyclosporine A, or mycophenolate mofetil) were administered at 2 dosages and in combination therapies. Mice were transplanted with SK-Hep1 cells (in the liver) and were monitored after 2 weeks.. The in vitro treatment with SRL showed a dose-dependent attenuation of cell proliferation and migration in case of the individual and IS combination treatments; further, decreased levels of pro-EMT proteins, namely, N-cadherin, transforming growth factor-β, ZEB1, Slug, and Snail were observed. In contrast, E-cadherin expression was upregulated after both the individual and IS combination treatments. These results were also observed in the samples from mice transplanted with the SK-Hep1 cells.. The present study demonstrated that SRL reduced HCC metastasis by inhibiting EMT. Thus, our findings provide a rationale for the use of SRL in combination with ISs in HCC LTx patients.

Topics: Animals; Calcineurin Inhibitors; Carcinoma, Hepatocellular; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Immunosuppressive Agents; Liver Neoplasms; Mice; Sirolimus

Topics: Carcinoma, Hepatocellular; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cell Proliferation; Humans; Liver Neoplasms; Phosphatidylinositols; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-akt; Sirolimus

Glycolysis caused by hypoxia-induced abnormal activation of hypoxia inducible factor-1α (HIF-1α) in the immune microenvironment promotes the progression of hepatocellular carcinoma (HCC), leading to enhanced drug resistance in cancer cells. Therefore, altering the immunosuppressive microenvironment by imp-roving the hypoxic state is a new goal in improving cancer treatment.. To analyse the role of HIF-1α, which is closely related to tumour proliferation, invasion, metastasis, and angiogenesis, in the proliferation and invasion of liver cancer, and to explore the HIF-1α pathway-mediated anti-cancer mechanism of sirolimus (SRL) combined with Huai Er.. Previous studies on HCC tissues identified the importance of HIF-1α, glucose transporter 1 (GLUT1), and lactate dehydrogenase A (LDHA) expression. In this study, HepG2 and Huh7 cell lines were treated, under hypoxic and normoxic conditions, with a combination of SRL and Huai Er. The effects on proliferation, invasion, cell cycle, and apoptosis were analysed. Proteomics and genomics techniques were used to analyze the HIF-1α-related signalling pathway during SRL combined with Huai Er treatment and its inhibition of the proliferation of HCC cells.. High levels of HIF-1α, LDHA, and GLUT-1 were found in poorly differentiated HCC, with lower patient survival rates. Hypoxia promoted the proliferation of HepG2 and Huh7 cells and weakened the apoptosis and cell cycle blocking effects of the SRL/Huai Er treatment. This was achieved by activation of HIF-1α and glycolysis in HCC, leading to the upregulation of LDHA, GLUT-1, Akt/mammalian target of rapamycin (mTOR), vascular endothelial growth factor (VEGF), and Forkhead box P3 and downregulation of phosphatase and tensin homolog deleted on chromosome ten (PTEN) and p27. The hypoxia-induced activation of HIF-1α showed the greatest attenuation in the SRL/Huai Er (S50 + H8) group compared to the drug treatments alone (. SRL increases the anti-cancer effect of Huai Er, which reduces the promotion of hypoxia-induced HIF-1α on the Warburg effect by inhibition of the PI3K/Akt/mTOR-HIF-1α and HIF-1α-PTEN signalling pathways in HCC.

Topics: Carcinoma, Hepatocellular; Glucose Transporter Type 1; Glycolysis; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Lactate Dehydrogenase 5; Liver Neoplasms; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RNA, Messenger; Sirolimus; Tensins; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor A

Sorafenib (SOR) is currently the first line molecular targeting agent for advanced liver cancer therapy. Unfortunately, the insensitivity of liver cancer patients to SOR relatively limits its effectiveness. Huaier (HUA), a natural medicinal parasitic fungus found on the Sophora japonica Linn., has been widely employed as an adjuvant medication for numerous malignancies due to its potent anti-tumoral properties. This study aims to elucidate the enhancing therapeutic efficacy of HUA on SOR treatment in hepatocellular carcinoma (HCC) cells and mouse models. The CCK-8, clone formation, flow cytometry, immunofluorescence, transmission electron microscopy, western blot, bioinformatic analysis, and xenograft tumor assays were performed to evaluate the synergistic anti-hepatoma efficacy and mechanisms of HUA-SOR combination treatment on HCC cells. The results revealed combination treatment further inhibited proliferation, promoted apoptosis, enhanced autophagy of HCC cells, and suppressed the growth of transplanted tumors in mice, compared with either HUA or SOR treatment alone. For Hep3B and Huh7 cells, the optimal synergistic doses of HUA in combination with SOR were 8 mg/mL + 4 μM and 4 mg/mL + 2 μM, with combination index values of 0.646 and 0.588, respectively. Additionally, the underlying mechanisms might be related to biological processes that are mediated by mammalian target of rapamycin (mTOR). The combination treatment downregulated the protein expression levels of p-mTOR, p-p70S6K, p62, and upregulated the protein expression levels of Beclin-1 and LC3B-II. The mTOR activator MHY1485 attenuated the effect of HUA-SOR combination by inhibiting autophagy, suggesting HUA may potentiate the sensitivity of HCC cells to SOR by partially inducing mTOR-mediated autophagic cell death. These findings might provide a rationale experimental foundation for clinical applications of HUA with SOR.

Topics: Animals; Autophagic Cell Death; Beclin-1; Carcinoma, Hepatocellular; Complex Mixtures; Fungi; Humans; Liver Neoplasms; Mammals; Mice; Ribosomal Protein S6 Kinases, 70-kDa; Sincalide; Sirolimus; Sorafenib; TOR Serine-Threonine Kinases; Trametes

The incidence of primary liver tumors, hepatocellular carcinoma (HCC), intrahepatic cholangiocellular carcinoma (ICC), and combined HCC/ICC (cHCC/CC) is increasing. For ICC, targeted therapy exists only for a small subpopulation of patients, while for HCC, Sorafenib and Lenvatinib are in use. Diagnosis of cHCC/CC is a great challenge and its incidence is underestimated, bearing the risk of unintended non-treatment of ICC. Here, we investigated effects of targeted inhibitors on human ICC cell lines (HUH28, RBE, SSP25), in comparison to extrahepatic (E)CC lines (EGI1, CCC5, TFK1), and HCC/hepatoblastoma cell lines (HEP3B, HUH7, HEPG2). Cells were challenged with: AKT inhibitor MK-2206; multikinase inhibitors Sorafenib, Lenvatinib and Dasatinib; PI3-kinase inhibitors BKM-120, Wortmannin, LY294002, and CAL-101; and mTOR inhibitor Rapamycin. Dosage of the substances was based on the large number of published data of recent years. Proliferation was analyzed daily for four days. All cell lines were highly responsive to MK-2206. Thereby, MK-2206 reduced expression of phospho(p)-AKT in all ICC, ECC, and HCC lines, which mostly corresponded to reduction of p-mTOR, whereas p-ERK1/2 was upregulated in many cases. Lenvatinib showed inhibitory effects on the two HCC cell lines, but not on HEPG2, ICCs and ECCs. Sorafenib inhibited proliferation of all cells, except the ECC line CCC5. However, at reduced dosage, we observed increased cell numbers in some ICC experiments. Dasatinib was highly effective especially in ICC cell lines. Inhibitory effects were observed with all four PI3-kinase inhibitors. However, cell type-specific differences were also evident here. Rapamycin was most effective in the two HCC cell lines. Our studies show that the nine inhibitors differentially target ICC, ECC, and HCC/hepatoblastoma lines. Caution should be taken with Lenvatinib and Sorafenib administration in patients with cHCC/CC as the drugs may have no effects on, or might even stimulate, ICC.

Topics: Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Carcinoma, Hepatocellular; Cholangiocarcinoma; Dasatinib; Hepatoblastoma; Humans; Liver Neoplasms; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Sirolimus; Sorafenib

p53 mutations occur frequently in human HCC. Activation of the mammalian target of rapamycin (mTOR) pathway is also associated with HCC. However, it is still unknown whether these changes together initiate HCC and can be targeted as a potential therapeutic strategy.. We generated mouse models in which mTOR was hyperactivated by loss of tuberous sclerosis complex 1 (Tsc1) with or without p53 haplodeficiency. Primary cells were isolated from mouse livers. Oncogenic signalling was assessed in vitro and in vivo, with or without targeted inhibition of a single molecule or multiple molecules. Transcriptional profiling was used to identify biomarkers predictive of HCC. Human HCC materials were used to corroborate the findings from mouse models.. p53 haploinsufficiency facilitates mTOR signalling via the PTEN/PI3K/Akt axis, promoting HCC tumorigenesis and lung metastasis. Inhibition of PI3K/Akt reduced mTOR activity, which effectively enhanced the anticancer effort of an mTOR inhibitor. ATP-binding cassette subfamily C member 4 (Abcc4) was found to be responsible for p53 haploinsufficiency- and Tsc1 loss-driven HCC tumorigenesis. Moreover, in clinical HCC samples, Abcc4 was specifically identified an aggressive subtype. The mTOR inhibitor rapamycin significantly reduced hepatocarcinogenesis triggered by Tsc1 loss and p53 haploinsufficiency in vivo, as well as the biomarker Abcc4.. Our data advance the current understanding of the activation of the PTEN/PI3K/Akt/mTOR axis and its downstream target Abcc4 in hepatocarcinogenesis driven by p53 reduction and Tsc1 loss. Targeting mTOR, an unexpected vulnerability in p53 (haplo)deficiency HCC, can be exploited therapeutically to treat Abcc4-positive patients with HCC.. Tsc1 loss facilitates the p53 (haplo)insufficiency-mediated activation of the PTEN/Akt/mTOR axis, leading to the elevated expression of Abcc4 to drive HCC tumorigenesis and metastasis in mice. Inhibition of mTOR protects against p53 haploinsufficiency and Tsc1 loss-triggered tumour-promoting activity, providing a new approach for treating an aggressive subtype of HCC exhibiting high Abcc4 expression.

Topics: Animals; Biomarkers, Tumor; Carcinoma, Hepatocellular; Gene Expression Regulation, Neoplastic; Haploinsufficiency; Humans; Liver Neoplasms; Mice; MTOR Inhibitors; Multidrug Resistance-Associated Proteins; Pyrazoles; Pyrimidines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Protein p53

The use of the immunosuppressive agent sirolimus (SRL) following liver transplantation (LT) in patients with hepatocellular carcinoma (HCC) is controversial. Sirolimus is a typical mammalian target of rapamycin (mTOR) inhibitor, and tuberous sclerosis 1-tuberous sclerosis 2 complex (TSC1/TSC2) is an important negative effector in the mTOR pathway. In this study, we investigated the effect of SRL-based immunosuppression on the prognosis of LT recipients with HCC beyond the Milan criteria based on TSC1/2 expression and explored the effect of TSC1 on HCC in vitro and in vivo.. We retrospectively analyzed 120 HCC patients who underwent LT in our hospital between January 1, 2015 and December 30, 2018. All patients had HCC beyond the Milan criteria and were divided into the SRL group (n = 50) and non-SRL group (n = 70). TSC1/2 expression levels in paraffin-embedded tissues were determined by immunohistochemistry (IHC) and then analyzed as subgroups. Overall survival (OS) and disease-free survival (DFS) were analyzed using the Kaplan-Meier method. TSC1 expression was silenced in Huh-7 and Bel-7402 cell lines for further cell function experiments.. 88.3% of patients were HBV LT recipients. The SRL group exhibited better DFS and OS compared to the non-SRL group (P = 0.02, P = 0.003). Subgroup (TSC1-based or TSC2-based) analyses revealed that patients with low TSC1 or TSC2 expression benefited from sirolimus (DFS: P = 0.046, OS: P = 0.006 for TSC1; DFS: P = 0.05, OS: P = 0.003 for TSC2) compared with patients with high expression. TSC1 knockdown in Huh-7 and Bel-7402 HCC cell lines activated the mTORC1 pathway and enhanced cell proliferation, migration and sensitivity to SRL in vitro and in vivo.. TSC1/2 expression could be used to predict the prognosis of patients with HCC beyond the Milan criteria who underwent SRL-based immunosuppression following LT. TSC1 knockdown promoted HCC malignancy and enhanced sensitivity to SRL.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease-Free Survival; Female; Gene Silencing; Humans; Immunosuppressive Agents; Kaplan-Meier Estimate; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Retrospective Studies; Sirolimus; Survival Rate; Tuberous Sclerosis Complex 1 Protein; Tuberous Sclerosis Complex 2 Protein

To investigate the efficacy of celastrol treatment of hepatocellular carcinoma (HCC) cells in vitro and in vivo and to propose a mechanism of action.. A human HepG2 liver cancer cell line and a xenograft tumor model were used to investigate the effects of celastrol on HCC in vitro and in vivo. A CCK-8 kit was used to detect cell viability. Flow cytometry and terminal-deoxynucleoitidyl transferase mediated nick end labeling staining were used to detect apoptosis. Western blotting and immunohistochemistry were used to detect the expression of cleaved-caspase-3, cleaved-caspase-8, cleaved-caspase-9, cleaved-PARP, mammalian target of rapamycin (mTOR), and p-mTOR. Hematoxylin-eosin staining was used to observe the tissue morphology.. Celastrol decreased the viability of HepG2 cells and induced apoptosis. Western blot assays indicated that celastrol up-regulated cleaved-caspase-3, cleaved-caspase-8, cleaved-caspase-9, and cleaved-PARP by inhibiting the phosphorylation of mTOR in HepG2 cells. Moreover, celastrol inhibited the tumor growth in a xenograft model. Celastrol also induced caspase-dependent apoptosis (up-regulation of cleaved-caspase- 3, -8, -9, and cleaved-PARP) and inhibited the activation of mTOR in vivo.. Celastrol induces caspase-dependent apoptosis in HCC cells by inhibiting the activation of mTOR.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Cell Proliferation; Humans; Liver Neoplasms; Pentacyclic Triterpenes; Sirolimus; TOR Serine-Threonine Kinases

The treatment of hepatocellular carcinoma after liver transplantation (LT) is controversial because of its high recurrence rate and low survival rate. Here, we report a case of early diffuse bilateral lung metastasis after LT beyond the Milan transplantation criteria (d = 18 cm, α-fetoprotein >24,000 ng/mL) that successfully achieved 1-year tumor-free remission survival with sirolimus combined with regorafenib. The donor source of the liver is legal, and this study followed the guidelines of the Helsinki Congress in this LT. To the best of our knowledge, this is the first report of the use of regorafenib as a first-line agent combined with sirolimus to treat recurrent hepatocellular carcinoma after LT, and this case expands the indications for LT.

Topics: Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Liver Transplantation; Lung Neoplasms; Male; Middle Aged; Phenylurea Compounds; Pyridines; Sirolimus; Survival Rate; Treatment Outcome

Methionine-1 (M1)-linked polyubiquitin chains conjugated by the linear ubiquitin chain assembly complex (LUBAC) control NF-κB activation, immune homoeostasis, and prevents tumour necrosis factor (TNF)-induced cell death. The deubiquitinase OTULIN negatively regulates M1-linked polyubiquitin signalling by removing the chains conjugated by LUBAC, and OTULIN deficiency causes OTULIN-related autoinflammatory syndrome (ORAS) in humans. However, the cellular pathways and physiological functions controlled by OTULIN remain poorly understood. Here, we show that OTULIN prevents development of liver disease in mice and humans. In an ORAS patient, OTULIN deficiency caused spontaneous and progressive steatotic liver disease at 10-13 months of age. Similarly, liver-specific deletion of OTULIN in mice leads to neonatally onset steatosis and hepatitis, akin to the ORAS patient. OTULIN deficiency triggers metabolic alterations, apoptosis, and inflammation in the liver. In mice, steatosis progresses to steatohepatitis, fibrosis and pre-malignant tumour formation by 8 weeks of age, and by the age of 7-12 months the phenotype has advanced to malignant hepatocellular carcinoma. Surprisingly, the pathology in OTULIN-deficient livers is independent of TNFR1 signalling. Instead, we find that steatohepatitis in OTULIN-deficient livers is associated with aberrant mTOR activation, and inhibition of mTOR by rapamycin administration significantly reduces the liver pathology. Collectively, our results reveal that OTULIN is critical for maintaining liver homoeostasis and suggest that M1-linked polyubiquitin chains may play a role in regulation of mTOR signalling and metabolism in the liver.

Topics: Animals; Animals, Newborn; Carcinogenesis; Carcinoma, Hepatocellular; Cell Death; Cell Proliferation; Endopeptidases; Fatty Liver; Female; Gene Deletion; Hematopoiesis; Humans; Inflammation; Liver; Liver Cirrhosis; Liver Neoplasms; Male; Mice; Receptors, Tumor Necrosis Factor, Type I; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Factors affecting outcomes in liver transplant (LTx) recipients with hepatocellular carcinoma (HCC) and hepatitis C viral (HCV) infection include the choice of immunosuppression. Here, we analyzed the HCV

Topics: Carcinoma, Hepatocellular; Hepatitis C; Humans; Liver Neoplasms; Liver Transplantation; Neoplasm Recurrence, Local; Silver; Sirolimus

Abnormal activation of mTORC1 signaling occurs at high frequency in hepatocellular carcinoma (HCC). However, the underlying causes of this aberrant activation remain elusive. In this study, we identified ventricular zone expressed pleckstrin homology domain-containing 1 (VEPH1) as a novel tumor suppressor that acts via the mTORC1 axis.. We performed quantitative reverse-transcription PCR (92 pairs), western blot (30 pairs), and immunostaining (225 cases) assays in HCC tissue samples to evaluate VEPH1 expression. We explored the functional effects of VEPH1 on tumor growth and metastasis. Molecular and biochemical strategies were used to gain insight into mechanisms underlying the tumor-suppressive function of VEPH1.. VEPH1 is frequently silenced in HCC tissues, primarily resulting from let-7d upregulation. Decreased VEPH1 expression is associated with poor prognosis and aggressive tumor phenotypes in patients with HCC. VEPH1 mediates its tumor-suppressing activity through regulation of cell proliferation, migration and invasion in vitro and in vivo. The VEPH1 fragments 580-625aa and 447-579 aa bind directly to TSC1 (719-1,164aa) and TSC2 (1-420 aa), respectively, enhancing TSC1/TCS2 binding and promoting translocation of TSC2 to the membrane, which leads to increased TSC2 Ser. The loss of VEPH1 leads to aberrantly activated mTORC1 signaling in HCC; rapamycin (or rapalogs) may serve as an effective treatment option for patients with HCC and dampened VEPH1 expression.. Abnormally activated mammalian target of rapamycin (mTOR) signaling is associated with poor tumor differentiation, early tumor recurrence and worse overall survival in patients with hepatocellular carcinoma. Herein, we identify low VEPH1 expression as a potential cause of abnormally activated mTOR signaling in hepatocellular carcinoma tissues. mTOR inhibitors could thus be an effective treatment option for patients with HCC and low VEPH1 expression.

Topics: Antibiotics, Antineoplastic; Carcinoma, Hepatocellular; Humans; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Mechanistic Target of Rapamycin Complex 1; Neoplasm Staging; Pleckstrin Homology Domains; Prognosis; Signal Transduction; Sirolimus; Tuberous Sclerosis Complex 1 Protein; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

Topics: Carcinoma, Hepatocellular; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Sirolimus

SOCS5 is a member of the suppressor of cytokine signaling (SOCS) protein family with important yet incompletely understood biological functions in cancer. In hepatocellular carcinoma (HCC), controversial tumor-promoting and tumor-suppressive roles of SOCS5 have been reported. Our study aims to unravel novel functions of SOCS5 in HCC, especially that affecting metastasis. We examined the expression levels of SOCS5 in HCC using publicly available datasets, and in our patient cohort, using quantitative real-time PCR, western blotting, and immunohistochemistry. The association of SOCS5 expression with clinical pathological data of HCC patients was examined and that with the mTOR pathway was predicted. We further studied the effects of SOCS5 on PI3K/Akt/mTOR activity; HCC cell autophagy, migration, and invasion; and HCC cell metastasis in vitro and in vivo. We observed that SOCS5 was significantly overexpressed in HCC tissues, compared to adjacent non-tumor liver tissues, in both the public datasets and in our patient cohort. SOCS5 overexpression was significantly and inversely correlated with HCC patient prognosis. Moreover, SOCS5 overexpression promoted HCC cell migration and invasion in vitro by inactivating PI3K/Akt/mTOR-mediated autophagy. Conversely, SOCS5 inhibition suppressed HCC cell migration and invasion in vitro by activating PI3K/Akt/mTOR-mediated autophagy. Dual inhibition of SOCS5 and mTOR further enhanced autophagy and the subsequent anti-metastatic effects on HCC cells. In vivo, stable knockdown of SOCS5 reduced HCC cell metastasis. Overall, our study revealed a novel metastasis-promoting function of SOCS5 in HCC, acting via the PI3K/Akt/mTOR-mediated autophagy pathway. Combined inhibition of SOCS5 and mTOR may be a potential therapeutic approach to inhibit HCC metastasis and prolong patient survival.

Topics: Animals; Autophagy; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Dimethyl Sulfoxide; Female; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Male; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Models, Biological; Multivariate Analysis; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphatidylinositol 3-Kinases; Prognosis; Proto-Oncogene Proteins c-akt; RNA, Messenger; Sirolimus; Suppressor of Cytokine Signaling Proteins; TOR Serine-Threonine Kinases

Activation of the PI3K/Akt/mTOR pathway is an important signaling mechanism involved in the development and the progression of liver cancer stem cell (LCSC) population during acquired Sorafenib resistance in advanced hepatocellular carcinoma (HCC). Therefore, identification of novel therapeutic targets involving this pathway and acting on LCSCs is highly essential. Here, we analyzed the bioactivities and the molecular pathways involved in the action of small-molecule PI3K/Akt/mTOR pathway inhibitors in comparison with Sorafenib, DNA intercalators, and DAPT (CSC inhibitor) on CD133/EpCAM-positive LCSCs. Sorafenib and DNA intercalators lead to the enrichment of LCSCs, whereas Rapamycin and DAPT significantly reduced CD133/EpCAM positivity. Sequential treatment with Rapamycin followed by Sorafenib decreased the ratio of LCSCs as well as their sphere formation capacity, as opposed to Sorafenib alone. Under the stress of the inhibitors, differential expression analysis of 770 cancer pathway genes using network-based systems biology approach singled out

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Humans; Interleukin-8; Liver Neoplasms; Neoplastic Stem Cells; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; Sorafenib; Sulfonamides; TOR Serine-Threonine Kinases

Sirtuin 4 (SIRT4) has been reported to play a vital role in the maintenance of glutamine catabolism and adenosine triphosphate (ATP) homeostasis, but its character in hepatocellular carcinomas (HCCs) remains obscure. In this study, we observed low expression of SIRT4 in both HCC cell lines and HCCs from patients. Decreased disease-free survival time is associated with low tumor levels of SIRT4 in patients. Deficiency of SIRT4 facilitated liver tumor development and lung metastasis in xenografts and knockout (KO) mice by promoting colony formation and migration of hepatoma cells and enhancing sphere formation of HCCs. Mechanistically, SIRT4 deletion augmented mammalian target of rapamycin (mTOR) signaling by inactivating adenosine-monophosphate (AMP)-activated protein kinase alpha (AMPKα) through regulation of glutamine catabolism and subsequent AM)/liver kinase B1 (LKB1) axis. Blockage of mTOR by rapamycin or inhibition of glutaminolysis abolished the discrepancy in tumorigenic capacity between SIRT4-depleted hepatoma cells and control cells. Suppression of LKB1 or promotion of AMP by metformin also abrogated the hyperproliferative phenotype caused by SIRT4 loss, which further confirmed that the LKB1/AMPKα/mTOR axis is required in SIRT4-deficiency-promoted HCC tumorigenesis. Conclusion: SIRT4 could exert its tumor suppressive function in HCC by inhibiting glutamine metabolism and thereby increasing the adenosine diphosphate (ADP)/AMP levels to phosphorylate AMPKα by LKB1, which blocks the mTOR signaling pathway.

Topics: Adenosine Monophosphate; AMP-Activated Protein Kinases; Animals; Carcinogenesis; Carcinoma, Hepatocellular; Down-Regulation; Glutamine; Hep G2 Cells; Humans; Liver Neoplasms, Experimental; Mice, Knockout; Mitochondrial Proteins; Protein Serine-Threonine Kinases; Signal Transduction; Sirolimus; Sirtuins; TOR Serine-Threonine Kinases

Based on their lobule location, hepatocytes display differential gene expression, including pericentral hepatocytes that surround the central vein, which are marked by Wnt-β-catenin signaling. Activating β-catenin mutations occur in a variety of liver tumors, including hepatocellular carcinoma (HCC), but no specific therapies are available to treat these tumor subsets. Here, we identify a positive relationship between β-catenin activation, its transcriptional target glutamine synthetase (GS), and p-mTOR-S2448, an indicator of mTORC1 activation. In normal livers of mice and humans, pericentral hepatocytes were simultaneously GS and p-mTOR-S2448 positive, as were β-catenin-mutated liver tumors. Genetic disruption of β-catenin signaling or GS prevented p-mTOR-S2448 expression, while its forced expression in β-catenin-deficient livers led to ectopic p-mTOR-S2448 expression. Further, we found notable therapeutic benefit of mTORC1 inhibition in mutant-β-catenin-driven HCC through suppression of cell proliferation and survival. Thus, mTORC1 inhibitors could be highly relevant in the treatment of liver tumors that are β-catenin mutated and GS positive.

Topics: Acetates; Animals; beta Catenin; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Survival; Child; Child, Preschool; Disease Models, Animal; Female; Glutamate-Ammonia Ligase; Glutamine; Hepatocytes; Humans; Infant; Liver Neoplasms; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred C57BL; Mice, Knockout; Mutation; Phenols; Retrospective Studies; Sirolimus; TOR Serine-Threonine Kinases; Transfection; Wnt Signaling Pathway

Liver cancer is a poor prognosis cancer with limited treatment options. To develop a new therapeutic approach, we derived HCC cells from a known model of murine hepatocellular carcinoma (HCC). We treated adiponectin (APN) knock-out mice with the carcinogen diethylnitrosamine, and the resulting tumors were 7-fold larger than wild-type controls. Tumors were disassociated from both genotypes and their growth characteristics evaluated. A52 cells from APN KO mice had the most robust growth in vitro and in vivo, and presented with pathology similar to the parental tumor. All primary tumors and cell lines exhibited activity of the mammalian target of Rapamycin (mTOR) and Src pathways. Subsequent combinatorial treatment, with the mTOR inhibitor Rapamycin and the Src inhibitor Dasatinib reduced A52 HCC growth 29-fold in vivo. Through protein and histological analyzes we observed activation of these pathways in human HCC, suggesting that targeting both mTOR and Src may be a novel approach for the treatment of HCC.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Dasatinib; Drug Delivery Systems; Humans; Liver Neoplasms, Experimental; Mice; Mice, Knockout; Proto-Oncogene Proteins pp60(c-src); Sirolimus; TOR Serine-Threonine Kinases

Topics: Anastomosis, Surgical; Carcinoma, Hepatocellular; Disease Progression; Fatal Outcome; Female; Hematoma; Hepatic Artery; Humans; Immunosuppressive Agents; Liver Neoplasms; Middle Aged; Patient Readmission; Peritoneal Cavity; Postoperative Care; Reoperation; Risk Assessment; Rupture, Spontaneous; Sirolimus; Tacrolimus; Tomography, X-Ray Computed

Topics: beta Catenin; Carcinoma, Hepatocellular; Glutamine; Humans; Liver Neoplasms; Mechanistic Target of Rapamycin Complex 1; Mutation; Sirolimus

Topics: Carcinoma, Hepatocellular; Everolimus; Humans; Liver Neoplasms; Liver Transplantation; Neoplasm Recurrence, Local; Sirolimus

Topics: Carcinoma, Hepatocellular; Everolimus; Humans; Liver Neoplasms; Liver Transplantation; Neoplasm Recurrence, Local; Sirolimus

Topics: Carcinoma, Hepatocellular; Everolimus; Humans; Liver Neoplasms; Liver Transplantation; Sirolimus

Rapamycin is employed as an immunosuppressant following organ transplant and, in patients with hepatocellular carcinoma, to inhibit cancer cell regrowth following liver surgery. Preconditioning the liver with rapamycin to induce the expression of antioxidant enzymes is a potential strategy to reduce ischemia reperfusion (IR) injury. However, pre-treatment with rapamycin inhibits bile flow, especially following ischemia. The aim was to investigate the mechanisms involved in this inhibition. In a rat model of segmental hepatic ischemia and reperfusion, acute administration of rapamycin by intravenous injection did not inhibit the basal rate of bile flow. Pre-treatment of rats with rapamycin for 24 h by intraperitoneal injection inhibited the expression of mRNA encoding the sinusoidal influx transporters Ntcp, Oatp1 and 2 and the canalicular efflux transporter Bsep, and increased expression of canalicular Mrp2. Dose-response curves for the actions of rapamycin on the expression of Bsep and Ntcp in cultured rat hepatocytes were biphasic, and monophasic for effects on Oatp1. In cultured tumorigenic H4IIE liver cells, several bile acid transporters were not expressed, or were expressed at very low levels compared to hepatocytes. In H4IIE cells, rapamycin increased expression of Ntcp, Oatp1 and Mrp2, but decreased expression of Oatp2. It is concluded that the inhibition of bile flow recovery following ischemia observed in rapamycin-treated livers is principally due to inhibition of the expression of sinusoidal bile acid transporters. Moreover, in tumorigenic liver tissue the contribution of tumorigenic hepatocytes to total liver bile flow is likely to be small and is unlikely to be greatly affected by rapamycin.

Topics: Animals; Bile; Bile Ducts; Carcinoma, Hepatocellular; Carrier Proteins; Cell Line, Tumor; Cholestasis; Disease Models, Animal; Dose-Response Relationship, Drug; Hepatocytes; Humans; Immunosuppressive Agents; Ischemia; Liver; Liver Neoplasms; Liver Transplantation; Male; Membrane Glycoproteins; Rats; Rats, Sprague-Dawley; Rats, Zucker; Reperfusion Injury; Sirolimus; Transplantation Conditioning

Topics: Carcinoma, Hepatocellular; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Neoplasm Recurrence, Local; Sirolimus

Topics: Carcinoma, Hepatocellular; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Neoplasm Recurrence, Local; Sirolimus

Amplification and/or activation of the c-Myc proto-oncogene is one of the leading genetic events along hepatocarcinogenesis. The oncogenic potential of c-Myc has been proven experimentally by the finding that its overexpression in the mouse liver triggers tumor formation. However, the molecular mechanism whereby c-Myc exerts its oncogenic activity in the liver remains poorly understood. Here, we demonstrate that the mammalian target of rapamycin complex 1 (mTORC1) cascade is activated and necessary for c-Myc-dependent hepatocarcinogenesis. Specifically, we found that ablation of Raptor, the unique member of mTORC1, strongly inhibits c-Myc liver tumor formation. Also, the p70 ribosomal S6 kinase/ribosomal protein S6 and eukaryotic translation initiation factor 4E-binding protein 1/eukaryotic translation initiation factor 4E signaling cascades downstream of mTORC1 are required for c-Myc-driven tumorigenesis. Intriguingly, microarray expression analysis revealed up-regulation of multiple amino acid transporters, including solute carrier family 1 member A5 (SLC1A5) and SLC7A6, leading to robust uptake of amino acids, including glutamine, into c-Myc tumor cells. Subsequent functional studies showed that amino acids are critical for activation of mTORC1 as their inhibition suppressed mTORC1 in c-Myc tumor cells. In human hepatocellular carcinoma specimens, levels of c-Myc directly correlate with those of mTORC1 activation as well as of SLC1A5 and SLC7A6.. Our current study indicates that an intact mTORC1 axis is required for c-Myc-driven hepatocarcinogenesis; thus, targeting the mTOR pathway or amino acid transporters may be an effective and novel therapeutic option for the treatment of hepatocellular carcinoma with activated c-Myc signaling. (Hepatology 2017;66:167-181).

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Biopsy, Needle; Carcinogenesis; Carcinoma, Hepatocellular; Cell Cycle Proteins; Disease Models, Animal; Genes, myc; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Liver Neoplasms; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Knockout; Multiprotein Complexes; Phosphoproteins; Phosphorylation; Proportional Hazards Models; Proto-Oncogene Mas; Random Allocation; Signal Transduction; Sirolimus; Statistics, Nonparametric; TOR Serine-Threonine Kinases

Topics: Animals; Antioxidants; Apoptosis; Autophagy; Benzodioxoles; Carcinoma, Hepatocellular; Cell Proliferation; Hep G2 Cells; Humans; Liver Neoplasms; Male; Membrane Potential, Mitochondrial; Mice, Nude; Mitochondria; Phenols; Sirolimus; Xenograft Model Antitumor Assays

Hepatocellular carcinoma (HCC) is the third most frequent cause of cancer deaths worldwide. The standard of care for intermediate HCC is transarterial chemoembolization, which combines tumour embolization with locoregional delivery of the chemotherapeutic doxorubicin. Embolization therapies induce hypoxia, leading to the escape and proliferation of hypoxia-adapted cancer cells. The transcription factor that orchestrates responses to hypoxia is hypoxia-inducible factor 1 (HIF-1). The aim of this work is to show that targeting HIF-1 with combined drug therapy presents an opportunity for improving outcomes for HCC treatment. HepG2 cells were cultured under normoxic and hypoxic conditions exposed to doxorubicin, rapamycin and combinations thereof, and analyzed for viability and the expression of hypoxia-induced HIF-1α in response to these treatments. A pilot study was carried out to evaluate the antitumour effects of these drug combinations delivered from drug-eluting beads in vivo using an ectopic xenograft murine model of HCC. A therapeutic doxorubicin concentration that inhibits the viability of normoxic and hypoxic HepG2 cells and above which hypoxic cells are chemoresistant was identified, together with the lowest effective dose of rapamycin against normoxic and hypoxic HepG2 cells. It was shown that combinations of rapamycin and doxorubicin are more effective than doxorubicin alone. Western Blotting indicated that both doxorubicin and rapamycin inhibit hypoxia-induced accumulation of HIF-1α. Combination treatments were more effective in vivo than either treatment alone. mTOR inhibition can improve outcomes of doxorubicin treatment in HCC.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Hepatocellular; Cell Hypoxia; Cell Survival; Chemoembolization, Therapeutic; Doxorubicin; Drug Resistance, Neoplasm; Female; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Microspheres; Random Allocation; Sirolimus; Xenograft Model Antitumor Assays

Hepatitis B virus (HBV) infection plays a crucial role and is a major cause of hepatocellular carcinoma (HCC) in China. microRNAs (miRNAs) have emerged as key players in hepatic steatosis and carcinogenesis. We found that down-regulation of miR-384 expression was a common event in HCC, especially HBV-related HCC. However, the possible function of miR-384 in HBV-related HCC remains unclear. The oncogene pleiotrophin (PTN) was a target of miR-384. HBx inhibited miR-384, increasing PTN expression. The PTN receptor N-syndecan was highly expressed in HCC. PTN induced by HBx acted as a growth factor via N-syndecan on hepatocytes and further promoted cell proliferation, metastasis and lipogenesis. PTN up-regulated sterol regulatory element-binding protein 1c (SREBP-1c) through the N-syndecan/PI3K/Akt/mTORC1 pathway and the expression of lipogenic genes, including fatty acid synthesis (FAS). PTN-mediated de novo lipid synthesis played an important role in HCC proliferation and metastasis. PI3K/AKT and an mTORC1 inhibitor diminished PTN-induced proliferation, metastasis and lipogenesis. Taken together, these data strongly suggest that the dysregulation of miR-384 could play a crucial role in HBV related to HCC, and the target gene of miR-384, PTN, represents a new potential therapeutic target for the prevention of hepatic steatosis and further progression to HCC after chronic HBV infection.

Topics: Adult; Carcinoma, Hepatocellular; Carrier Proteins; Cell Proliferation; Chromones; Cytokines; fas Receptor; Female; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Hepatitis B; Hepatitis B virus; Hepatocytes; Host-Pathogen Interactions; Humans; Lipogenesis; Liver Neoplasms; Lymphatic Metastasis; Male; Mechanistic Target of Rapamycin Complex 1; MicroRNAs; Middle Aged; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; Sterol Regulatory Element Binding Protein 1; Syndecan-3; Trans-Activators; Viral Regulatory and Accessory Proteins

Mechanistic target of rapamycin (mTOR) signaling is typically increased in hepatocellular carcinoma (HCC). A panel of HCC cell lines (HepG2, Hep3B and HuH6) was exposed to various concentrations of the mTOR inhibitors, everolimus and temsirolimus, in order to investigate their effects on cell growth, clonal formation, cell cycle progression, and adhesion and chemotactic migration using MTT and clonal cell growth assays, fluorometric detection of cell cycle phases and a Boyden chamber assay. In addition, integrin α and β adhesion receptors were analyzed by flow cytometry and blocking studies using function blocking monoclonal antibodies were conducted to explore functional relevance. The results demonstrated that everolimus and temsirolimus significantly suppressed HCC cell growth and clonal formation, at 0.1 or 1 nM (depending on the cell line). In addition, the number of cells in G0/G1 phase was increased in response to drug treatment, whereas the number of G2/M phase cells was decreased. Drug treatment also considerably suppressed HCC cell adhesion to immobilized collagen. Integrin profiling revealed strong expression of integrin α1, α2, α6 and β1 subtypes; and integrin α1 was upregulated in response to mTOR inhibition. Suppression of integrin α1 did not affect cell growth; however, it did significantly decrease adhesion and chemotaxis, with the influence on adhesion being greater than that on motility. Due to a positive association between integrin α1 expression and the extent of adhesion, whereby reduced receptor expression was correlated to decreased cell adhesion, it may be hypothesized that the adhesion‑blocking effects of mTOR inhibitors are not associated with mechanical contact inhibition of the α1 receptor but with integrin α1‑dependent suppression of oncogenic signaling, thus preventing tumor cell‑matrix interaction.

Topics: Antibodies, Monoclonal; Carcinoma, Hepatocellular; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Chemotaxis; Everolimus; G1 Phase Cell Cycle Checkpoints; Hep G2 Cells; Humans; Integrin alpha1; Integrins; Liver Neoplasms; Sirolimus; TOR Serine-Threonine Kinases; Up-Regulation

Topics: Animals; Awards and Prizes; Biomedical Research; Carcinoma, Hepatocellular; Clinical Trials, Phase III as Topic; Diet, High-Fat; Graft Rejection; Heart Transplantation; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Obesity; Organ Transplantation; Randomized Controlled Trials as Topic; Sirolimus

The oncogenic PI3K/Akt/mTOR pathway is frequently activated in hepatocellular carcinoma (HCC). The aim of this study is to investigate the anti-HCC effect of combination of temsirolimus, an mTOR inhibitor, and adriamycin, a routinely used drug for treating HCC.. Proliferation of HCC cells exposure to temsirolimus, adriamycin, and their combination was determined using MTT assay in vitro as well as in a nude mice model in vivo. Cell apoptosis was examined using flow cytometry. Expressions of apoptosis-related proteins including caspase-9, -3, PARP, Bax, and Bcl-2 were determined using Western blotting.. Temsirolimus plus adriamycin showed an enhanced inhibitory effect on cell proliferation compared to temsirolimus or adriamycin in HCC cells PLC/PRF/5, BEL7402, and HuH7 in vitro. The drug combination solicited a higher percentage of apoptosis cells and induced higher levels of cleaved caspase-9, -3, and PARP than temsirolimus or adriamycin used alone. The ratio of Bax/Bcl-2 was increased in cells exposed to the combination treatment. The enhanced anti-tumor effect of this drug combination was verified in a nude mice model. We also observed that half doses of temsirolimus and adriamycin used in combination achieved a comparable tumor growth inhibitor rate with full dose of temsirolimus or adriamycin used alone.. Temsirolimus plus adriamycin exhibited an enhanced antitumor effect in HCC and this drug combination might have a potential value in treatment of HCC. Studies are warranted to comprehensively evaluate the efficacy and safety of this regimen in the future.

Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Doxorubicin; Humans; Liver Neoplasms; Mice; Mice, Nude; Poly(ADP-ribose) Polymerases; Sirolimus; TOR Serine-Threonine Kinases

We investigated the mutational landscape of mammalian target of rapamycin (mTOR) signalling cascade in hepatocellular carcinomas (HCCs) with chronic HBV background, aiming to evaluate and delineate mutation-dependent mechanism of mTOR hyperactivation in hepatocarcinogenesis.. We performed next-generation sequencing on human HCC samples and cell line panel. Systematic mutational screening of mTOR pathway-related genes was undertaken and mutant genes were evaluated based on their recurrence. Protein expressions of tuberous sclerosis complex (TSC)1, TSC2 and pRPS6 were assessed by immunohistochemistry in human HCC samples. Rapamycin sensitivity was estimated by colony-formation assay in HCC cell lines and the treatment was further tested using our patient-derived tumour xenograft (PDTX) models.. We identified and confirmed multiple mTOR components as recurrently mutated in HBV-associated HCCs. Of significance, we detected frequent (16.2%, n=18/111) mutations of. Taken together, our findings suggest the significance of previously undocumented mutation-dependent mTOR hyperactivation and frequent

Topics: Adult; Aged; Animals; Antibiotics, Antineoplastic; Axin Protein; beta Catenin; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; DNA Mutational Analysis; DNA-Binding Proteins; Female; Humans; Liver Neoplasms; Male; Mice; Middle Aged; Mutation Rate; Neoplasm Transplantation; Nuclear Proteins; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Tuberous Sclerosis Complex 1 Protein; Tuberous Sclerosis Complex 2 Protein; Tumor Stem Cell Assay; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Young Adult

Topics: Adult; Carcinoma, Hepatocellular; Esophagitis; Fatal Outcome; Humans; Liver Neoplasms; Male; Neuroblastoma; Radiation Injuries; Radiation-Sensitizing Agents; Sirolimus

Topics: Carcinoma, Hepatocellular; Female; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Male; Sirolimus

Deregulation of mTOR and IGF pathways is frequent in hepatocellular carcinoma (HCC), thus mTOR and IGF1R represent suitable therapeutic targets in HCC. The aim of this study was to evaluate the effects of mTOR inhibitors (mTORi) and OSI-906, blocker of IGF1R/IR, on HCC cell proliferation, viability, migration and invasion, and alpha-fetoprotein (α-FP) secretion. In HepG2 and HuH-7 we evaluated, the expression of mTOR and IGF pathway components; the effects of Sirolimus, Everolimus, Temsirolimus and OSI-906 on cell proliferation; the effects of Sirolimus, OSI-906, and their combination, on cell secretion, proliferation, viability, cell cycle, apoptosis, invasion and migration. Moreover, intracellular mechanisms underlying these cell functions were evaluated in both cell lines. Our results show that HepG2 and HuH-7 present with the same mRNA expression profile with high levels of IGF2. OSI-906 inhibited cell proliferation at high concentration, while mTORi suppressed cell proliferation in a dose-time dependent manner in both cell lines. The co-treatment showed an additive inhibitory effect on cell proliferation and viability. This effect was not related to induction of apoptosis, but to G0/G1 phase block. Moreover, the co-treatment prevented the Sirolimus-induced AKT activation as escape mechanism. Both agents demonstrated to be differently effective in inhibiting α-FP secretion. Sirolimus, OSI-906, and their combination, blocked cell migration and invasion in HuH-7. These findings indicate that, co-targeting of IGF1R/IR and mTOR pathways could be a novel therapeutic approach in the management of HCC, in order to maximize antitumoral effect and to prevent the early development of resistance mechanisms.

Topics: alpha-Fetoproteins; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Drug Combinations; Everolimus; Extracellular Signal-Regulated MAP Kinases; G1 Phase Cell Cycle Checkpoints; Hep G2 Cells; Humans; Imidazoles; Insulin; Liver Neoplasms; Proto-Oncogene Proteins c-akt; Pyrazines; Receptor, IGF Type 1; Receptors, Somatomedin; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Messenger; Sirolimus; TOR Serine-Threonine Kinases

For recipients of liver transplantations (LTs) for hepatocellular carcinoma (HCC), HCC recurrence after transplantation remains a major concern. Sirolimus (SRL), an immunosuppressant with anticarcinogenic properties, may reduce HCC recurrence and improve survival. In our study, the US Scientific Registry of Transplant Recipients was linked to pharmacy claims. For liver recipients transplanted for HCC, Cox regression was used to estimate associations of early SRL use with recurrence, cancer-specific mortality, and all-cause mortality, adjusting for recipient ethnicity, calendar year of transplant, total tumor volume, alpha-fetoprotein, transplant center size, use of interleukin 2 induction therapy, and allocated and calculated Model for End-Stage Liver Disease score. We performed stratified analyses among recipients who met Milan criteria, among those without renal failure, among those with deceased liver donors, by age at transplantation, and by tumor size. Among the 3936 included HCC LTs, 234 (6%) were SRL users. In total, there were 242 recurrences and 879 deaths, including 261 cancer-related deaths. All-cause mortality was similar in SRL users and nonusers (adjusted hazard ratio [aHR], 1.01; 95% CI, 0.73-1.39). HCC recurrence and cancer-specific mortality rates appeared lower in SRL users, but associations were not statistically significant (recurrence aHR, 0.86; 95% CI, 0.45-1.65; cancer-specific mortality aHR, 0.80; 95% CI, 0.43-1.50). Among recipients >55 years old, associations were suggestive of better outcomes for SRL users (all-cause mortality aHR, 0.62; 95% CI, 0.38-1.01; recurrence aHR, 0.52; 95% CI, 0.19-1.44; cancer-specific mortality aHR, 0.34; 95% CI, 0.11-1.09), whereas among recipients ≤55 years old, SRL users had worse outcomes (all-cause mortality aHR, 1.76; 95% CI, 1.12-2.75; recurrence aHR, 1.49; 95% CI, 0.62-3.61; cancer-specific mortality aHR, 1.54; 95% CI, 0.71-3.32). In conclusion, among HCC liver recipients overall, SRL did not appear beneficial in reducing all-cause mortality. However, there were suggestions of reductions in recurrence and cancer-specific mortality, and effects appeared to be modified by age at transplantation. Liver Transplantation 22 627-634 2016 AASLD.

Topics: Carcinoma, Hepatocellular; Drug Administration Schedule; End Stage Liver Disease; Female; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Proportional Hazards Models; Severity of Illness Index; Sirolimus; Transplant Recipients; Treatment Outcome

This study aimed to determine the expression of progranulin (PGRN) in hepatocellular carcinoma (HCC) cells in response to interleukin 6 (IL-6), a non-cellular component of the tumor microenvironment, and the molecular mechanism of PGRN oncogenic activity in hepatocarcinogenesis. Levels of IL-6 and PGRN were increased and positively correlated in HCC tissues. IL-6 dose- and time-dependently increased PGRN level in HCC cells. IL-6-driven PGRN expression was at least in part mediated by Erk/C/EBPβ signaling, and reduced expression of PGRN impaired IL-6-stimulated proliferation, migration and invasion of HepG2 cells. PGRN activated mammalian target of rapamycin (mTOR) signaling, as evidenced by increased phosphorylation of p70S6K, 4E-BP1, and Akt-Ser473/FoxO1. Inhibition of mTOR signaling with rapamycin, an mTOR signaling inhibitor, disturbed PGRN- or IL-6-mediated proliferation, migration and invasion of HCC cells in vitro. Persistent activation of mTOR signaling by knockdown of TSC2 restored PGRN-knockdown-attenuated pro-proliferation effects of IL-6 in HepG2 cells. In addition, rapamycin treatment in vivo in mice slowed tumor growth stimulated by recombinant human PGRN. Our findings provide a better understanding of the biological activities of the IL-6/PGRN/mTOR cascade in the carcinogenesis of HCC, which may suggest a novel target in the treatment of HCC.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Heterografts; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-6; Liver Neoplasms; Mice; Progranulins; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Burden

Topics: Animals; Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Liver Transplantation; Sirolimus; TOR Serine-Threonine Kinases

Matrix stiffness as an important physical attribute of extracellular matrix exerts significant impacts on biological behaviors of cancer cells such as growth, proliferation, motility, metabolism and invasion. However, its influence on cancer stemness still remains elusive. Here, we explore whether matrix stiffness-mediated effects on stemness characteristics occur in HCC cells. As the substrate stiffness increased, HCC cells exhibited high proportion of cells with CD133(+)/EpCAM(+), high expression levels of CD133, EpCAM, Nanog and SOX2, greater self-renewing ability and oxaliplatin resistance. Simultaneously, their phosphorylation levels of Akt and mTOR, as well as p-4E-BP and SOX2 expressions were also obviously upregulated. Conversely, knockdown of integrin β1 partially attenuated higher stiffness-mediated stemness characteristics in HCC cells, and reversed the phosphorylation levels of Akt and mTOR, and expressions of p-4E-BP and SOX2, suggesting that integrin β1 may deliver higher stiffness signal into HCC cells and activate mTOR signaling pathway. Additionally, mTOR inhibitor suppressed the mTOR phosphorylation level and expression levels of p-4E-BP and SOX2 in HCC cells grown on higher stiffness substrate, as well as depressed their stemness properties significantly, favoring a regulating role of mTOR signaling pathway in matrix stiffness-mediated effects on stemness. In summary, matrix stiffness may be involved in the process of stemness regulation via activating integrin β1/Akt/mTOR/SOX2 signaling pathway. To the best of our knowledge, this study first reveals a novel regulating pathway to direct the stemness characteristics in HCC cells.

Topics: Antineoplastic Agents; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Self Renewal; Drug Resistance, Neoplasm; Elasticity; Extracellular Matrix; Humans; Integrin beta1; Liver Neoplasms; Mechanotransduction, Cellular; Neoplastic Stem Cells; Organoplatinum Compounds; Oxaliplatin; Phenotype; Phosphorylation; Proto-Oncogene Proteins c-akt; RNA Interference; Sirolimus; SOXB1 Transcription Factors; TOR Serine-Threonine Kinases; Transfection

Topics: Animals; Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Liver Transplantation; Sirolimus; TOR Serine-Threonine Kinases

Topics: Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Liver Transplantation; Neoplasm Recurrence, Local; Sirolimus

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Although recent studies facilitate the identification of crucial genes and relevant regulatory pathways, therapeutic approaches against advanced HCC are insufficiently effective. Therefore, we aimed here to develop potent therapeutics to provide a reliable biomarker for the therapeutic efficacy in patients with HCC. To this end, we first compared the cytotoxic effects of various anti-cancer drugs between well differentiated (HAK-1A) and poorly differentiated (HAK-1B) cell lines established from a single HCC tumor. Of various drug screened, HAK-1B cells were more sensitive by a factor of 2,000 to the mTORC1 inhibitors (rapalogs), rapamycin and everolimus, than HAK-1A cells. Although rapalogs inhibited phosphorylation of mTOR Ser2448 in HAK-1A and HAK-1B cells, phosphorylation of mTOR Ser2481 was specifically inhibited only in HAK-1B cells. Silencing of Raptor induced apoptosis and inhibited the growth of only HAK-1B cells. Further, three other cell lines established independently from the tumors of three patients with HCC were also approximately 2,000-fold times more sensitive to rapamycin, which correlated closely with the inhibition of mTOR Ser2481 phosphorylation by rapamycin. Treatment with everolimus markedly inhibited the growth of tumors induced by poorly differentiated HAK-1B and KYN-2 cells and phosphorylation of mTOR Ser2481 in vivo. To our knowledge, this is the first study showing that the phosphorylation of mTOR Ser2481 is selectively inhibited by rapalogs in mTORC1-addicted HCC cells and may be a potential reliable biomarker for the therapeutic efficacy of rapalogs for treating HCC patients.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Everolimus; Humans; Liver Neoplasms; Mechanistic Target of Rapamycin Complex 1; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Sirolimus; TOR Serine-Threonine Kinases

To evaluated patterns and outcomes of hepatocellular carcinoma (HCC) recurrence after living donor liver transplantation (LDLT).. From 2001 to 2014, 293 patients underwent LDLT for HCC at our transplant center. We retrospectively reviewed 54 (18.4%) patients with HCC recurrence after LDLT. We evaluated patterns and outcomes of HCC recurrence after LDLT, with particular attention to the Milan criteria at transplantation, treatments for HCC-recurrent patients, and factors related to survival after HCC recurrence. Furthermore, we evaluated the efficacy of combination treatment of sorafenib and an mTOR inhibitor.. The 1-, 2-, and 3-year overall survival rates after HCC recurrence were 41.1%, 20.5%, and 15.4%, respectively. The median time interval between LDLT and HCC recurrence was 6.5 mo. Although recurrence rates according to the Milan criteria at LDLT were significantly different, HCC recurrence patterns and survival rates after HCC recurrence were not significantly different between the two groups. Time to recurrence < 12 mo (P = 0.048), multiple recurrences at HCC recurrence (P = 0.038), and palliative treatment for recurrent tumors (P = 0.003) were significant independent prognostic factors for poor survival after HCC recurrence in a multivariate analysis. The combination treatment of sorafenib and sirolimus showed survival benefits in the palliative treatment group (P = 0.005).. Curative treatment for recurrent HCC after LDLT is the most important factor in survival rates after HCC recurrence and combination treatments of sorafenib and an mTOR inhibitor could have survival benefits in patients with HCC recurrence after LT in the palliative treatment group.

Topics: Adult; Antineoplastic Agents; Bone Neoplasms; Carcinoma, Hepatocellular; Female; Humans; Liver Neoplasms; Liver Transplantation; Living Donors; Lung Neoplasms; Male; Middle Aged; Multivariate Analysis; Neoplasm Recurrence, Local; Niacinamide; Phenylurea Compounds; Retrospective Studies; Sirolimus; Sorafenib

The presented study aimed to investigate the antitumor efficacy of combination of oxaliplatin with rapamycin, an mTOR inhibitor, in hepatocellular carcinoma (HCC). The activation status of mTOR pathway was first examined in HCC cell lines HepG2, BEL7402, and HuH7 using Western blotting. Effects of rapamycin, oxaliplatin, and their combination on the proliferation of HCC cells were determined in vitro using MTT assay and in vivo using a nude mice model bearing HepG2 xenografts. Drug-induced cell apoptosis was examined by flow cytometry. Expression of apoptosis-related protein was determined by Western blotting. We observed that mTOR pathway was activated in all three cell lines used in the current study. MTT assay demonstrated that oxaliplatin in combination with rapamycin synergistically inhibited the proliferation of HCC cells. The combination regimen reduced terminal tumor burden more efficiently than the corresponding monotherapy. The percentages of apoptotic cells and the expression levels of apoptosis-related proteins including cleaved caspase-9, -3, and PARP were significantly higher in combination-treatment groups than those in mono-drug-treatment groups. The ratios of Bax/Bcl-2 in cells exposed to both oxaliplatin and rapamycin were significantly increased compared to those in cells subjected to oxaliplatin or rapamycin alone treatment. Results obtained in the presented study suggested that combination of oxaliplatin and rapamycin was superior to mono-drug and may have a potential value in treatment of HCC.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma, Hepatocellular; Caspase 9; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Mice, Nude; Oxaliplatin; Proto-Oncogene Proteins c-bcl-2; Sirolimus; TOR Serine-Threonine Kinases

Immunosuppressive agents used postoperatively after liver transplantation (LT) for hepatocellular carcinoma (HCC) favor recurrence and metastasis. Therefore, new effective immunosuppressants are needed. This retrospective study assessed combined sirolimus and metformin on survival of HCC patients after LT. In 2001-2013, 133 HCC patients with LT were divided into four groups: sirolimus and metformin combination (Sir+Met), sirolimus monotherapy (Sir), other immunosuppressants in diabetes mellitus (DM) patients without metformin (No Sir with DM), and other immunosuppressants in patients without DM (No Sir without DM). Kaplan-Meier and Log-rank tests were used to assess survival. Cell proliferation and tumor xenograft assays were performed to disclose the mechanisms underlying the sirolimus and metformin effects. The Sir+Met group showed significantly prolonged survival compared to the other groups. The most significant cytotoxicity was seen in the Sir+Met group, with significantly decreased levels of phosphorylated PI3K, AKT, AMPK, mTOR, 4EBP1 and S6K, compared with the other groups. In agreement, Sir+Met had the highest suppressive effect on tumor growth among all groups (P<0.01). In summary, Sir+Met treatment significantly prolonged survival, likely by suppressing cell proliferation. Therefore, this combination could represent a potential routine-regimen for patients post LT.

Topics: Adult; Animals; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Synergism; Female; Hep G2 Cells; Hepatitis B; Hepatitis B virus; Humans; Immunosuppressive Agents; Liver Cirrhosis; Liver Neoplasms; Liver Transplantation; Male; Metformin; Mice; Middle Aged; Neoplasm Metastasis; Neoplasm Recurrence, Local; Neoplasm Transplantation; Phosphorylation; Sirolimus; Treatment Outcome

Rapamycin, a specific inhibitor of mTOR used extensively as an immunosuppressant, has been expanded recently to cancer therapy, because the mTOR signal is known to be up-regulated in various cancer cells including hepatocellular carcinoma (HCC) cells. In spite of extensive efforts to employ mTOR inhibitors as anti-HCC therapy, they have not yet been approved by the FDA. Because of the heterogeneity and complexity of molecular signaling in HCC, suitable biomarkers should be identified or discovered to improve clinical efficacy of mTOR-specific inhibitors to HCC cells. In this study, the effect of rapamycin was investigated on two different HCC cell lines, Huh7 cells and HepG2 cells. Rapamycin was found to inhibit the proliferation of Huh7 cells but not of HepG2 cells. Moreover, it was found that rapamycin can up-regulate p53 at the protein level, but not affect its transcript. To understand the critical role of p53 in the rapamycin effect, knock-down experiments were performed using small-interfering RNAs (siRNAs). The anti-proliferative effect of rapamycin on Huh7 cells clearly disappeared after blocking p53 production with siRNA, which indicates that p53 is a critical factor in the anti-proliferative effect of rapamycin in HCC cells. The over-expression system of p53 was also employed to mimic the effect of rapamycin and found that cell proliferation was clearly down-regulated by p53 over-expression. Finally, we found that the extracellular signal-regulated kinase 1/2 (ERK1/2) signal was regulated by p53 whose expression was induced by rapamycin. Overall, this study demonstrates that rapamycin inhibited the proliferation of Huh7 cells by up-regulating the expression of p53 and down-regulating the ERK1/2 signal, indicating that p53 is a useful biomarker for anti-cancer therapy using the specific inhibitor of mTOR signal, rapamycin, against hepatocellular carcinoma cells.

Topics: Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Immunosuppressive Agents; Liver Neoplasms; MAP Kinase Signaling System; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Sirolimus; TOR Serine-Threonine Kinases; Tumor Suppressor Protein p53; Up-Regulation

Hepatocellular carcinoma is the most prevalent type of tumor among primary tumors affecting the liver. Rapamycin is currently used as a basis for chemotherapy in the treatment of cancers, including the liver. Because it shows several adverse effects, minimizing these effects without compromising efficacy is important. In this sense other drugs may be used concomitantly. One of these drugs is fructose-1,6-bisphosphate (FBP), which has shown therapeutic effect in various pathological situations, having antioxidant and anti-inflammatory proprieties. The objective of the present study was to evaluate the activity of rapamycin in combination with the FBP in HepG2 cell proliferation and the mechanisms involved. HepG2 cells were analyzed after 72 h of treatment with both drugs. Cell proliferation, cytotoxicity, cytokines, apoptosis, senescence, autophagy and oxidative stress were accessed. Ιt was demonstrated that the combination is more efficient than the single use of substances, because subtherapeutic doses of rapamycin, when associated to FBP become effective, reducing cell proliferation, through a significant increase in the production of tiobarbituric acid reactive substances (TBARS), suggesting that this might be the cause of death by apoptosis. According to these results, we believe that the association of both drugs may be a promising choice for the treatment of hepatocarcinoma.

Topics: Antineoplastic Combined Chemotherapy Protocols; Antioxidants; Apoptosis; Autophagy; Carcinoma, Hepatocellular; Cell Proliferation; Cellular Senescence; Free Radicals; Fructose-Bisphosphatase; Hep G2 Cells; Humans; Liver Neoplasms; Sirolimus

To investigate the effect of a sirolimus-based immunosuppressive protocol on tumor recurrence and survival after liver transplantation (LT) in hepatocellular carcinoma (HCC) patients.. We retrospectively analyzed 142 HCC patients who underwent LT in our hospital from January 2006 to January 2012. The patients were divided into the sirolimus (SRL) group (62 cases) and non-sirolimus (control) group (80 cases). Disease-free survival (DFS) and tumor-bearing survival after tumor recurrence were compared using the Kaplan-Meier method.. No significant difference in DFS was observed between the two groups. Furthermore, DFS showed no significant differences in the subgroups of patients who met the Milan criteria, exceeded the Milan criteria but met the University of California, San Francisco (UCSF) criteria, or exceeded the UCSF criteria between the two groups. In the control group, 21 patients who were administered SRL after tumor recurrence had a median tumor-bearing survival time of 12months (3-34months), while 14 patients who did not experience a change in their immunosuppressive protocol after tumor recurrence had a median tumor-bearing survival time of 8months (6-22months). There was a significant difference in the tumor-bearing survival time between these patients (P=0.039).. Not all HCC patients benefited from the sirolimus-based immunosuppressive protocol after LT. However, sirolimus may prolong the survival time of patients after tumor recurrence.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Case-Control Studies; China; Female; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Neoplasm Recurrence, Local; Retrospective Studies; Sirolimus; Young Adult

It has been reported that non-selective autophagy of infected hepatocytes could facilitate the development of malaria in the liver stage, but the fate of parasites following selective autophagy of infected hepatocytes is still not very clear. Here, we confirmed that sporozoite infection can induce a selective autophagy-like process targeting EEFs (exo-erythrocytic forms) in Hepa1-6. Rapamycin treatment greatly enhanced this process in EEFs and non-selective autophagy of infected Hepa1-6 cells and enhanced the development of the malaria liver stage in vivo. Although rapamycin promoted the fusion of autophagosomes containing the malaria parasite with lysosomes, some parasites inside the autophagosome survived and replicated normally. Further study showed that the maturation of affected autolysosomes was greatly inhibited. Therefore, in addition to the previously described positive role of rapamycin-induced nonselective autophagy of hepatocytes, we provide evidence that the survival of EEFs in the autophagosome of the infected hepatocytes also contributes to rapamycin-enhanced development of the malaria liver stage, possibly due to the suppression of autolysosome maturation by EEFs. These data suggest that the inhibition of autolysosome maturation might be a novel escape strategy used by the malaria liver stage.

Topics: Animals; Antibiotics, Antineoplastic; Autophagosomes; Autophagy; Carcinoma, Hepatocellular; Cell Line, Tumor; Hepatocytes; Host-Parasite Interactions; Liver; Liver Neoplasms; Malaria; Mice; Plasmodium yoelii; Sirolimus; Sporozoites

Concomitant expression of activated forms of v-akt murine thymoma viral oncogene homolog (AKT) and Ras in mouse liver (AKT/Ras) leads to rapid tumor development through strong activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway. mTORC1 functions by regulating p70S6K/ribosomal protein S6 (RPS6) and eukaryotic translation initiation factor 4E-binding protein 1/ eukaryotic translation initiation factor 4E (4EBP1/eIF4E) cascades. How these cascades contribute to hepatocarcinogenesis remains unknown. Here, we show that inhibition of the RPS6 pathway by rapamycin effectively suppressed, whereas blockade of the 4EBP1/eIF4E cascade by 4EBP1A4, an unphosphorylatable form of 4EBP1, significantly delayed, AKT/Ras-induced hepatocarcinogenesis. Combined treatment with rapamycin and 4EBP1A4 completely inhibited AKT/Ras hepatocarcinogenesis. This strong antineoplastic effect was successfully recapitulated by ablating regulatory associated protein of mTORC1, the major subunit of mTORC1, in AKT/Ras-overexpressing livers. Furthermore, we demonstrate that overexpression of eIF4E, the proto-oncogene whose activity is specifically inhibited by 4EBP1, resulted in hepatocellular carcinoma (HCC) development in cooperation with activated Ras. Mechanistically, we identified the ectonucleoside triphosphate diphosphohydrolase 5/ adenylate kinase 1/cytidine monophosphate kinase 1 axis and the mitochondrial biogenesis pathway as targets of the 4EBP1/eIF4E cascade in AKT/Ras and Ras/eIF4E livers as well as in human HCC cell lines and tissues.. Complete inhibition of mTORC1 is required to suppress liver cancer development induced by AKT and Ras proto-oncogenes in mice. The mTORC1 effectors, RPS6 and eIF4E, play distinct roles and are both necessary for AKT/Ras hepatocarcinogenesis. These new findings might open the way for innovative therapies against human HCC.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Carcinoma, Hepatocellular; Carrier Proteins; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Eukaryotic Initiation Factor-4E; Eukaryotic Initiation Factors; Humans; Liver Neoplasms, Experimental; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; Oncogene Proteins; Phosphoproteins; Proto-Oncogene Mas; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins p21(ras); Pyrophosphatases; Ribosomal Protein S6; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; TOR Serine-Threonine Kinases

Autophagy is a cellular degradation process for the clearance of damaged or superfluous proteins and organelles, the recycling of which serves as an alternative energy source during periods of metabolic stress to maintain cell homeostasis and viability. The anti-necrotic function of autophagy is critical for tumorigenesis of many tumor cells, including hepatocellular carcinoma (HCC). However, the underlying mechanism is not clarified yet. Ammonium chloride (NH4Cl) is a well-known autophagy inhibitor, whereas its interaction with SMAD2 signaling pathway has not been reported previously. Here, we show that NH4Cl significantly inhibited rapamycin-induced autophagy in HCC cells through decreasing the levels of Beclin-1, autophagy-related protein 7 (ATG7), p62, and autophagosome marker LC3 and significantly decreased the level of phosphorylated SMAD2 in rapamycin-treated HCC cells. In order to find out whether NH4Cl may inhibit the autophagy in rapamycin-treated HCC cells through inhibition of SMAD2 signaling, we used transforming growth factor β1 (TGFβ1) to induce phosphorylation of SMAD2 in HCC cells. We found that induction of SMAD2 in HCC cells completely abolished the inhibitory effect of NH4Cl on rapamycin-induced autophagy in HCC cells, suggesting that NH4Cl inhibits autophagy of HCC cells through inhibiting SMAD2 signaling.

Topics: Ammonium Chloride; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Protein 7; Beclin-1; Carcinoma, Hepatocellular; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Membrane Proteins; RNA-Binding Proteins; Signal Transduction; Sirolimus; Smad2 Protein; Ubiquitin-Activating Enzymes

The use of sirolimus-based immune suppression in liver transplantation, particularly in hepatitis C virus (HCV)-infected recipients, remains contentious. There is some evidence that sirolimus retards hepatic fibrosis, is renal sparing and may be of benefit in preventing hepatocellular carcinoma (HCC) recurrence. Sirolimus has not been adopted by many transplant centres because of persistent concerns regarding an increased risk of hepatic artery thrombosis, graft loss and death with de novo sirolimus.. To review the impact of switching to sirolimus monotherapy in HCV-infected liver recipients with respect to survival, graft loss and hepatic fibrosis.. A retrospective review of 190 patients from a single centre undergoing first liver transplantation for HCV over 15 years. 113 patients were switched from calcineurin inhibitor (CNI)-based therapy to low-dose sirolimus monotherapy at a median of 15 months after transplantation for HCV-related fibrosis (72%), renal impairment (14%) or high-risk HCC (5%).. Patients switched to sirolimus had improved survival (P < 0.001) and slower progression to cirrhosis (P = 0.001). In patients with HCC (n = 91), sirolimus duration rather than strategy was an independent predictor of survival (P = 0.001) and extended time to HCC recurrence (33 vs. 16 months). Patients switched for renal dysfunction showed improvement in serum creatinine (140-108 μmol/L, P = 0.001). Those remaining on CNI-therapy were more likely to develop post-transplant diabetes (P = 0.03).. These data suggest selective switching to low-dose sirolimus monotherapy in HCV-positive liver recipients improves clinical outcome.

Topics: Adult; Aged; Calcineurin Inhibitors; Carcinoma, Hepatocellular; Comorbidity; Disease Progression; Female; Hepacivirus; Humans; Immunosuppressive Agents; Liver Cirrhosis; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Renal Insufficiency; Retrospective Studies; Sirolimus

Systemic therapy has proven only marginal effects in hepatocellular carcinoma (HCC) so far. The aim of this study was to evaluate the effect of targeting fibroblast growth factor receptor (FGFR) on tumour and stromal cells in HCC models.. Human and murine HCC cells, endothelial cells (ECs), vascular smooth muscle cells (VSMCs), hepatic stellate cells (HSCs), human HCC samples, FGFR inhibitor BGJ398 and mammalian target of rapamycin (mTOR) inhibitor rapamycin were used. Effects on growth, motility, signalling and angiogenic markers were determined. In vivo subcutaneous and syngeneic orthotopic tumour models were used.. In tumour cells and ECs, targeting FGFR showed significant inhibitory effects on signalling and motility. Minor effects of FGFR inhibition were observed on VSMCs and HSCs, which were significantly enhanced by combining FGFR and mTOR blockade. In vivo daily (5 mg kg(-1)) treatment with BGJ398 led to a significant growth inhibition in subcutaneous tumour models, but only a combination of FGFR and mTOR blockade impaired tumour growth in the orthotopic model. This was paralleled by reduced tumour cell proliferation, vascularisation, pericytes and increased apoptosis.. Targeting FGFR with BGJ398 affects tumour cells and ECs, whereas only a combination with mTOR inhibition impairs recruitment of VSMCs and HSCs. Therefore, this study provides evidence for combined FGFR/mTOR inhibition in HCC.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Drug Synergism; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Mice, Nude; Neoplasm Transplantation; Phenylurea Compounds; Pyrimidines; Receptor, Fibroblast Growth Factor, Type 1; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Levels of the Golgi protein 73 (GP73) increase during development of hepatocellular carcinoma (HCC); GP73 is a serum marker for HCC. However, little is known about the mechanisms or effects of GP73 during hepatic carcinogenesis.. GP73 was overexpressed from a retroviral vector in HepG2 cells, which were analyzed in proliferation and migration assays. Xenograft tumors were grown from these cells in nude mice. The effects of monoclonal antibodies against GP73 were studied in mice and cell lines. GP73(-/-), GP73(+/-), and GP73(+/+) mice were given injections of diethylnitrosamine to induce liver injury. Levels of GP73 were reduced in MHCC97H, HCCLM3, and HepG2.215 cell lines using small hairpin RNAs; xenograft tumors were grown in mice from MHCC97H-small hairpin GP73 or MHCC97H-vector cells. We used microarray analysis to compare expression patterns between GP73-knockdown and control MHCC97H cells. We studied the effects of the mechanistic target of rapamycin (mTOR) inhibitor rapamycin on GP73 expression in different cancer cell lines and on growth of tumors in mice. Levels of GP73 and activated mTOR were quantified in human HCC tissues.. Xenograft tumors grown from HepG2 cells that expressed GP73 formed more rapidly and more metastases than control HepG2 cells in mice. A monoclonal antibody against GP73 reduced proliferation of HepG2 cells and growth of xenograft tumors in mice. GP73(-/-) mice had less liver damage after administration of diethylnitrosamine than GP73(+/-) or GP73(+/+) mice. In phosphatase and tensin homolog-null mouse embryonic fibroblasts with constitutively activated mTOR, GP73 was up-regulated compared with control mouse embryonic fibroblasts; this increase was reversed after incubation with rapamycin. Expression of GP73 also was reduced in HCC and other cancer cell lines incubated with rapamycin. mTORC1 appeared to regulate expression of GP73 in cell lines. Activated mTOR correlated with the level of GP73 in human HCC tissues. Injection of rapamycin slowed the growth of xenograft tumors from MHCC97H-vector cells, compared with MHCC97H-short hairpin GP73 cells.. Increased expression of GP73 promotes proliferation and migration of HCC cell lines and growth of xenograft tumors in mice. mTORC1 regulates the expression of GP73, so GP73 up-regulation can be blocked with rapamycin. mTOR inhibitors or other reagents that reduce the level or activity of GP73 might be developed for the treatment of HCC.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Mechanistic Target of Rapamycin Complex 1; Membrane Proteins; Mice, Inbred BALB C; Mice, Knockout; Mice, Nude; Multiprotein Complexes; Neoplasm Invasiveness; Phosphoproteins; RNA Interference; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Transfection; Xenograft Model Antitumor Assays

To explore the impact of triple anti-tumor therapy based on thymosin α1 (Tα1) combined with Huaier granule(HG) and sirolimus on the level of serum alpha-fetoprotein (AFP) in rat models of liver cancer.. Ninety Sprague-Dawley rats were randomly divided into triple anti-tumor therapy group, Tα1 group, HG group, sirolimus group, positive control and blank control groups, with 15 rats in each group. Except the blank control group, the rats in the other groups were induced using diethylnitrosamine (DEN) to establish liver cancer models. After DEN treatment, the triple therapy group underwent 0.8 mg/kg Tα1 subcutaneous injection (from once a day for two weeks to twice a week since the third week), 0.35 g/kg HG gavage (three times a day) and 1 mg/kg sirolimus gavage (once a day). The dose of the rest single drug groups were the same with that of the triple therapy group. The positive control and blank control groups were not treated with the drugs. The treatment lasted 20 weeks. Then, the behavior of the rats were observed at the different time points, and the level of serum AFP in the rats were detected at 6, 16, 18, 20 weeks, respectively.. The typical symptoms of liver cancer were seen in the DEN-induced rats at 16 weeks. Since the tenth week, 6 rats died one after another. Pathological section of rat liver tissue suggested that the rat models were established successfully. According to the incidence rate of liver cancer and the survival rate at 20 weeks, the triple anti-tumor therapy was significantly superior to the single drug treatments. In addition, the triple anti-tumor therapy significantly reduced the level of serum AFP in the rats.. The triple anti-tumor therapy can significantly prolong the survival time of rats with liver cancer, decrease the cancer incidence rate and the level of serum AFP.

Topics: alpha-Fetoproteins; Animals; Antineoplastic Combined Chemotherapy Protocols; Behavior, Animal; Carcinoma, Hepatocellular; Disease Models, Animal; Liver; Liver Neoplasms; Male; Rats; Rats, Sprague-Dawley; Sirolimus; Survival Analysis; Thymalfasin; Thymosin

mTORC1 is a master regulator of cell growth and proliferation, and an established anticancer drug target. Aberrant mTORC1 signaling is common in hepatocellular carcinoma (HCC), but the underlying mechanism remains elusive. Rab1A is a newly identified mTORC1 activator that mediates an alternative amino acid (AA) signaling branch to Rag GTPases. Because liver is a physiological hub for nutrient sensing and metabolic homeostasis, we investigated the possible role of Rab1A in HCC. We found that Rab1A is frequently overexpressed in HCC, which enhances hyperactive AA-mTORC1 signaling, promoting malignant growth and metastasis of HCC in vitro and in vivo. Moreover, aberrant Rab1A expression is closely associated with poor prognosis. Strikingly, aberrant Rab1A overexpression leads to increased rapamycin sensitivity, indicating that inappropriate activation of AA signaling is a cancer-driving event in HCC. Our findings further suggest that Rab1A is a valuable biomarker for prognosis and personalized mTORC1-targeted therapy in liver cancer.

Topics: Aged; Amino Acids; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Survival; Cohort Studies; Female; Gene Dosage; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Liver Neoplasms; Male; Mechanistic Target of Rapamycin Complex 1; Middle Aged; Multiprotein Complexes; Neoplasm Invasiveness; Neoplasm Metastasis; Prognosis; rab1 GTP-Binding Proteins; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Wound Healing

To observe enhanced effects of polypeptide extract from scorpion venom (PESV) combined Rapamycin on autophagy of H22 hepatoma cells in mice and to explore its possible mechanism.. The H22 hepatocarcinoma cell suspension was subcutaneously inoculated into 40 Kunming mice. Then tumor-bearing mice were randomly divided into four groups, i.e., the control group,the high dose PESV group, the low dose PESV group, and the combination group (high dose PESV + Rapamycin), 10 in each group. Mice in high and dose PESV groups were administered with 20 mg/kg and 10 mg/kg PESV respectively by gastrogavage. Mice in the combination group were administered with 2 mg/kg rapamycin and 20 mg/kg PESV by gastrogavage. The intervention lasted for 14 successive days. The tumor volume was measured once every other day, the tumor growth curve was drawn, and then the tumor inhibitory rate calculated. Pathological changes of the tumor tissue were observed by HE staining. Protein expression levels of mammal target of rapamycin (mTOR), UNC-51-like kinase-1 (ULK1), microtubule-associated protein1 light chain3 (MAPILC3A), and Beclin1 were detected by immunohistochemical assay.. The growth of H22 hepatoma transplantation tumor was inhibited in high and low dose PESV groups and the combination group (P < 0.05). And there was statistical difference in tumor weight and tumor volume between the combination group and high and low dose PESV groups (P < 0.05). There was no statistical difference in tumor weight or tumor volume between the high dose PESV group and the low dose PESV group (P > 0.05). lmmunohistochemical assay showed that the protein expression of mTOR was higher, but protein expressions of ULK1, MAP1LC3A, Beclin1 were lower in the control group than in the rest 3 groups (P < 0.05, P < 0.01). Compared with the high dose PESV group, protein expressions of ULK1, MAP1LC3A, and Beclin1 were obviously lower (P < 0.05).. PESV combined Rapamycin might inhibit the development of H22 hepatoma transplantation tumor in mice possibly through inhibiting the activity of mTOR, enhancing expressions of ULK1, MAP1LC3A, and Beclin1.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Autophagy; Carcinoma, Hepatocellular; Cell Line, Tumor; Liver Neoplasms; Mice; Neoplasm Transplantation; Peptides; Scorpion Venoms; Sirolimus

There are no established prognostic factors or standardized therapies for hepatocellular carcinoma (HCC) recurrence in liver transplantation (LT). The aim of this study was to investigate impact of underlying patient condition on treatment and outcomes of recurrence of HCC after LT. The medical records of 268 LT patients with HCC were evaluated. Potential prognostic factors for survival after recurrence were evaluated, including recurrent tumor characteristics, medical/radiological/surgical therapies for recurrence, and an inflammatory marker (neutrophil/lymphocyte ratio). Laboratory tests at recurrence, including albumin, absolute lymphocyte count (ALC), prognostic nutritional index (PNI: ALC(/μL) × 0.005 + Albumin(g/dL) × 10), were evaluated as surrogate markers for underlying patient conditions. A total of 51 (19%) patients developed HCC recurrence. The use of sirolimus and sorafenib significantly improved outcome (p = 0.007 and 0.04), and better nutritional status (PNI ≥ 40) enhanced their efficacy. On multivariate analysis, low ALC (<500/μL) and albumin (<2.8 g/L) remained independent prognostic factors (p = 0.03 and 0.02; hazard ratio = 3.61 [Ref. >1000/μL] and 4.97 [Ref. >3.5 g/dL], respectively). Low PNI (<40) showed significantly lower survival rate after adjusting the risk (p = 0.006, hazard ratio = 3.29). Underlying patient conditions and nutritional status, represented by ALC and albumin, are important to successful cancer treatment and strong prognostic markers for survival after HCC recurrence.

Topics: Antineoplastic Agents; Biomarkers, Tumor; Carcinoma, Hepatocellular; Combined Modality Therapy; Follow-Up Studies; Graft Survival; Humans; Liver Neoplasms; Liver Transplantation; Lymphocytes; Multivariate Analysis; Neoplasm Recurrence, Local; Neutrophils; Niacinamide; Phenylurea Compounds; Postoperative Complications; Prognosis; Retrospective Studies; Risk Factors; Sirolimus; Sorafenib; Survival Rate

Progression of liver fibrosis to HCC (hepatocellular carcinoma) is a very complex process which involves several pathological phenomena, including hepatic stellate cell activation, inflammation, fibrosis and angiogenesis. Therefore inhibiting multiple pathological processes using a single drug can be an effective choice to curb the progression of HCC. In the present study, we used the mTOR inhibitor everolimus to observe its effect on the in vitro activation of hepatic stellate cells and angiogenesis. The results of the present study demonstrated that everolimus treatment blocked the functions of the immortalized human activated hepatic stellate cell line LX-2 without affecting the viability and migration of primary human stellate cells. We also observed that treatment with everolimus (20 nM) inhibited collagen production by activated stellate cells, as well as cell contraction. Everolimus treatment was also able to attenuate the activation of primary stellate cells to their activated form. Angiogenesis studies showed that everolimus blocked angiogenesis in a rat aortic ring assay and inhibited the tube formation and migration of liver sinusoidal endothelial cells. Finally, everolimus treatment reduced the load of tumoral myofibroblasts in a rat model of HCC. These data suggest that everolimus targets multiple mechanisms, making it a potent blocker of the progression of HCC from liver fibrosis.

Topics: Actins; Angiogenesis Inhibitors; Animals; Carcinoma, Hepatocellular; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cell Survival; Collagen; Disease Models, Animal; Dose-Response Relationship, Drug; Everolimus; Hepatic Stellate Cells; Humans; Immunosuppressive Agents; Liver; Liver Cirrhosis; Liver Neoplasms; Male; Muscle, Smooth; Myofibroblasts; Neoplasm Transplantation; Neovascularization, Pathologic; Rats; Rats, Wistar; Sirolimus

Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide. It is well known that the activation of PI3K/AKT/mTOR and the Ras/MAPK signaling pathway plays a critical role in cellular metabolism, growth and proliferation, and its inhibitors have been used as therapeutic drugs for hepatocellular carcinoma. Cetuximab, a chimerical monoclonal EGFR IgG1 antibody, can block the binding of EGF or other ligands to EGFR and thus inhibit ligands-induced receptor phosphorylation. In the present study, we found that rapamycin could enhance the antiproliferation effect of cetuximab in both HepG2 cells and Huh-7 cells and arrest the cell cycle. Cetuximab in combination with rapamycin had synergistic effects on inhibiting the phosphrylation of proteins in PI3K/AKT/mTOR and Ras/MAPK signaling pathway. Combination of cetuximab with rapamycin treatment significantly suppressed the HCC development in HepG2 cells-xenografted mice and improved the survival. Cetuximab and rapamycin inhibited the growth of HCC both in vitro and in vivo. These results suggest that the combination therapy using the inhibitors for both EGFR and PI3K/AKT/mTOR signaling pathways may be a novel therapeutic approach for HCC.

Topics: Animals; Antibodies, Monoclonal, Humanized; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cetuximab; Disease Models, Animal; Drug Synergism; Female; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins p21(ras); Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

In this study, we demonstrate that EGF inhibits the TGF-β1-induced apoptosis of Huh7 cells. TGF-β1 up-regulates the expression of PDCD4 causing apoptosis, by stimulating the synthesis of PDCD4 mRNA via the Smad signaling pathway. TGF-β1 also inhibits the activation of S6 kinase 1 which phosphorylates the serine 67 residue of PDCD4 and leads to the phosphorylation of serine 71 and serine 76 in the β-TRCP binding sequence. This phosphorylation sequence causes the protein to be degraded in the ubiquitin-proteasome system. EGF activates S6 kinase 1 via the PI3K-Akt-mTOR signaling pathway and stimulates the degradation of PDCD4. EGF also suppresses PDCD4 mRNA levels. As the mTOR inhibitor rapamycin up-regulated PDCD4 mRNA levels, the PI3K-Akt-mTOR signaling pathway may control the transcription of the PDCD4 gene as well as the degradation of the protein. TPA also inhibited the TGF-β1-induced apoptosis of Huh7 cells, stimulating the degradation of the PDCD4-protein. Analyses using PDCD4 mutants with changes of serines 67, 71 and 76 to alanine revealed that the phosphorylation of serine 67 is not essential for the TPA-induced suppression of the protein. The mitogens could not suppress the PDCD4-mutant proteins with changes of serine 71 and/or serine 76 to alanine, however, indicating that phosphorylations at these residues are necessary for the proteasome-mediated degradation of PDCD4. The phosphor-mimic S71/D and S76/D mutants were able to be degraded in the ubiquitin-proteasome system unlike the mutants with changes of serine to alanine. The expression of S71/D mutant was suppressed with EGF but that of S76/D mutant was not indicating that at least partly the phosphorylation of both sites was mediated by different enzymes.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Hepatocellular; Cell Line, Tumor; Epidermal Growth Factor; Humans; Liver Neoplasms; Mutation; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; RNA-Binding Proteins; RNA, Messenger; Signal Transduction; Sirolimus; Smad Proteins; TOR Serine-Threonine Kinases; Transcription, Genetic; Transcriptional Activation; Transforming Growth Factor beta1

To explore whether combining inhibitors that target the insulin-like growth factor receptor (IGFR)/PI3K/Akt/mTOR signaling pathway (vertical blockade) can improve treatment efficacy for hepatocellular carcinoma (HCC).. HCC cell lines (including Hep3B, Huh7, and PLC5) and HUVECs (human umbilical venous endothelial cells) were tested. The molecular targeting therapy agents tested included NVP-AEW541 (IGFR kinase inhibitor), MK2206 (Akt inhibitor), BEZ235 (PI3K/mTOR inhibitor), and RAD001 (mTOR inhibitor). Potential synergistic antitumor effects were tested by median dose-effect analysis in vitro and by xenograft HCC models. Apoptosis was analyzed by flow cytometry (sub-G1 fraction analysis) and Western blotting. The activities of pertinent signaling pathways and expression of apoptosis-related proteins were measured by Western blotting.. Vertical blockade induced a more sustained inhibition of PI3K/Akt/mTOR signaling activities in all the HCC cells and HUVEC tested. Synergistic apoptosis-inducing effects, however, varied among different cell lines and drug combinations and were most prominent when NVP-AEW541 was combined with MK2206. Using an apoptosis array, we identified survivin as a potential downstream mediator. Over-expression of survivin in HCC cells abolished the anti-tumor synergy between NVP-AEW541 and MK2206, whereas knockdown of survivin improved the anti-tumor effects of all drug combinations tested. In vivo by xenograft studies confirmed the anti-tumor synergy between NVP-AEW541 and MK2206 and exhibited acceptable toxicity profiles.. Vertical blockade of the IGFR/PI3K/Akt/mTOR pathway has promising anti-tumor activity for HCC. Survivin expression may serve as a biomarker to predict treatment efficacy.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biomarkers, Tumor; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Drug Synergism; Enzyme Inhibitors; Everolimus; Flow Cytometry; Heterocyclic Compounds, 3-Ring; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Liver Neoplasms; Male; Mice, Inbred BALB C; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyrimidines; Pyrroles; Quinolines; Receptors, Somatomedin; Signal Transduction; Sirolimus; Survivin; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Topics: Blindness, Cortical; Blood Group Incompatibility; Calcineurin Inhibitors; Carcinoma, Hepatocellular; Confusion; Drug Substitution; Drug Therapy, Combination; Emergencies; Everolimus; Graft Rejection; Hepatitis B, Chronic; Humans; Immunosuppressive Agents; Liver Cirrhosis; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Photopheresis; Plasmapheresis; Sirolimus; Syndrome; Tacrolimus

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Drug Interactions; Everolimus; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Molecular Targeted Therapy; Neoplasm Recurrence, Local; Niacinamide; Phenylurea Compounds; Protein Kinase Inhibitors; Sirolimus; Sorafenib; TOR Serine-Threonine Kinases; Treatment Outcome

The synergistic effect of combined drug therapy provides an enhanced treatment for advanced liver cancer. We aimed to investigate the underlying mechanism of cetuximab sensitization by rapamycin in hepatoma cells. Four hepatoma cell lines, HepG2, HuH7, SNU-387, and SNU-449, were treated with cetuximab or cetuximab plus rapamycin and growth inhibition was evaluated by measuring relative cell viability and cell proliferation. The cell phenotype was determined for each hepatoma cell line by western blot analysis of E-cadherin and vimentin expression and mTOR activation status. To identify the role of mTOR signaling in cetuximab sensitization, we used deferoxamine-mediated hypoxia to induce epithelial-mesenchymal transition (EMT) in HuH7 and HepG2 cells and measured mTOR activity after rapamycin treatment. Rapamycin significantly increased cetuximab cytotoxicity in hepatoma cell lines with differential sensitivities. Phenotypic differences among hepatoma cell lines, specifically epithelial (HuH7 and HepG2) and mesenchymal (SNU-387 and SNU-449), correlated with the efficacy of rapamycin cotreatment, although rapamycin treatment did not affect cell phenotype. We further showed that rapamycin inhibits mTOR in mesenchymal SNU-387 and SNU-449 cells. In addition, the induction of EMT in HuH7 and HepG2 cells significantly decreased cetuximab cytotoxicity; however, rapamycin treatment significantly restored cetuximab sensitivity and decreased mTOR signaling in these cells. In conclusion, we identified significant differences in rapamycin-induced cetuximab sensitization between epithelial and mesenchymal hepatoma cells. We therefore report that rapamycin cotreatment enhances cetuximab cytotoxicity by inhibiting mTOR signaling in mesenchymal cells.

Topics: Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Cadherins; Carcinoma, Hepatocellular; Cell Hypoxia; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cetuximab; Drug Resistance, Neoplasm; Drug Synergism; Epithelial-Mesenchymal Transition; Humans; Liver Neoplasms; Phenotype; Sirolimus; TOR Serine-Threonine Kinases; Vimentin

Obesity can result in insulin resistance, hepatosteatosis, and nonalcoholic steatohepatitis (NASH) and increases liver cancer risk. Obesity-induced insulin resistance depends, in part, on chronic activation of mammalian target of rapamycin complex 1 (mTORC1), which also occurs in human and mouse hepatocellular carcinoma (HCC), a frequently fatal liver cancer. Correspondingly, mTORC1 inhibitors have been considered as potential NASH and HCC treatments. Using a mouse model in which high-fat diet enhances HCC induction by the hepatic carcinogen DEN, we examined whether mTORC1 inhibition attenuates liver inflammation and tumorigenesis. Notably, rapamycin treatment or hepatocyte-specific ablation of the specific mTORC1 subunit Raptor resulted in elevated interleukin-6 (IL-6) production, activation of signal transducer and activator of transcription 3 (STAT3), and enhanced HCC development, despite a transient reduction in hepatosteatosis. These results suggest that long-term rapamycin treatment, which also increases IL-6 production in humans, is unsuitable for prevention or treatment of obesity-promoted liver cancer.

Topics: Adaptor Proteins, Signal Transducing; Animals; Carcinoma, Hepatocellular; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Diet, High-Fat; Diethylnitrosamine; DNA Damage; Fatty Liver; Glucose Tolerance Test; Hepatocytes; Humans; Inflammation; Interleukin-6; Liver; Liver Neoplasms; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Obese; Mitosis; Multiprotein Complexes; Reactive Oxygen Species; Regulatory-Associated Protein of mTOR; Sirolimus; STAT3 Transcription Factor; TOR Serine-Threonine Kinases

To evaluate the influence of sirolimus on the long-term survival of patients after orthotopic liver transplantation (OLT) for hepatocellular carcinoma (HCC).. Clinic data of 165 consecutive patients who underwent OLT for HCC from February 2005 to March 2012 was analyzed retrospectively. Among them, 94 patients were treated with a sirolimus-based immunosuppressive protocol after OLT, while the other 71 patients with a FK506-based protocol. Postoperative survival time, survival, disease-free survival (DFS) and tumor recurrence rates between the two groups were compared.. The 2 groups were comparable in all clinicopathologic parameters. The sirolimus-based group had higher patient survival rates than the control group at 1-year (87% vs. 97%, P = 0.03), 2-year (80% vs. 88%), 3-year (76% vs. 85%) and 5-year (63% vs. 75%). The 1-year, 2-year, 3-year and 5-year recurrence rates were 12% vs. 3%, 17% vs. 9%, 21% vs. 9% (P = 0.04) and 31% vs. 16% (P = 0.03). Early and mid-HCC (I - II stage) of 131 cases (control group 61 cases, sirolimus-based group of 70 patients). The 1-year, 2-year, 3-year and 5-year survival rates were 90% vs. 97% , 80% vs. 90%, 78% vs. 86% and 65% vs. 82% (P = 0.04) and recurrence rates were 10% vs. 3%, 16% vs. 8%, 18% vs. 8% and 29% vs. 11% (P = 0.01).. The sirolimus-based immunosuppressive protocol reduce long-term postoperative recurrence rate and improve the survival rate of patients after OLT for HCC significantly (especially early-mid HCC).

Topics: Adult; Carcinoma, Hepatocellular; Female; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Neoplasm Recurrence, Local; Retrospective Studies; Sirolimus; Survival Rate; Tacrolimus

Hepatocellular carcinoma is a difficult-to-treat cancer and, after numerous phase III trials assessing kinase inhibitors have failed to meet their end points, sorafenib is the only accepted treatment for advanced stages of the disease. Now, the trial EVOLVE-1 has shown a lack of benefit for everolimus in the second-line treatment setting.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Clinical Trials, Phase III as Topic; Everolimus; Humans; Liver Neoplasms; Patient Selection; Randomized Controlled Trials as Topic; Sirolimus; TOR Serine-Threonine Kinases; Treatment Failure

Rapamycin is clinically used as an immunosuppressant. Increasing evidence suggests that rapamycin has an important inhibitory role in the development and progression of different types of cancer and that it is a promising candidate for cancer chemotherapy. Berberine is an isoquinoline alkaloid isolated from medicinal plant species, which has been used in traditional Chinese medicine with no significant side effects. Recent research has demonstrated that berberine has anticancer activity against various types of cancer, mediated through the suppression of mammalian target of rapamycin (mTOR). The present study aimed to investigate the in vitro synergistic anticancer effect of combined treatment of rapamycin at various concentrations (0, 10, 50, 100 and 200 nM) and berberine (62.5 µM) in SMMC7721 and HepG2 hepatocellular carcinoma (HCC) cell lines, and the potential underlying molecular mechanism. The combined use of rapamycin and berberine was found to have a synergistic cytotoxic effect, with berberine observed to maintain the cyotoxic effect of rapamycin on HCC cells at a lower rapamycin concentration. Moreover, the cells treated with the combination of the two agents exhibited significantly decreased protein levels of phosphorylated (p)‑p70S6 kinase 1 (Thr389), the downstream effector of mTOR, compared with the cells treated with rapamycin or berberine alone. Furthermore, overexpression of cluster of differentiation (CD) 147, a transmembrance glycoprotein associated with the anticancer effects of berberine, was found to upregulate p‑mTOR expression and inhibit cell death in SMMC7721 cells co‑treated with rapamycin and berberine. In conclusion, the findings of the present study suggest that the combined use of rapamycin and berberine may improve HCC therapy through synergistically inhibiting the mTOR signaling pathway, which is at least in part, mediated through CD147.

Topics: Berberine; Carcinoma, Hepatocellular; Cell Death; Cell Line, Tumor; Drug Synergism; Hep G2 Cells; Humans; Liver Neoplasms; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Liver transplantation offers the most effective treatment in patients with hepatocellular carcinoma (HCC). However, transplant patients outside the Milan criteria have a high risk of tumor recurrence, which has been linked to standard immunosuppression regimens. Everolimus is a mammalian target of rapamycin inhibitor that has been used for immunosuppression, but its effect on recurrence and survival in HCC patients with a high risk of tumor recurrence has not been examined. We compared long-term survival and cumulative recurrence in high-risk patients receiving everolimus-based immunosuppression after liver transplantation for HCC with an historic control group.. The everolimus group comprised 21 patients receiving a liver transplant at our center from February 2005 to December 2010. The control group comprised 31 patients receiving a liver transplant from May 1994 to January 2005. All patients received cyclosporine or tacrolimus as initial post-transplant immunosuppression. Patients in the everolimus group switched to everolimus 2 weeks later.. There were no differences between the two groups in number of rejection episodes or of infectious or surgical complications. Five-year survival was 60.2% in the everolimus group and 32.3% in the control group (P = .05). Five-year cumulative recurrence rate was 61.3% in the control group and 41.3% in the everolimus group. Treatment with everolimus was identified as an independent predictor of longer survival (hazard ratio = 0.34; P = .02).. Patients receiving liver transplantation for HCC with a high risk of tumor recurrence may well benefit from everolimus-based immunosuppression, with no added risks of rejection or other post-transplant complications.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Everolimus; Female; Humans; Immunosuppression Therapy; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Neoplasm Recurrence, Local; Sirolimus; Treatment Outcome

To evaluate first-generation rapamycin analogs (everolimus, temsirolimus, and rapamycin) and second-generation drugs inhibiting mTOR kinase (AZD-8055), PI3K (BKM-120) or both (BEZ-235 and GDC-0980) in hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC) cells characterized for acquired resistance to sorafenib or sunitinib.. Anti-proliferative (MTT assay) and cell signaling (Western blot) effects of rapamycin analogs (1-20 μM) and second-generation drugs (0.03-20.0 μM) were assessed in human HCC SK-HEP1, RCC 786-0, and sorafenib- (SK-Sora) or sunitinib-resistant (786-Suni) cells.. In SK-HEP1 cells displaying high PTEN and Bcl2 expression, rapamycin analogs had poor anti-proliferative effects. However, SK-Sora cells were more sensitive to rapamycin analogs (≥1 μM) than SK-HEP1 cells. In 786-0 cells, lacking PTEN and Bcl2 expression, ≥1 μM rapamycin analogs blocked mTORC1 signaling, transiently activated Akt, and inhibited cell proliferation. Protracted sunitinib exposure in 786-Suni cells yielded an increase in p27 expression and a decreased sensitivity to rapamycin analogs, although mTORC1 function could be inhibited with rapamycin analogs. Second-generation drugs induced more potent growth inhibition than rapamycin analogs at concentrations >0.03 μM in parental cells, SK-Sora, and 786-Suni cells. Growth inhibitory concentrations of these new drugs also blocked mTORC1 downstream targets.. Rapamycin analogs inhibited mTORC1 downstream targets and yielded anti-proliferative effects in HCC and RCC cells. Second-generation drugs also appeared to be potent inhibitors of mTORC1 signaling; however, they appeared to be far more potent in inhibiting cellular proliferation in parental HCC and RCC cells and in cells developing resistance to sorafenib or sunitinib.

Topics: Aminopyridines; Antineoplastic Agents; Bridged Bicyclo Compounds, Heterocyclic; Carcinoma, Hepatocellular; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Everolimus; Humans; Imidazoles; Indoles; Kidney Neoplasms; Liver Neoplasms; Mechanistic Target of Rapamycin Complex 1; Morpholines; Multiprotein Complexes; Niacinamide; Phenylurea Compounds; Phosphoinositide-3 Kinase Inhibitors; Pyrimidines; Pyrroles; Quinolines; Signal Transduction; Sirolimus; Sorafenib; Sunitinib; TOR Serine-Threonine Kinases

Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide with only few therapeutic options for patients with advanced disease. There is growing evidence indicating that activation of the PI3K/Akt/mTOR pathway plays an important role in HCC and therefore represents a promising target for novel therapeutic approaches. The aim of this study was to evaluate and compare the antitumour activity of the mTOR inhibitor RAD001, the dual mTOR and PI3-kinase inhibitor BEZ235 and the PI3-kinase inhibitor BKM120 in vitro and in vivo.. The antitumour effects of RAD001, BEZ235 and BKM120 were analysed in seven hepatoma cell lines as mono and combination therapy with Doxorubicin, Cisplatin, Irinotecan or 5-Flourouracil in vitro and in xenografts. Cell-cycle progression, apoptosis, and autophagy were analysed. Furthermore, effects on mitochondrial respiration and glycolysis were assessed.. Treatment with RAD001, BEZ235 and BKM120 markedly reduced tumour cell viability. Combination of PI3K inhibitors with chemotherapy was most effective. RAD001, BEZ235 and BKM120 reduced tumour growth mainly by inhibiting cell-cycle progression rather than by inducing apoptosis. Interestingly, the antitumour effects were strongly associated with a reduction of mitochondrial respiration. BKM120, which exhibited the strongest antiproliferative effect, most strongly impaired oxidative phosphorylation compared with the other drugs.. In this study, BKM120 showed the strongest antitumour activity. Our findings suggest impairment of mitochondrial function as a relevant mechanism of BKM120. Moreover, combination of PI3K and mTOR inhibitors with cytotoxic agents could be promising option for non-cirrhotic HCC patients.

Topics: Aminopyridines; Analysis of Variance; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autophagy; Camptothecin; Carcinoma, Hepatocellular; Cell Cycle; Cell Line; Cell Respiration; Cisplatin; Doxorubicin; Everolimus; Humans; Imidazoles; Immunoblotting; Immunohistochemistry; In Situ Nick-End Labeling; Irinotecan; Liver Neoplasms; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Quinolines; Sirolimus; TOR Serine-Threonine Kinases

RAD001 targets at the mammalian target of rapamycin (mTOR), while TKI-258 is a potent tyrosine kinase inhibitor targeting at fibroblast growth factor receptor, vascular endothelial growth factor receptor, platelet-derived growth factor receptor and c-kit. We aim to study the activity of combined RAD001 and TKI-258 in cell lines and xenograft model of hepatocellular carcinoma (HCC), with reference to the parallel and upstream pathways of Akt-mTOR axis.. A panel of 4 human HCC cell lines HepG2, Hep3B, PLC/PRF/5 and Huh7 and the Hep3B-derived xenograft were treated with TKI-258 or/and RAD001, respectively. Related mechanistic studies (including apoptosis and angiogenesis) were conducted.. There was an enhanced increase in suppression of cell proliferation with combined TKI-258 and RAD001 compared with either drug alone. The combination could significantly suppress the phosphorylation of mTOR, MEK1/2 and p38 MAPK. Although the addition of the TKI258 only slightly suppressed the phosphorylation of AKT induced by RAD001, the pi-mTOR and its downstream signaling pathways including pi-p70S6K, pi-S6 and pi-4EBP1 were lowered in the combination. In Hep3B-derived xenograft, TKI-258 and RAD001 had shown an enhanced inhibition of tumor growth without impact on the weight of animals. There was a reduction in microvessel density in the xenograft with the combination, which indicated an enhanced inhibition on angiogenesis. Pro-caspases-3 and PARP cleavage were slightly detected at 48 h after treatment, suggesting that the combination mainly increased the cytostatic arrest ability.. The combination of RAD001 and TKI-258 was active in HCC via inhibition of both mTOR-mediated signaling and its parallel pathways.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzimidazoles; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Survival; Everolimus; Humans; Liver Neoplasms; Mice; Mice, Nude; Neovascularization, Pathologic; Proto-Oncogene Proteins c-akt; Quinolones; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide, with limited treatment options. AKT/mTOR and Ras/MAPK pathways are frequently deregulated in human hepatocarcinogenesis. Recently, we generated an animal model characterized by the co-expression of activated forms of AKT and Ras in the mouse liver. We found that concomitant activation of AKT/mTOR and Ras/MAPK cascades leads to rapid liver tumor development in AKT/Ras mice, mainly through mTORC1 induction. To further define the role of mTORC1 cascade in AKT/Ras induced HCC development, the mTORC1 inhibitor Rapamycin was administered to AKT/Ras mice at the time when small tumors started to emerge in the liver. Of note, Rapamycin treatment significantly delayed hepatocarcinogenesis in AKT/Ras mice. However, some microscopic lesions persisted in the livers of AKT/Ras mice despite the treatment and rapidly gave rise to HCC following Rapamycin withdrawal. Mechanistically, Rapamycin inhibited mTORC1 and mTORC2 pathways, lipogenesis and glycolysis, resulting in inhibition of proliferation in the treated livers. However, activated ERK and its downstream effectors, Mnk1 and eIF4E, were strongly upregulated in the residual lesions. Concomitant suppression of AKT/mTOR and Ras/MAPK pathways was highly detrimental for the growth of AKT/Ras cells in vitro. The study indicates the existence of a complex interplay between AKT/mTOR and Ras/MAPK pathways during hepatocarcinogenesis, with important implications for the understanding of HCC pathogenesis as well as for its prevention and treatment.

Topics: Animals; Antibiotics, Antineoplastic; Carcinoma, Hepatocellular; Drug Screening Assays, Antitumor; Humans; Liver; Liver Neoplasms, Experimental; MAP Kinase Signaling System; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Transgenic; Molecular Targeted Therapy; Multiprotein Complexes; Proto-Oncogene Proteins c-akt; ras Proteins; Receptor Cross-Talk; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

Insulin is known to regulate multiple cellular functions and is used for the treatment of diabetes. MicroRNAs have been demonstrated to be involved in many human diseases, including Type 2 diabetes. In this study, we showed that insulin decreased miR-99a expression levels, but induced glucose consumption and lactate production, and increased the expression of mTOR, HIF-1α and PKM2 in HepG2 and HL7702 cells. Forced expression of miR-99a or rapamycin treatment blocked insulin-induced PKM2 and HIF-1α expression, and glucose consumption and lactate production. Meanwhile, knockdown of HIF-1α inhibited PKM2 expression and insulin-induced glucose consumption. Taken together, these findings will reveal the role and mechanism of insulin in regulating glycolytic activities via miR-99a/mTOR.

Topics: Antibiotics, Antineoplastic; Blotting, Western; Carcinoma, Hepatocellular; Carrier Proteins; Cells, Cultured; Gene Expression Regulation, Neoplastic; Glucose; Humans; Hypoglycemic Agents; Hypoxia-Inducible Factor 1, alpha Subunit; Insulin; Lactates; Liver; Liver Neoplasms; Luciferases; Membrane Proteins; MicroRNAs; Phosphorylation; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Sirolimus; Thyroid Hormone-Binding Proteins; Thyroid Hormones; TOR Serine-Threonine Kinases

Everolimus is a potent immunosuppressant with several advantages over calcineurin inhibitors, such as good tolerance, preventive effects on cardiovascular morbidity, and mortality and cancer prevention as it inhibits cell proliferation.. Between April 1986 and December 2010, we performed 1500 liver transplants (OLT) in 1341 recipients, including 57 patients who were prescribed everolimus 24 (42.1%) as monotherapy and 33 (57.9%) as treatments combined with other immunosuppressants. We performed a retrospective analysis of our experience with conversion to everolimus in OLT recipients.. The 43 men and 14 women had a mean overall age at transplantation of 59.1 ± 10 years. The most frequent indication for OLT was hepatocellular carcinoma (HCC; 53.8%). Everolimus was introduced to prevent HCC recurrence (53%), development of de novo tumors (33%), address renal dysfunction (7%), or overcome side effects of other immunosuppressants (7%). We observed a significant improvement in renal function using the estimated glomerular filtration rate (Crockcroft-Gault formula) from 68.5 mL/min before to 74.5 mL/min after switching to everolimus. The 72% of recipients who developed ≥1 adverse event, most frequently showed hyperlipidemia (34.4%).. Both monotherapy and combined everolimus regimens were well-tolerated immunosuppressive regimens in liver transplant recipients with recurrent or de novo malignancies. Everolimus improved renal function. The most common side effects were hyperlipidemia, edema, and mouth ulcerations, which were well controlled with anti-lipidemic agents or decreased everolimus dosages.

Topics: Aged; Carcinoma, Hepatocellular; Drug Therapy, Combination; Everolimus; Female; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Sirolimus

Despite careful patient selection, hepatocellular carcinoma (HCC) recurs in 10-20% of cases after liver transplantation, and the use of potent adjuvant anticancer drugs would be welcome. The aim of this study was to evaluate the efficiency of a combined therapy of rapamycin (sirolimus) and anti-death receptor (DR)5 monoclonal antibody (mAb) on HCC.. We first assessed the side effects of anti-DR5 mAb administration in vivo by giving various doses of anti-DR5 mAb. Cell proliferation assays were then performed using mouse Hepa1-6 cells or human Huh7 cells to quantify the relative cell viability under various concentrations of sirolimus, anti-DR5 mAb or a combination. Next, one million Hepa1-6 cells were transplanted into C.B17-SCID-beige mice subcutaneously, and four groups were created: (1) untreated, (2) anti-DR5 mAb alone, (3) sirolimus alone and (4) anti-DR5 mAb + sirolimus.. Anti-DR5 mAb (200 and 300 μg/day) induced liver dysfunction with partial necrosis of the liver, but 100 μg/day was well tolerated with transaminitis, but normal bilirubin and only minor histological liver damage. In vitro, anti-DR5 mAb lysed Hepa1-6 and Huh7 cells in a dose-dependent manner, and combinations of sirolimus and anti-DR5 mAb demonstrated an additive effect. In vivo studies demonstrated that tumour sizes were significantly smaller in the combined therapy group than in the monotherapy groups.. Combining sirolimus and low-dose anti-DR5 mAb has a significant effect against HCC. This strategy represents a potential novel approach for the management of HCC.

Topics: Analysis of Variance; Animals; Antibodies, Monoclonal; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Hepatocellular; Cell Line; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Drug Synergism; Humans; Liver Neoplasms; Mice; Mice, Inbred C57BL; Receptors, TNF-Related Apoptosis-Inducing Ligand; Sirolimus; Tetrazolium Salts; Thiazoles

Tumor-initiating stem-like cells (TICs) are resistant to chemotherapy and associated with hepatocellular carcinoma (HCC) caused by HCV and/or alcohol-related chronic liver injury. Using HCV Tg mouse models and patients with HCC, we isolated CD133(+) TICs and identified the pluripotency marker NANOG as a direct target of TLR4, which drives the tumor-initiating activity of TICs. These TLR4/NANOG-dependent TICs were defective in the TGF-β tumor suppressor pathway. Functional oncogene screening of a TIC cDNA library identified Yap1 and Igf2bp3 as NANOG-dependent genes that inactivate TGF-β signaling. Mechanistically, we determined that YAP1 mediates cytoplasmic retention of phosphorylated SMAD3 and suppresses SMAD3 phosphorylation/activation by the IGF2BP3/AKT/mTOR pathway. Silencing of both YAP1 and IGF2BP3 restored TGF-β signaling, inhibited pluripotency genes and tumorigenesis, and abrogated chemoresistance of TICs. Mice with defective TGF-β signaling (Spnb2(+/-) mice) exhibited enhanced liver TLR4 expression and developed HCC in a TLR4-dependent manner. Taken together, these results suggest that the activated TLR4/NANOG oncogenic pathway is linked to suppression of cytostatic TGF-β signaling and could potentially serve as a therapeutic target for HCV-related HCC.

Topics: AC133 Antigen; Adaptor Proteins, Signal Transducing; Animals; Antigens, CD; Antineoplastic Agents; Base Sequence; Carcinoma, Hepatocellular; Cell Separation; Drug Resistance, Neoplasm; Flow Cytometry; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glycoproteins; Homeodomain Proteins; Humans; Inhibitory Concentration 50; Liver Neoplasms; Mice; Mice, Inbred NOD; Mice, SCID; Mice, Transgenic; Nanog Homeobox Protein; Neoplastic Stem Cells; Niacinamide; Oncogenes; Peptides; Phenylurea Compounds; Phosphoproteins; RNA-Binding Proteins; RNA, Small Interfering; Signal Transduction; Sirolimus; Smad Proteins; Sorafenib; Spheroids, Cellular; Toll-Like Receptor 4; Transcription Factors; Transcriptional Activation; Transforming Growth Factor beta; Tumor Burden; Tumor Cells, Cultured; Xenograft Model Antitumor Assays; YAP-Signaling Proteins

Topics: Aminopyridines; Carcinoma, Hepatocellular; Humans; Imidazoles; Liver Neoplasms; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Quinolines; Sirolimus; TOR Serine-Threonine Kinases

Recurrent hepatocellular carcinoma (HCC) after liver transplantation (LT) is a rare but challenging condition. In most cases, the recurrent tumor is presented with extrahepatic spread. Therefore, systemic treatment with sorafenib has to be assessed. Because of a plethora of possible drug interactions, e.g., with immunosuppressant or anti-infective therapy, safety and feasibility of sorafenib treatment requires special attention.. We retrospectively analyzed 18 patients who suffered from recurrent advanced HCC after LT between January 2002 and December 2010 at the University Hospital Heidelberg regarding safety of sorafenib treatment and survival.. Results showed that 8 patients were eligible for treatment with sorafenib showing a median time to progression (TTP) of 4.5 months and an overall survival of 9 months. Most common side effects were grades I and II diarrhea and hand-foot syndrome (HFS) which could be managed by sorafenib dose reduction. No grade III or IV adverse events (AEs) were noticed. No patient had to discontinue treatment due to AEs. The ten patients not amenable for sorafenib treatment, due to initial poor performance status or its deterioration after first line treatment, were treated with surgical resection (n = 3), locoregional therapies (n = 1), or palliative radiation therapy (n = 1). They showed a median overall survival of 2.3 months.. Sorafenib may represent a therapeutic option for recurrent HCC after LT with manageable side effects. The clinical benefit of sorafenib in this setting is promising but needs to be confirmed in a prospective randomized trial.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Female; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Neoplasm Recurrence, Local; Niacinamide; Phenylurea Compounds; Postoperative Complications; Retrospective Studies; Sirolimus; Sorafenib; Survival Rate; Treatment Outcome

Liver cancer is associated with chronic inflammation, which is linked to immune dysregulation, disordered metabolism, and aberrant cell proliferation. Nucleoside triphosphate diphosphohydrolase-1; (CD39/ENTPD1) is an ectonucleotidase that regulates extracellular nucleotide/nucleoside concentrations by scavenging nucleotides to ultimately generate adenosine. These properties inhibit antitumor immune responses and promote angiogenesis, being permissive for the growth of transplanted tumors. Here we show that Cd39 deletion promotes development of both induced and spontaneous autochthonous liver cancer in mice. Loss of Cd39 results in higher concentrations of extracellular nucleotides, which stimulate proliferation of hepatocytes, abrogate autophagy, and disrupt glycolytic metabolism. Constitutive activation of Ras-mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR)-S6K1 pathways occurs in both quiescent Cd39 null hepatocytes in vitro and liver tissues in vivo. Exogenous adenosine 5'-triphosphate (ATP) boosts these signaling pathways, whereas rapamycin inhibits such aberrant responses in hepatocytes.. Deletion of Cd39 and resulting changes in disordered purinergic signaling perturb hepatocellular metabolic/proliferative responses, paradoxically resulting in malignant transformation. These findings might impact adjunctive therapies for cancer. Our studies indicate that the biology of autochthonous and transplanted tumors is quite distinct.

Topics: Animals; Antigens, CD; Apyrase; Autophagy; Carcinoma, Hepatocellular; Cell Proliferation; Gene Deletion; Gene Expression Regulation, Neoplastic; Glycolysis; Hepatocytes; Liver; Liver Neoplasms, Experimental; Male; Mice; Mice, Inbred C57BL; Phosphatidylinositol 3-Kinases; Receptors, Purinergic; Ribosomal Protein S6 Kinases, 90-kDa; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Topics: Adult; Antineoplastic Agents; Benzenesulfonates; Carcinoma, Hepatocellular; Combined Modality Therapy; Hepacivirus; Hepatitis C; Humans; Immunosuppressive Agents; Liver Cirrhosis; Liver Transplantation; Male; Neoadjuvant Therapy; Niacinamide; Phenylurea Compounds; Pyridines; Sirolimus; Sorafenib; Treatment Outcome

Glycine N-methyltransferase (GNMT) is a tumor suppressor for hepatocellular carcinoma (HCC). High rates of Gnmt knockout mice developed HCC. Epigenetic alteration and dysregulation of several pathways including wingless-type MMTV integration site (Wnt), mitogen-activated protein kinase (MAPK) and Janus kinase and signal transducer and activator of transcription (JAK-STAT) are associated with HCC development in Gnmt knockout mice. We hypothesized that GNMT may regulate signal transduction through interacting with other proteins directly. In this report, we identified a mammalian target of rapamycin (mTOR) inhibitor (DEP domain containing MTOR-interacting protein [DEPDC6/DEPTOR]) as a GNMT-binding protein by using yeast two-hybrid screening. Fluorescence resonance energy transfer assay demonstrated that the C-terminal half of GNMT interact with the PSD-95/Dlg1/ZO-1 (PDZ) domain of DEPDC6/DEPTOR. Immunohistochemical staining showed that 27.5% (14/51) of HCC patients had higher expression levels of DEPDC6/DEPTOR in the tumorous tissues than in tumor-adjacent tissues, especially among HCC patients with hepatitis B viral infection (odds ratio 10.3, 95% confidence interval [CI] 1.05-11.3) or patients with poor prognosis (death hazard ratio 4.51, 95% CI 1.60-12.7). In terms of molecular mechanism, knockdown of DEPDC6/DEPTOR expression in HuH-7 cells caused S6K and 4E-BP activation, but suppressed Akt. Overexpression of DEPDC6/DEPTOR activated Akt and increased survival of HCC cells. Overexpression of GNMT caused activation of mTOR/raptor downstream signaling and delayed G2/M cell cycle progression, which altogether resulted in cellular senescence. Furthermore, GNMT reduced proliferation of HuH-7 cells and sensitized them to rapamycin treatment both in vitro and in vivo. In conclusion, GNMT regulates HCC growth in part through interacting with DEPDC6/DEPTOR and modulating mTOR/raptor signaling pathway. Both GNMT and DEPDC6/DEPTOR are potential targets for developing therapeutics for HCC.

Topics: Adult; Aged; Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Everolimus; Female; Glycine N-Methyltransferase; HEK293 Cells; Hepatitis B; Hepatitis C; Humans; Immunosuppressive Agents; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Male; Mice; Mice, SCID; Middle Aged; Sirolimus; TOR Serine-Threonine Kinases; Two-Hybrid System Techniques; Xenograft Model Antitumor Assays

Initiation, a major rate-limiting step of host protein translation, is a critical target in many viral infections. Chronic hepatitis C virus (HCV) infection results in hepatocellular carcinoma. Translation initiation, up-regulated in many cancers, plays a critical role in tumorigenesis. mTOR is a major regulator of host protein translation. Even though activation of PI3K-AKT-mTOR by HCV non-structural protein 5A (NS5A) is known, not much is understood about the regulation of host translation initiation by this virus. Here for the first time we show that HCV up-regulates host cap-dependent translation machinery in Huh7.5 cells through simultaneous activation of mTORC1 and eukaryotic translation initiation factor 4E (eIF4E) by NS5A. NS5A, interestingly, overexpressed and subsequently hyperphosphorylated 4EBP1. NS5A phosphorylated eIF4E through the p38 MAPK-MNK pathway. Both HCV infection and NS5A expression augmented eIF4F complex assembly, an indicator of cap-dependent translation efficiency. Global translation, however, was not altered by HCV NS5A. 4EBP1 phosphorylation, but not that of S6K1, was uniquely resistant to rapamycin in NS5A-Huh7.5 cells, indicative of an alternate phosphorylation mechanism of 4EBP1. Resistance of Ser-473, but not Thr-308, phosphorylation of AKT to PI3K inhibitors suggested an activation of mTORC2 by NS5A. NS5A associated with eIF4F complex and polysomes, suggesting its active involvement in host translation. This is the first report that implicates an HCV protein in the up-regulation of host translation initiation apparatus through concomitant regulation of multiple pathways. Because both mTORC1 activation and eIF4E phosphorylation are involved in tumorigenesis, we propose that their simultaneous activation by NS5A might contribute significantly to the development of hepatocellular carcinoma.

Topics: Adaptor Proteins, Signal Transducing; Anti-Bacterial Agents; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Line, Tumor; Cell Transformation, Viral; Drug Resistance; Eukaryotic Initiation Factor-4E; Eukaryotic Initiation Factor-4F; Hepacivirus; Hepatitis C; Humans; Liver Neoplasms; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Peptide Chain Initiation, Translational; Phosphoproteins; Phosphorylation; Polyribosomes; Proteins; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Up-Regulation; Viral Nonstructural Proteins

Deregulated Ras/Raf/MAPK and PI3K/AKT/mTOR signaling pathways are found in hepatocellular carcinoma (HCC). This study aimed to test the inhibitory effects of PKI-587 and sorafenib as single agents or in combination on HCC (Huh7 cell line) proliferation.. (3)H-thymidine incorporation and MTT assay were used to assess Huh7 cell proliferation. Phosphorylation of the key enzymes in the Ras/Raf/MAPK and PI3K/AKT/mTOR pathways was detected by Western blot.. We found that PKI-587 is a more potent PI3K/mTOR inhibitor than PI-103. Combination of PKI-587 and sorafenib was a more effective inhibitor of Huh7 proliferation than the combination of PI-103 and sorafenib. Combination of PKI-587 and sorafenib synergistically inhibited epidermal growth factor (EGF)-stimulated Huh7 proliferation compared with monodrug therapy. EGF increased phosphorylation of Ras/Raf downstream signaling proteins MEK and ERK; EGF-stimulated activation was inhibited by sorafenib. However, sorafenib, as a single agent, increased AKT (Ser473) phosphorylation. EGF-stimulated AKT (ser473) activation was inhibited by PKI-587. PKI-587 is a potent inhibitor of AKT (Ser473), mTOR (Ser2448), and S6K (Thr389) phosphorylation; in contrast, rapamycin stimulated mTOR complex 2 substrate AKT(Ser473) phosphorylation although it inhibited mTOR complex 1 substrate S6K phosphorylation. PKI-587, as a single agent, stimulated MEK and ERK phosphorylation. However, when PKI-587 and sorafenib were used in combination, they inhibited all the tested kinases in the Ras/Raf /MAPK and PI3K/AKT/mTOR pathways.. The combination of PKI-587 and sorafenib has the advantage over monodrug therapy on inhibition of HCC cell proliferation by blocking both PI3K/AKT/mTOR and Ras/Raf/MAPK signaling pathways.

Topics: Antibiotics, Antineoplastic; Antineoplastic Agents; Benzenesulfonates; Carcinoma, Hepatocellular; Cell Division; Cell Line, Tumor; Drug Synergism; Feedback, Physiological; Furans; Humans; Liver Neoplasms; MAP Kinase Signaling System; Mechanistic Target of Rapamycin Complex 1; Morpholines; Multiprotein Complexes; Niacinamide; Phenylurea Compounds; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proteins; Proto-Oncogene Proteins c-akt; Pyridines; Pyrimidines; Sirolimus; Sorafenib; TOR Serine-Threonine Kinases; Transcription Factors; Triazines

Recurrence of hepatocellular carcinoma after orthotopic liver transplantation not amenable to surgical approaches is associated with poor outcome.. Retrospective evaluation of the safety and efficacy of sorafenib in patients with post-transplant hepatocellular carcinoma recurrence.. Patients with post-transplant hepatocellular carcinoma recurrence were treated with sorafenib. Adverse events were assessed using National Cancer Institute Common Toxicity Criteria of AEs version 3.0, tumour response was evaluated according to Response Evaluation Criteria in Solid Tumours.. First-line therapy after recurrence was surgery (n=6), radiation therapy (n=1), chemotherapy (n=1), and sorafenib (n=3). Finally, 11 patients were treated with sorafenib. Nine patients (82%) received an additionally targeted therapy with sirolimus as part of their immunosuppressive regimen. Most common grade 3 adverse events included diarrhoea (46%), hand-foot skin reaction (27%), nausea, fatigue, and leucopoenia (all 18%). Sorafenib had to be discontinued in two patients due to adverse events and six additional patients required a dose adjustment. No deterioration of liver graft function occurred. Median time to progression was 4.1 months; however, patients were treated with ongoing sorafenib in case of clinical benefit (median 8.9 months). Median overall survival after initiation of sorafenib treatment was 20.1 months.. Sorafenib in combination with immunosuppression including sirolimus may be administered to patients with post-transplant hepatocellular carcinoma recurrence with acceptable toxicity and without deterioration of liver graft function.

Topics: Adult; Aged; Antineoplastic Agents; Benzenesulfonates; Carcinoma, Hepatocellular; Female; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Neoplasm Recurrence, Local; Niacinamide; Phenylurea Compounds; Pyridines; Retrospective Studies; Sirolimus; Sorafenib; Tacrolimus

Mounting epidemiological evidence supports a role for insulin-signaling deregulation and diabetes mellitus in human hepatocarcinogenesis. However, the underlying molecular mechanisms remain unknown. To study the oncogenic effect of chronically elevated insulin on hepatocytes in the presence of mild hyperglycemia, we developed a model of pancreatic islet transplantation into the liver. In this model, islets of a donor rat are transplanted into the liver of a recipient diabetic rat, with resulting local hyperinsulinism that leads to the development of preneoplastic lesions and hepatocellular carcinoma (HCC). Here, we investigated the metabolic and growth properties of the v-akt murine thymoma viral oncogene homolog/mammalian target of rapamycin (AKT/mTOR) pathway, a major downstream effector of insulin signaling, in this model of insulin-induced hepatocarcinogenesis. We found that activation of insulin signaling triggers a strong induction of the AKT/mTOR cascade that is paralleled by increased synthesis of fatty acids, cholesterol, and triglycerides, induction of glycolysis, and decrease of fatty acid oxidation and gluconeogenesis in rat preneoplastic and neoplastic liver lesions, when compared with the healthy liver. AKT/mTOR metabolic effects on hepatocytes, after insulin stimulation, were found to be mTORC1 dependent and independent in human HCC cell lines. In these cells, suppression of lipogenesis, glycolysis, and the pentose phosphate pathway triggered a strong growth restraint, despite insulin administration. Noticeably, metabolic abnormalities and proliferation driven by insulin were effectively reverted using the dual PI3K/mTOR inhibitor, NVP-BEZ235, both in vitro and in vivo.. The present results indicate that activation of the AKT/mTOR cascade by unconstrained insulin signaling induces a defined module of metabolic alterations in hepatocytes contributing to aberrant cell growth. Thus, inhibition of AKT/mTOR and related metabolic changes might represent a novel preventive and therapeutic approach to effectively inhibit insulin-induced hepatocarcinogenesis.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Diabetes Mellitus, Experimental; Disease Models, Animal; Fatty Acids; Hyperinsulinism; Immunoblotting; Lipogenesis; Liver Neoplasms; Male; Mice; Proto-Oncogene Proteins c-akt; Random Allocation; Rats; Rats, Inbred Lew; Sensitivity and Specificity; Sirolimus; Streptozocin; Thymoma; TOR Serine-Threonine Kinases; Transfection

The mammalian target of rapamycin (mTOR) and the microtubules are prominent druggable targets for hepatocellular carcinoma (HCC). PI3K/Akt/mTOR activation is associated with resistance to microtubule inhibitors. Here, we hypothesized that co-targeting of mTOR (by mTOR inhibitor temsirolimus) and the microtubule (by microtubule-destabilizing agent vinblastine) would be more efficacious than single targeting in HCC models. In vitro studies showed that effective inhibition of mTOR signaling with temsirolimus alone was able to suppress HCC cell growth in a dose-dependent manner. Among five cell lines tested, Huh7 was the most temsirolimus-sensitive (IC(50)=1.27±0.06μM), while Hep3B was the most temsirolimus-resistant (IC(50)=52.95±17.14μM). We found that co-targeting of mTOR (by temsirolimus) and the microtubule (by vinblastine, at low nM) resulted in marked growth inhibition in Huh7 cells and synergistic growth inhibition in Hep3B cells (achieving maximal growth inhibition of 80-90%), demonstrating potent antitumor activity of this novel combination. In vivo studies showed that temsirolimus treatment alone for 1 week was able to inhibit the growth of Huh7 xenografts. Strikingly, the temsirolimus/vinblastine combination induced a significant and sustained antitumor activity (up to 27 days post-treatment), with effective reduction of tumor vessel density in both Huh7 and Hep3B xenograft models. Mechanistic investigation revealed that this marked antitumor effect was accompanied by specific and concerted down-regulation of several key anti-apoptotic/survival proteins (survivin, Bcl-2, and Mcl-1), which was not observed in single agent treatments. Our findings demonstrated that the potent anti-cancer activity of this co-targeting strategy was indeed mediated in parts by inhibition of these key survival/anti-apoptotic proteins.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis Regulatory Proteins; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Humans; Inhibitor of Apoptosis Proteins; Liver Neoplasms; Male; Mice; Mice, Nude; Microtubules; Myeloid Cell Leukemia Sequence 1 Protein; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Sirolimus; Survivin; TOR Serine-Threonine Kinases; Vinblastine; Xenograft Model Antitumor Assays

The PI3K/Akt/mTOR and JAK/STAT3 signaling pathways are important for regulating apoptosis, and are frequently activated in cancers. In this study, we targeted STAT3 and mTOR in human hepatocellular carcinoma Bel-7402 cells and examined the subsequent alterations in cellular apoptosis. The expression of STAT3 was silenced with small interfering RNA (siRNA)-expressing plasmid. The activity of mTOR was inhibited using rapamycin. Following treatment, Annexin V/propidium iodide staining followed by flow cytometry and Hoechst33258 immunofluorescence staining was used to examine cellular apoptosis. JC-1 staining was used to monitor depolarization of mitochondrial membrane (ΔΨm). Furthermore, the expression of activated caspase 3 protein was analyzed by Western blotting. Compared to non-treated or control siRNA-transfected cells, significantly higher levels of apoptosis were detected in siSTAT3-transfected or rapamycin-treated cells (P < 0.05), which was further enhanced in cells targeted for both molecules (P < 0.05). The pro-apoptotic effects were accompanied with concomitant depolarization of mitochondrial membrane and up-regulation of activated caspase 3. Combined treatments using rapamycin and STAT3 gene silencing significantly increases apoptosis in Bel-7402 cells, displaying more dramatic effect than any single treatment. This study provides evidence for targeting multiple molecules in cancer therapy.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Gene Silencing; Humans; Liver Neoplasms; Membrane Potential, Mitochondrial; RNA Interference; Sirolimus; STAT3 Transcription Factor; TOR Serine-Threonine Kinases

Rapamycin is an attractive approach for the treatment and prevention of HCC recurrence after liver transplantation. However, the objective response rates of rapamycin achieved with single-agent therapy were modest, supporting that rapamycin resistance is a frequently observed characteristic of many cancers. Some studies have been devoted to understanding the mechanisms of rapamycin resistance, however, the mechanisms are cell-type-dependent and studies on rapamycin resistance in HCC are extremely limited.. The anti-tumor sensitivity of rapamycin was modest in vitro and in vivo. In both human and rat HCC cells, rapamycin up-regulated the expression and phosphorylation of PDGFRβ in a time and dose-dependent manner as assessed by RT-PCR and western blot analysis. Using siRNA mediated knockdown of PDGFRβ, we confirmed that subsequent activation of AKT and ERK was PDGFRβ-dependent and compromised the anti-tumor activity of rapamycin. Then, blockade of this PDGFRβ-dependent feedback loop by sorafenib enhanced the anti-tumor sensitivity of rapamycin in vitro and in an immunocompetent orthotopic rat model of HCC.. Activation of PI3K/AKT and MAPK pathway through a PDGFRβ-dependent feedback loop compromises the anti-tumor activity of rapamycin in HCC, and blockade of this feedback loop by sorafenib is an attractive approach to improve the anti-tumor effect of rapamycin, particularly in preventing or treating HCC recurrence after liver transplantation.

Topics: Animals; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Drug Resistance, Neoplasm; Enzyme Activation; Feedback, Physiological; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Immunohistochemistry; Liver Neoplasms; Male; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats; Receptor, Platelet-Derived Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Signal Transduction; Sirolimus; Tetrazolium Salts; Thiazoles

The mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is a nutrient-sensitive protein kinase that is aberrantly activated in many human cancers. Whether dysregulation of mTORC1 signaling in normal tissues increases the risk for cancer, however, is unknown. We focused on hepatocellular carcinoma, which has been linked to environmental factors that affect mTORC1 activity, including diet. Ablation of the gene encoding TSC1 (tuberous sclerosis complex 1), which as part of the TSC1-TSC2 complex is an upstream inhibitor of mTORC1, results in constitutively increased mTORC1 signaling, an effect on this pathway similar to that of obesity. We found that mice with liver-specific knockout of Tsc1 developed sporadic hepatocellular carcinoma with heterogeneous histological and biochemical features. The spontaneous development of hepatocellular carcinoma in this mouse model was preceded by a series of pathological changes that accompany the primary etiologies of this cancer in humans, including liver damage, inflammation, necrosis, and regeneration. Chronic mTORC1 signaling led to unresolved endoplasmic reticulum stress and defects in autophagy, factors that contributed to hepatocyte damage and hepatocellular carcinoma development. Therefore, we conclude that increased activation of mTORC1 can promote carcinogenesis and may thus represent a key molecular link between cancer risk and environmental factors, such as diet.

Topics: Animals; Antibiotics, Antineoplastic; Autophagy; Carcinoma, Hepatocellular; Cells, Cultured; Disease Progression; Endoplasmic Reticulum Stress; Female; Hepatocytes; Immunoblotting; Immunohistochemistry; Liver; Liver Neoplasms; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred C57BL; Mice, Knockout; Multiprotein Complexes; Proliferating Cell Nuclear Antigen; Proteins; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins

Oval cell activation occurs under conditions of severe liver injury when normal hepatocyte proliferation is blocked. Recent studies have shown that a subset of hepatocellular carcinomas expresses oval cell markers, suggesting that these cells are targets of hepatocarcinogens. However, the signaling pathways that control oval cell activation and proliferation are not well characterized. Based on the role of the nutrient signaling kinase complex, mTORC1, in liver development, we investigated the role of this pathway in oval cell activation. Oval cell proliferation was induced in male Fisher rats by a modification of the traditional choline deficient plus ethionine model (CDE) or by 2-acetylaminoflourene treatment followed by 2/3 partial hepatectomy with or without initiation by diethylnitrosamine. To assess the role of mTOR in the oval cell response and development of preneoplastic foci, the effect of the mTORC1 inhibitor, rapamycin, was studied in all models. Rapamycin induced a significant suppression of the oval cell response in both models, an effect that coincided with a decrease in oval cell proliferation. Rapamycin administration did not affect the abundance of neutrophils or natural killer cells in CDE-treated liver or the expression of key cytokines. Gene expression studies revealed the fetal hepatocyte marker MKP-4 to be expressed in oval cells. In an experimental model of hepatic carcinogenesis, rapamycin decreased the size of preneoplastic foci and the rate of cell proliferation within the foci. mTORC1 signaling plays a key role in the oval cell response and in the development of preneoplastic foci. This pathway may be a target for the chemoprevention of hepatocellular carcinoma.

Topics: Animals; Antibiotics, Antineoplastic; Carcinoma, Hepatocellular; Cell Proliferation; Cell Shape; Cell Transformation, Neoplastic; Choline Deficiency; Diethylnitrosamine; Dual-Specificity Phosphatases; Ethionine; Fluorenes; Gene Expression Profiling; Hepatectomy; Hepatocytes; Liver Neoplasms, Experimental; Male; Mechanistic Target of Rapamycin Complex 1; Mitogen-Activated Protein Kinase Phosphatases; Multiprotein Complexes; Precancerous Conditions; Proteins; Rats; Sirolimus; TOR Serine-Threonine Kinases

Hepatocellular carcinoma (HCC) affects more than half a million people worldwide and is the third most common cause of cancer deaths. Because mammalian target of rapamycin (mTOR) signaling is up-regulated in 50% of HCCs, we compared the effects of the U.S. Food and Drug Administration-approved mTOR-allosteric inhibitor, RAD001, with a new-generation phosphatidylinositol 3-kinase/mTOR adenosine triphosphate-site competitive inhibitor, BEZ235. Unexpectedly, the two drugs acted synergistically in inhibiting the proliferation of cultured HCC cells. The synergistic effect closely paralleled eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) dephosphorylation, which is implicated in the suppression of tumor cell proliferation. In a mouse model approximating human HCC, the drugs in combination, but not singly, induced a marked regression in tumor burden. However, in the tumor, BEZ235 alone was as effective as the combination in inhibiting 4E-BP1 phosphorylation, which suggests that additional target(s) may also be involved. Microarray analyses revealed a large number of genes that reverted to normal liver tissue expression in mice treated with both drugs, but not either drug alone. These analyses also revealed the down-regulation of autophagy genes in tumors compared to normal liver. Moreover, in HCC patients, altered expression of autophagy genes was associated with poor prognosis. Consistent with these findings, the drug combination had a profound effect on UNC51-like kinase 1 (ULK1) dephosphorylation and autophagy in culture, independent of 4E-BP1, and in parallel induced tumor mitophagy, a tumor suppressor process in liver. These observations have led to an investigator-initiated phase 1B-2 dose escalation trial with RAD001 combined with BEZ235 in patients with HCC and other advanced solid tumors.

Topics: Animals; Antineoplastic Agents; Autophagy; Autophagy-Related Protein-1 Homolog; Carcinoma, Hepatocellular; Everolimus; Humans; Imidazoles; Immunoblotting; Liver Neoplasms; Male; Mice; Mice, Inbred C57BL; Protein Serine-Threonine Kinases; Quinolines; Sirolimus; TOR Serine-Threonine Kinases

Despite recent advances in the treatment of hepatocellular carcinoma (HCC), the chemotherapy efficacy against HCC is still unsatisfactory. The mammalian target of rapamycin (mTOR) has been emerged as an important cancer therapeutic target. However, HCC cells often resistant to rapamycin because of the paradoxical activation of Akt by rapamycin. In this study, we investigated whether bortezomib could enhance the antitumor effects of rapamycin.. The effects of rapamycin and bortezomib on HCC proliferation, apoptosis, migration, and invasiveness in vitro were assessed by CCK-8 analysis, flow cytometry, Hoechst 33342 staining and transwell assays, respectively. Total and phosphorylated protein levels of Akt were detected by Western blotting. The effects of rapamycin and/or bortezomib on the mRNA expression levels of p53, p27, p21 and Bcl-2 family in HCCLM3 cells were evaluated by RT-PCR. The roles of rapamycin and bortezomib on HCC growth and metastasis in xenograft models were evaluated by tumor volumes and fluorescent signals. The effects of rapamycin and bortezomib on cell proliferation and apoptosis in vivo were test by PCNA and TUNEL staining.. Bortezomib synergized with rapamycin to reduce cell growth, induce apoptosis, and inhibit cell mobility in vitro. Further mechanistic studies showed that bortezomib inhibited rapamycin-induced phosphorylated Akt, which in turn enhanced apoptosis of HCC cell lines. The alteration of the mRNA expression of cell cycle inhibitors p53, p27, p21 and apoptosis associated genes Bcl-2, Bax were also involved in the synergistic antitumor effects of rapamycin and bortezomib. P53 inhibitor PFT-α significantly attenuate the effect of rapamycin and bortezomib on cell apoptosis, which indicated that the pro-apoptotic effect of rapamycin and bortezomib may be p53-dependent. Treatment of HCCLM3-R bearing nude mice with rapamycin and bortezomib significantly enhanced tumor growth inhibition (72.4%), comparing with either rapamycin- (54.7%) or bortezomib-treated mice (22.4%). In addition, the lung metastasis was significantly suppressed in mice received the combination treatment (16.6%). The combination treatment of rapamycin and bortezomib significantly inhibited tumor cell proliferation and tumor angiogenesis in vivo.. The combination of rapamycin with bortezomib could be a novel and promising therapeutic approach to the treatment of HCC.

Topics: Animals; Antineoplastic Agents; Apoptosis; Boronic Acids; Bortezomib; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Drug Synergism; Humans; Liver Neoplasms; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neovascularization, Pathologic; Proto-Oncogene Proteins c-akt; Pyrazines; Sirolimus; TOR Serine-Threonine Kinases; Tumor Burden; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Treatment of hepatocellular carcinoma (HCC) is a major concern for physicians as its response to chemotherapy and radiotherapy remains generally poor, due, in part, to intrinsic resistance to either form of treatment. We previously reported that an irradiation with fast neutrons, which are high-linear energy transfer (LET) particles, massively induced autophagic cell death in the human HCC SK-Hep1 cell line. In the present study, we tested the capacity of the mammalian target of rapamycin (mTOR) inhibitor RAD001 to augment the cytotoxicity of low and high-LET radiation in these cells. As mTOR is a key component in a series of pathways involved in tumor growth and development, it represents a potential molecular target for cancer treatment. Results indicate that RAD001, at clinically relevant nanomolar concentrations, enhances the efficacy of both high- and low-LET radiation in SK-Hep1 cells, and that the induction of autophagy may account for this effect. However, fast neutrons were found to be more efficient at reducing tumor cell growth than low-LET radiation.

Topics: Autophagy; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Everolimus; Growth Inhibitors; Humans; Linear Energy Transfer; Liver Neoplasms; Neutrons; Radiation-Sensitizing Agents; Sirolimus; TOR Serine-Threonine Kinases

Topics: Carcinoma, Hepatocellular; Cytochrome P-450 CYP3A; Drug Interactions; Enzyme Induction; Everolimus; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Rifabutin; Rifampin; Sirolimus; Tuberculosis, Pulmonary

Sirolimus has immunosuppressive properties and antitumor effects. It was prescribed in liver transplantation initially in association with calcineurin inhibitors because of its lower nephrotoxic and neurotoxic effects and its potential antitumor effects. The aim of this study was to analyze the use of sirolimus as rescue therapy for liver transplant patients.. We retrospectively analyzed all 15 patients treated with sirolimus from 2009 to 2011 among 150 liver transplantations. We analyzed pre- and postconversion data. With statistical analysis using the Student's t-test.. Sirolimus was the immunosuppressant therapy in 15 of 150 (10%) patients. Their average age was 56.2 years (range, 42-69) including 9 men (60%). The mean time between liver transplantation and the introduction of sirolimus was 24.6 months (range, 1-120). Sirolimus remained as the sole medication for 4 patients (26.6%). The overall time of sirolimus thereby averaged 14.3 months (range, 1-18). The reasons for the introduction of sirolimus were acute rejection (n = 8; 53.3%), chronic rejection (n = 2; 13.3%), development of malignancy (n = 3; 20%) or prior hepatocellular carcinoma (n = 2; 13.3%). Among 9 patients who initiated sirolimus because of rejection, 7 (77.7%) showed improvement in serum liver enzymes. Among the 3 (33.3%) patients who displayed renal insufficiency before the introduction of sirolimus (creatinine level > 1.5 mg/dL) 1 showed improvement with a decrease of ≥50%. The average follow-up was 18 months (range, 1-36). The average sirolimus level during the first 3 months was 10.3 ng/mL (range, 6.1-19.3). All patients developed side effects such as anemia, hypertriglyceridemia, hypercholesterolemia, and infection. In conclusion, sirolimus was useful as rescue therapy.

Topics: Acute Disease; Adult; Aged; Carcinoma, Hepatocellular; Chronic Disease; Drug Substitution; Female; Graft Rejection; Graft Survival; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Neoplasm Recurrence, Local; Retrospective Studies; Salvage Therapy; Sirolimus; Time Factors; Treatment Outcome

Due to the frequent dysregulation of the PI3K/AKT/mTOR signaling pathway, mTOR represents a suitable therapeutic target in hepatocellular carcinoma (HCC). However, emerging data from clinical trials of HCC patients indicate that mTOR inhibition by RAD001 (Everolimus) alone has only moderate antitumor efficacy which may be due to the feedback activation of AKT after mTOR inhibition. In this study, we analyzed the effects of dual inhibition of mTOR and AKT on the proliferation of HCC cell lines. In addition, we measured the feedback activation of each of the AKT isoforms after mTOR inhibition in HCC cell lines and their enzymatic activity in primary samples from HCC patients.. The activation status of specific AKT isoforms in human HCC samples and corresponding healthy liver tissue was analyzed using an AKT isoform specific in vitro kinase assay. AKT isoform activation after mTOR inhibition was analyzed in three HCC cell lines (Hep3B, HepG2 and Huh7), and the impact of AKT signaling on proliferation after mTOR inhibition was investigated using the novel AKT inhibitor MK-2206 and AKT isoform specific knockdown cells.. AKT isoforms become differentially activated during feedback activation following RAD001 treatment. The combination of mTOR inhibition and AKT isoform knockdown showed only a weak synergistic effect on proliferation of HCC cell lines. However, the combinatorial treatment with RAD001 and the pan AKT inhibitor MK-2206 resulted in a strong synergism, both in vitro and in vivo. Moreover, by analyzing primary HCC tissue samples we were able to demonstrate that a hotspot mutation (H1047R) of PI3KCA, the gene encoding the catalytic subunit of PI3K, was associated with increased in vitro kinase activity of all AKT isoforms in comparison to healthy liver tissue of the patient.. Our results demonstrate that dual targeting of mTOR and AKT by use of RAD001 and the pan AKT inhibitor MK-2206 does effectively inhibit proliferation of HCC cell lines. These data suggest that combined treatment with RAD001 and MK-2206 may be a promising therapy approach in the treatment of hepatocellular carcinoma.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Drug Delivery Systems; Drug Synergism; Everolimus; Feedback, Physiological; Female; Gene Knockdown Techniques; Hep G2 Cells; Heterocyclic Compounds, 3-Ring; Humans; Isoenzymes; Liver Neoplasms; Mice; Mice, SCID; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Radiation-induced gastritis is an infrequent cause of gastrointestinal bleeding. It is a serious complication arising from radiation therapy, and the standard treatment method has not been established. The initial injury is characteristically acute inflammation of gastric mucosa. We presented a 46-year-old male patient with hemorrhagic gastritis induced by external radiotherapy for metastatic retroperitoneal lymph node of hepatocellular carcinoma. The endoscopic examination showed diffuse edematous hyperemicmucosa with telangiectasias in the whole muscosa of the stomach and duodenal bulb. Multiple hemorrhagic patches with active oozing were found over the antrum. Anti-secretary therapy was initiated for hemostasis, but melena still occurred off and on. Finally, he was successfully treated by prednisolone therapy. We therefore strongly argue in favor of perdnisolone therapy to effectively treat patients with radiation-induced hemorrhagic gastritis.

Topics: Carcinoma, Hepatocellular; Endoscopy; Gastritis; Gastrointestinal Hemorrhage; Hemostasis; Humans; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Neoplasm Metastasis; Prednisolone; Radiation Injuries; Sirolimus

No standard therapies have been established for the treatment of recurrent hepatocellular carcinoma (HCC) after liver transplantation.. Sirolimus is a mTOR inhibitor which has been used as an immunosuppressive medication in patients who are at high risk of tumor reoccurrence after liver transplantation. Sorafenib is a multikinase inhibitor approved for the treatment of advanced HCC. However the role of sorafenib in patients with HCC reoccurrence after liver transplantation is unclear.. Combination of sirolimus and sorafenib appears to have synergistic effect when treating HCC in preclinical settings. We report a case of a post-liver transplant patient treated with sorafenib and sirolimus for hepatic HCC recurrence who exhibited complete radiologic response after 5A months of therapy.

Topics: Benzenesulfonates; Carcinoma, Hepatocellular; Diagnostic Imaging; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Neoplasm Recurrence, Local; Niacinamide; Phenylurea Compounds; Protein Kinase Inhibitors; Pyridines; Sirolimus; Sorafenib; Tomography, X-Ray Computed; Treatment Outcome

Topics: Antineoplastic Combined Chemotherapy Protocols; Benzenesulfonates; Carcinoma, Hepatocellular; Combined Modality Therapy; Everolimus; Humans; Liver Neoplasms; Liver Transplantation; Neoplasm Recurrence, Local; Niacinamide; Phenylurea Compounds; Pyridines; Sirolimus; Sorafenib; TOR Serine-Threonine Kinases

Highly effective antiretroviral therapy in the last decade has increased the survival rates of HIV-positive patients, yielding a greater number of HIV patients suffering from liver-related disease. Liver transplantation (LT) is the only curative treatment for end-stage liver disease (ESLD) associated or not with hepatocellular carcinoma (HCC).. From June 2003 to September 2010, 23 patients underwent cadaveric donor LT for ESLD at our institution. Inclusion criteria followed the Italian Protocol for LT in HIV-positive patients. Immunosuppressive regimens were based on cyclosporine or tacrolimus, eventually switched to Rapamycin.. The median CD4 T-cell count was 275/mmc (range=119-924). All patients were affected by ESLD, which was associated with HCC in 14 cases. Ten patients were within the Milan criteria and four patients exceeded them but were within the San Francisco criteria. Conversion from calcineurin inhibitors (CNI) to rapamycin occurred in ten cases. Hepatitis C virus (HCV) recurrence occurred in 13/21 HCV-positive patients. Acute cellular rejection occurred in eight patients with one developing chronic cellular rejection. Overall patient and graft survivals at 80 months were 50% and 45% respectively.. LT in HIV-positive patients is a feasible procedure, even if in our experience was burdened by a greater incidence of complications including HCV recurrence and infection compared with HIV-negative patients.

Topics: Adult; Antiretroviral Therapy, Highly Active; Carcinoma, Hepatocellular; CD4 Lymphocyte Count; Cyclosporine; Drug Substitution; End Stage Liver Disease; Female; Graft Rejection; Graft Survival; Hepatitis C, Chronic; HIV; HIV Infections; Hospitals, University; Humans; Immunosuppressive Agents; Italy; Kaplan-Meier Estimate; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Recurrence; RNA, Viral; Severity of Illness Index; Sirolimus; Tacrolimus; Time Factors; Treatment Outcome; Viral Load

The mammalian target of rapamycin (mTOR) is a central regulator of cell growth and proliferation in response to growth factor and nutrient signaling. Consequently, this kinase is implicated in metabolic diseases including cancer and diabetes, so there is great interest in understanding the complete spectrum of mTOR-regulated networks. mTOR exists in two functionally distinct complexes, mTORC1 and mTORC2, and whereas the natural product rapamycin inhibits only a subset of mTORC1 functions, recently developed ATP-competitive mTOR inhibitors have revealed new roles for both complexes. A number of studies have highlighted mTORC1 as a regulator of lipid homeostasis. We show that the ATP-competitive inhibitor PP242, but not rapamycin, significantly down-regulates cholesterol biosynthesis genes in a 4E-BP1-dependent manner in NIH 3T3 cells, whereas S6 kinase 1 is the dominant regulator in hepatocellular carcinoma cells. To identify other rapamycin-resistant transcriptional outputs of mTOR, we compared the expression profiles of NIH 3T3 cells treated with rapamycin versus PP242. PP242 caused 1,666 genes to be differentially expressed whereas rapamycin affected only 88 genes. Our analysis provides a genomewide view of the transcriptional outputs of mTOR signaling that are insensitive to rapamycin.

Topics: Adaptor Proteins, Signal Transducing; Animals; Carcinoma, Hepatocellular; Carrier Proteins; Cell Cycle Proteins; Cell Line, Tumor; Cholesterol; Eukaryotic Initiation Factors; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Liver Neoplasms; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; NIH 3T3 Cells; Phosphoproteins; Proteins; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; Sterol Regulatory Element Binding Protein 2; TOR Serine-Threonine Kinases; Transcription, Genetic

The mammalian target of the rapamycin (mTOR) pathway, which drives cell proliferation, is frequently hyperactivated in a variety of malignancies. Therefore, the inhibition of the mTOR pathway has been considered as an appropriate approach for cancer therapy. In this study, we examined the roles of mTOR in the maintenance and differentiation of cancer stem-like cells (CSCs), the conversion of conventional cancer cells to CSCs and continuous tumor growth in vivo. In H-Ras-transformed mouse liver tumor cells, we found that pharmacological inhibition of mTOR with rapamycin greatly increased not only the CD133+ populations both in vitro and in vivo but also the expression of stem cell-like genes. Enhancing mTOR activity by over-expressing Rheb significantly decreased CD133 expression, whereas knockdown of the mTOR yielded an opposite effect. In addition, mTOR inhibition severely blocked the differentiation of CD133+ to CD133- liver tumor cells. Strikingly, single-cell culture experiments revealed that CD133- liver tumor cells were capable of converting to CD133+ cells and the inhibition of mTOR signaling substantially promoted this conversion. In serial implantation of tumor xenografts in nude BALB/c mice, the residual tumor cells that were exposed to rapamycin in vivo displayed higher CD133 expression and had increased secondary tumorigenicity compared with the control group. Moreover, rapamycin treatment also enhanced the level of stem cell-associated genes and CD133 expression in certain human liver tumor cell lines, such as Huh7, PLC/PRC/7 and Hep3B. The mTOR pathway is significantly involved in the generation and the differentiation of tumorigenic liver CSCs. These results may be valuable for the design of more rational strategies to control clinical malignant HCC using mTOR inhibitors.

Topics: AC133 Antigen; Animals; Antibiotics, Antineoplastic; Antigens, CD; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Glycoproteins; Humans; Liver; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Knockout; Mice, Nude; Neoplastic Stem Cells; Peptides; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sirolimus; TOR Serine-Threonine Kinases; Tumor Suppressor Protein p53

Resveratrol is a plant-derived polyphenol that extends lifespan and healthspan in model organism. Despite extensive investigation, the biological processes mediating resveratrol's effects have yet to be elucidated. Because repression of translation shares many of resveratrol's beneficial effects, we hypothesized that resveratrol was a modulator of protein synthesis. We studied the effect of the drug on the H4-II-E rat hepatoma cell line. Initial studies showed that resveratrol inhibited global protein synthesis. Given the role of the mammalian Target of Rapamycin (mTOR) in regulating protein synthesis, we examined the effect of resveratrol on mTOR signaling. Resveratrol inhibited mTOR self-phosphorylation and the phosphorylation of mTOR targets S6K1 and eIF4E-BP1. It attenuated the formation of the translation initiation complex eIF4F and increased the phosphorylation of eIF2α. The latter event, also a mechanism for translation inhibition, was not recapitulated by mTOR inhibitors. The effects on mTOR signaling were independent of effects on AMP-activated kinase or AKT. We conclude that resveratrol is an inhibitor of global protein synthesis, and that this effect is mediated through modulation of mTOR-dependent and independent signaling.

Topics: AMP-Activated Protein Kinases; Animals; Carcinoma, Hepatocellular; Cell Proliferation; Cell Survival; Enzyme Activation; Eukaryotic Initiation Factor-2; Eukaryotic Initiation Factor-4F; Liver; Liver Neoplasms; Phosphorylation; Protein Biosynthesis; Protein Kinase Inhibitors; Protein Stability; Proto-Oncogene Proteins c-akt; Rats; Resveratrol; Signal Transduction; Sirolimus; Stilbenes; TOR Serine-Threonine Kinases; Transcription Factors

Systemic immunosuppression represents the major factor for cancer recurrence after orthotopic liver transplantation for hepatocellular carcinoma (HCC). Rapamycin is an immunosuppressant with unique antitumoral properties. Although rapamycin has been successfully used in HCC patients after liver transplantation, the detailed mechanisms of rapamycin action on tumor cells are poorly understood.. Two HCC cell lines (PLC5 and HuH7) were used to evaluate the effect of rapamycin. Tumor cell proliferation was analyzed using cell counting and BrdU incorporation assay. Expression of phosphorylated Akt was studied using enzyme linked immunosorbent assay. Digital time-lapse microscopy was utilized to measure tumor cell migration in vitro.. Rapamycin induced a strongly dose-dependent inhibition of tumor cell proliferation in both HCC cell lines. Additionally, rapamycin inhibited activation of Akt phosphorylation and tumor cell migration after prolonged treatment.. Rapamycin suppresses tumor progression due to inhibition of phosphorylated Akt, cell proliferation, and migration. The data of the present study strengthen clinical implications of rapamycin after liver transplantation with HCC.

Topics: Antibiotics, Antineoplastic; Carcinoma, Hepatocellular; Cell Division; Cell Line, Tumor; Cell Movement; Humans; Immunosuppressive Agents; Liver Neoplasms; Sirolimus

The mammalian target of rapamycin (mTOR), which phosphorylates p70S6K and 4EBP1 and activates the protein translation process, is upregulated in cancers and its activation may be involved in cancer development.. In this study, we investigated the tumour-suppressive effects of rapamycin and its new analogue CCI-779 on hepatocellular carcinoma (HCC).. Rapamycin and its new analogue CCI-779 were applied to treat HCC cells. Cell proliferation, cell cycle profile and tumorigenicity were analysed.. In human HCCs, we observed frequent (67%, 37/55) overexpression of mTOR transcripts using real-time reverse transcriptase-polymerase chain reaction. Upon drug treatment, PLC/PRF/5 showed the greatest reduction in cell proliferation using the colony formation assay, as compared with HepG2, Hep3B and HLE. Rapamycin was a more potent antiproliferative agent than CCI-779 in HCC cell lines. Proliferation assays by cell counting showed that the IC(50) value of rapamycin was lower than that of CCI-779 in PLC/PRF/5 cells. Furthermore, flow cytometric analysis showed that both drugs could arrest HCC cells in the G(1) phase but did not induce apoptosis of these cells, suggesting that these mTOR inhibitors are cytostatic rather than cytotoxic. Upon rapamycin and CCI-779 treatment, the phosphorylation level of mTOR and p70S6K in HCC cell lines was significantly reduced, indicating that both drugs can suppress mTOR activity in HCC cells. In addition, both drugs significantly inhibited the growth of xenografts of PLC/PRF/5 cells in nude mice.. Our findings indicate that rapamycin and its clinical analogue CCI-779 possess tumour-suppressive functions towards HCC cells.

Topics: Animals; Antibiotics, Antineoplastic; Carcinoma, Hepatocellular; Cell Count; Cell Line, Transformed; Cell Line, Tumor; Cell Proliferation; Drug Screening Assays, Antitumor; Female; Humans; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Protein Serine-Threonine Kinases; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Tumor recurrence represents the main limitation of liver transplantation in patients with hepatocellular carcinoma (HCC) and can be favored by exposure to calcineurin inhibitors.. We investigated the effect of an immunosuppressant schedule that minimizes the exposure to calcineurin inhibitors on patients transplanted for HCC to ascertain whether this can reduce the tumor recurrence rate. For this purpose, we conducted a matched-cohort study: 31 patients with HCC transplanted between 2004 and 2007 who received sirolimus as part of their immunosuppression (group A) were compared with a control group of 31 patients (group B) transplanted in the same period who had the same prognostic factors but were given standard immunosuppression based on tacrolimus.. Three-year recurrence-free survival was 86% in group A and 56% in group B (P=0.04). Although the prevalence of microvascular invasion G3-G4 grading and alpha-fetoprotein more than 200 ng/mL was identical in the two groups, exposure to tacrolimus was significantly higher in patients of group B (median, 8.54; range, 5.5-13.5) in comparison with those of group A (median, 4.6; range, 1.8-9.1) (P=0.0001).. By using sirolimus, exposure to calcineurin inhibitors can be minimized, reducing the risk of HCC recurrence.

Topics: Adrenal Cortex Hormones; Adult; Aged; Carcinoma, Hepatocellular; Cohort Studies; Disease-Free Survival; Drug Administration Schedule; Female; Hepatitis B; Hepatitis C; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Neoplasm Recurrence, Local; Risk Factors; Sirolimus; Survivors; Tacrolimus; Young Adult

The protein kinase B (AKT)/mammalian target of rapamycin (AKT/mTOR) and mitogen activated protein kinase/extracellular regulated kinase kinase/extracellular regulated kinase (MEK/ERK) signaling pathways have been shown to play an important role in hepatocellular carcinoma (HCC) growth and angiogenesis, suggesting that inhibition of these pathways may have therapeutic potential.. We treated patient-derived HCC xenografts with 1) mTOR inhibitor rapamycin (RAPA); 2) MEK inhibitor AZD6244 (ARRY-142886); and 3) AZD6244 plus RAPA (AZD6244/RAPA). Western blotting was used to determine pharmacodynamic changes in biomarkers relevant to angiogenesis, mTOR pathway, and MEK signaling. Apoptosis, microvessel density, and cell proliferation were analyzed by immunohistochemistry.. We report here that pharmacological inhibition of the MEK/ERK pathway by AZD6244 enhanced the antitumor and antiangiogenic activities of mTOR inhibitor RAPA in both orthotopic and ectopic models of HCC. Such inhibition led to increased apoptosis, decreased angiogenesis and cell proliferation, reduced expression of positive cell cycle regulators, and increase in proapoptotic protein Bim.. Our findings indicate that the AZD6244/RAPA combination had antitumor and antiangiogenic effects in preclinical models of human HCC. Given the urgent need for effective therapies in HCC, clinical evaluating AZD6244/RAPA combination seems warranted.

Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Benzimidazoles; Carcinoma, Hepatocellular; Drug Synergism; Extracellular Signal-Regulated MAP Kinases; Humans; Intracellular Signaling Peptides and Proteins; Liver Neoplasms, Experimental; Mice; Protein Serine-Threonine Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Liver transplantation is an important treatment option for selected patients with nonresectable hepatocellular carcinoma (HCC). Several reports have suggested a lower risk of posttransplant tumor recurrence with the use of sirolimus and a higher one with calcineurin inhibitors, but the selection of an ideal immunosuppression protocol is still a matter of debate. The aim of this study was to define the immunosuppression associated with the best survival after liver transplantation for HCC. It was based on the Scientific Registry of Transplant Recipients and included 2,491 adult recipients of isolated liver transplantation for HCC and 12,167 for non-HCC diagnoses between March 2002 and March 2009. All patients remained on stable maintenance immunosuppression protocols for at least 6 months posttransplant. In a multivariate analysis, only anti-CD25 antibody induction and sirolimus-based maintenance therapy were associated with improved survivals after transplantation for HCC (hazard ratio [HR] 0.64, 95% confidence interval [CI]: 0.45-0.9, P < or = 0.01; HR 0.53, 95% CI: 0.31-0.92, P < or = 0.05, respectively). The other studied drugs, including calcineurin inhibitors, did not demonstrate a significant impact. In an effort to understand whether the observed effects were due to a direct impact of the drug on tumor or more on liver transplant in general, we conducted a similar analysis on non-HCC patients. Although anti-CD25 induction was again associated with a trend toward improved survival, sirolimus showed a trend toward lower rates of survival in non-HCC recipients, confirming the specificity of its beneficial impact to cancer patients.. According to these data, sirolimus-based immunosuppression has unique posttransplant effects on HCC patients that lead to improved survival.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Female; Humans; Immunosuppression Therapy; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Sirolimus

Mammalian target of rapamycin (mTOR) inhibitors behave as potent immunosuppressants, which have the advantages, with respect to calcineurin inhibitors (CNI; cyclosporine or tacrolimus), of no nephrotoxicity but inhibition of cell proliferation. They are particularly suitable for patients with renal insufficiency or neoplasias.. Twenty-eight liver transplant patients were immunosuppressed with everolimus or sirolimus as rescue therapy after CNI treatment: 8 hepatocellular carcinomas; 7 de novo malignancies; 6 renal insufficiencies; 3 chronic rejections; 3 acute rejection episodes; and 1 epilepsy.. There were 0% tumor recurrences, 50% improvements in 33% no change, and 17% worsening of renal function among cases of renal insufficiency; 0% improvement in cases of chronic rejection, and 33% improvement in acute rejection episodes. There was a 7% incidence of acute rejection episodes, but no kidney failure, gastrointestinal intolerance, hydrocarbon intolerance, hypertension, or arterial or venous thrombosis. Diarrhea occurred in 7%; hypercholesterolemi in 46% hypertriglyceridemia in 50% thrombocytopenia in 14%, leukopenia in 14%, and anemia in 39%. The 12% intercurrent infection rate included oral thrush in 11%. Lower limb edema occurred in 21%; 1 case displayed facial edema and 1, alopecia.. mTOR inhibitors were safe immunosuppressive drugs whose side effects were controlled and easily managed. They have advantages with respect to CNI due to their slight effects on kidney function and lack of promotion of diabetes mellitus. Although their long-term effectiveness for control of neoplastic diseases is yet to be seen, they can be used safely in these patients with a low incidence of rejection. Their effectiveness to control steroid-resistant acute rejection episodes or renal insufficiency seems significant, but they are of doubtful benefit for chronic rejection.

Topics: Carcinoma, Hepatocellular; Everolimus; Graft Rejection; Humans; Immunosuppressive Agents; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Liver Transplantation; Neoplasm Recurrence, Local; Protein Serine-Threonine Kinases; Safety; Sirolimus; TOR Serine-Threonine Kinases

The advent of molecular medicine that targets specific pathways is changing the therapeutic approach to hepatocellular carcinoma. For several aberrantly activated pathways in hepatocarcinoma, surrogate markers of activation can be assessed by immunohistochemistry, although associations with in vivo response to targeted therapies are still lacking.. A patient, who presented with hepatic and extra-hepatic hepatocarcinoma recurrence 11 years after liver transplantation, was assessed for beta-catenin, pERK, and pS6 in primary and secondary tumor specimens, in order to define a possible activation of the Wnt, Ras/MAPK and Akt/mTOR pathways and design a personalized targeted therapy in absence of alternative treatment options. Moreover, mutation analysis of the beta-catenin gene (CTNNB1) and DNA microsatellite analyses were performed.. The identification of the same mutation in the beta-catenin gene, as well as the same microsatellite pattern in tumor tissues taken 11 years apart, proved that the observed hepatocarcinoma was a true recurrence. Nuclear beta-catenin and pS6 in tumor cells were positive, whereas pERK was positive only in the peritumoral endothelium. This pattern of immunohistochemistry, after failure of sorafenib alone, lead to the choice to add the mTOR inhibitor, everolimus, to sorafenib. Three months later a 50% tumor reduction was observed, and after 6 months a further reduction of tumor vital components was confirmed, while a grade II gastrointestinal bleeding episode occurred.. A personalized approach aimed to treat recurrent hepatocarcinoma is possible through analysis of tumoral molecular pathways. Partial success of the selected combination of sorafenib and everolimus supports the pivotal role of mTOR signalling and highlights the importance of reliable biomarkers to route the best molecular-based therapeutic options in HCC.

Topics: alpha-Fetoproteins; Antineoplastic Agents; Antiviral Agents; Benzenesulfonates; beta Catenin; Carcinoma, Hepatocellular; Chemoembolization, Therapeutic; DNA Mutational Analysis; Everolimus; Hepatitis C; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Neoplasm Recurrence, Local; Niacinamide; Phenylurea Compounds; Point Mutation; Precision Medicine; Pyridines; Ribavirin; Sirolimus; Sorafenib

Tumor progression before orthotopic liver transplantation (OLT) is the main cause of dropouts from waiting lists among patients with hepatocellular carcinoma (HCC). Performing a porto-caval shunt (PCS) before parenchymal liver transection has the potential to allow an extended hepatectomy in patients with decompensated liver cirrhosis, reducing portal hyperflow and therefore the sinusoidal shear-stress on the remnant liver. We report the case of a 59-year-old man affected by hepatitis C virus (HCV)-related decompensated liver cirrhosis (Child Pugh score presentation, C-10; Model for End Stage Liver Disease score, 18) and HCC (2 lesions of 2 and 2.8 cm). The patient began the evaluation to join the OLT waiting list, but, in the 3 months required to complete the evaluation, he developed tumor progression: 3 HCC lesions, the largest 1 with a diameter of about 4.4 cm. These findings excluded transplantation criteria and the patient was referred to our center. After appropriate preoperative studies, the patient underwent a major liver resection (trisegmentectomy) after side-to-side PCS by interposition of an iliac vein graft from a cadaveric donor. The patient overcame the worsened severity of cirrhosis. After 6 months of follow-up, he developed 2 other HCC nodules. He was then included on the waiting list at our center, undergoing OLT from a cadaveric donor at 8 months after salvage treatment. At 36 months after OLT, he is alive and free from HCC recurrence. Associating a partial side-to-side PCS with hepatic resection may represent a potential salvage therapy for patients with decompensated cirrhosis and HCC progression beyond listing criteria for OLT.

Topics: Adult; Alcoholism; Aneurysm, False; Antibiotics, Antineoplastic; Carcinoma, Hepatocellular; Embolization, Therapeutic; Female; Humans; Hydrothorax; Immunosuppressive Agents; Liver Cirrhosis; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Portasystemic Shunt, Transjugular Intrahepatic; Sirolimus; Treatment Outcome; Varicose Veins

The PI3K/Akt/mTOR signal pathway is involved in hepatocarcinogenesis. Rapamycin (=sirolimus), a specific mTOR inhibitor, leads to G(1) arrest of many malignant cell lines and currently, analogues of rapamycin are being investigated as a cancer chemotherapeutic adjuvant.. To study the toxicity and tolerability of rapamycin therapy in patients with advanced hepatocellular carcinoma (HCC).. Between June 2005 and February 2007, patients with advanced HCC, not eligible for any established therapy, were included in the study.. Eighteen patients (F/M: 5/13) with compensated liver cirrhosis (Child A n = 11, Child B n = 5, Child C n = 2) and histologically proven HCC were included in this study. According to the BCLC staging system, most of the patients enrolled had an advanced HCC: BCLC stage B: n = 2, Barcelona Clinic Liver-Cancer (BCLC) stage C: n = 14, BCLC stage D: n = 2. Overall, therapy with rapamycin was well tolerated. Most common toxicities were thrombocytopaenia and anaemia. We did not observe any partial or complete tumour response. At 3 months, two patients had stable disease and at 6 months, all patients had progressed. The median overall survival was 5.27 months, median time to progression was 3 months.. Rapamycin is well tolerated in patients with advanced HCC, but only minimally effective.

Topics: Aged; Aged, 80 and over; Carcinoma, Hepatocellular; Disease Progression; Female; Humans; Immunosuppressive Agents; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Pilot Projects; Sirolimus; Treatment Outcome

Topics: Carcinoma, Hepatocellular; Humans; Immunosuppressive Agents; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Protein Serine-Threonine Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Sirolimus (SRL) is a newer immunosuppressant whose possible benefits and side effects in comparison to calcineurin inhibitors (CNIs) still have to be addressed in the liver transplantation setting. We report the results of the use of SRL in 86 liver transplant recipients, 38 of whom received SRL as the main immunosuppressant in a CNI-sparing regimen. Indications for the use of SRL were: impaired renal function (n = 32), CNI neurotoxicity (n = 16), hepatocellular carcinoma (HCC) at high risk of recurrence (n = 21), recurrence of HCC (n = 6), de novo malignancies (n = 4), cholangiocarcinoma (n = 1), and the need to reinforce immunosuppression (n = 6). Among patients on SRL-based treatment, four episodes of acute rejection were observed, three of which occurred during the first postoperative month. Renal function significantly improved when sirolimus was introduced within the third postoperative month, while no change was observed when it was introduced later. Neurological symptoms resolved completely in 14/16 patients. The 3-year recurrence-free survival of patients with HCC on SRL was 84%. Sixty-two patients developed side effects that required drug withdrawal in seven cases. There was a reduced prevalence of hypertension and new-onset diabetes among patients under SRL. In conclusion, SRL was an effective immunosuppressant even when used in a CNI-sparing regimen. It was beneficial for patients with recently developed renal dysfunction or neurological disorders.

Topics: Adult; Carcinoma, Hepatocellular; Disease-Free Survival; Drug Administration Schedule; Female; Glomerular Filtration Rate; Humans; Immunosuppressive Agents; Kidney Function Tests; Liver Cirrhosis; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Recurrence; Sirolimus; Time Factors

A case of hepatocellular carcinoma (HCC) with pulmonary recurrence after liver transplantation for HCC is presented in this report. The patient showed disease progression on sorafenib therapy demonstrated by computed tomography scans as well as serial serum α-fetoprotein (AFP) elevation. After his immunosuppression therapy was successfully transitioned to sirolimus and a continuation of sorafenib, he achieved partial remission based on RECIST criteria and normalization of AFP. Mammalian target of rapamycin inhibitors including sirolimus alone or in conjunction with sorafenib may be useful in the treatment of post transplant HCC.

Topics: Antineoplastic Agents; Benzenesulfonates; Carcinoma, Hepatocellular; Drug Therapy, Combination; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Lung Neoplasms; Male; Middle Aged; Niacinamide; Phenylurea Compounds; Pyridines; Sirolimus; Sorafenib; Treatment Outcome

Immunosuppressive therapy after liver transplantation for hepatocellular carcinoma (HCC) is one of the major contributory factors for HCC recurrence and metastasis. Sirolimus, a potent immunosuppressant, has been reported to be an effective inhibitor in a variety of tumors. The present study is designed to explore whether sirolimus could block the growth and metastatic progression of HCC.. MHCC97H cells were used as targets to explore the effect of sirolimus on cell cycle progression, apoptosis, proliferation, and its antiangiogenic mechanism. LCI-D20, a highly metastatic model of human HCC in nude mice, was also used as the model tumor to explore the effect of sirolimus on tumor growth and metastatic progression.. In vitro, sirolimus induced cell cycle arrest at the G1 checkpoint and blocked proliferation of MHCC97H cells but did not induce apoptosis. In vivo, sirolimus prevented tumor growth and metastatic progression in LCI-D20. Intratumoral microvessel density and circulating levels of VEGF in tumor-bearing mice were also significantly reduced in sirolimus treatment group. Quantitative RT-PCR showed that sirolimus down-regulated the mRNA expression of VEGF and HIF-1a, but not of bFGF, and TGF-b in MHCC97H cells. Furthermore, western blot analysis confirmed that sirolimus also decreased expression of HIF-1a at protein level, in parallel with the down-regulation of the levels of VEGF protein excretion in a time-dependent manner as compared to untreated control cells following anoxia.. The immunosuppressive macrolide sirolimus prevents the growth and metastatic progression of HCC, and suppresses VEGF synthesis and secretion by downregulating HIF-1a expression. Sirolimus may be useful for clinical application in patients who received a liver transplant for HCC.

Topics: Actins; Angiogenesis Inhibitors; Animals; Antibiotics, Antineoplastic; Apoptosis; Carcinoma, Hepatocellular; Cell Cycle; Cell Division; Cell Line, Tumor; DNA Primers; Fibroblast Growth Factor 2; Humans; Liver Neoplasms; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; RNA, Messenger; Sirolimus; Transforming Growth Factor beta; Transplantation, Heterologous; Vascular Endothelial Growth Factor A

Rapamycin (RAPA) inhibits tumor growth and angiogenesis in hepatocellular carcinoma (HCC). The molecular mechanism underlying the antitumoral effects of RAPA remains unclear. Here we established a chemical-induced rat HCC model to investigate the signaling pathways mediating RAPA's antitumor activity. We found that RAPA exposure significantly diminished tumor growth, angiogenesis, and metastasis of HCC. Meanwhile, the antitumor drug dramatically decreased expression of HIF-1alpha and VEGF, either at mRNA or protein levels. Moreover, the low-dose of RAPA (1.5 mg/kg/day) was effective enough to markedly inhibit tumor progression of HCC. The preliminary results suggested that the antitumoral effects of RAPA might be at least partially mediated through downregulation of HIF-1alpha and VEGF, and low-dose RAPA-based regimens exhibited a promising future in treatment of HCC.

Topics: Animals; Antibiotics, Antineoplastic; Blotting, Western; Carcinoma, Hepatocellular; Enzyme-Linked Immunosorbent Assay; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Liver Neoplasms, Experimental; Male; Neovascularization, Pathologic; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sirolimus; Vascular Endothelial Growth Factor A

Hepatocellular carcinoma (HCC) remains associated with a poor prognosis, but novel targeted therapies in combination with anti-angiogenic substances may offer new perspectives. We hypothesized that simultaneous targeting of tumor cells, endothelial cells, and pericytes would reduce growth and angiogenesis of HCC, which represents a highly vascularized tumor entity. Recently, because of their anti-angiogenic properties, inhibitors of mammalian target of rapamycin (mTOR) have entered clinical trials for therapy of HCC. However, treatment with mTOR inhibitors may lead to paradoxical activation of Akt signaling in tumor cells via insulin-like growth factor-I receptor (IGF-IR)-dependent and IGF-IR-independent mechanisms. Because we have recently identified heat shock protein 90 (Hsp90) antagonists to impair both oncogenic and angiogenic signaling cascades in tumor cells, including Akt and IGF-IR, we sought to investigate whether Hsp90 blockade could improve growth-inhibitory and anti-angiogenic effects of the mTOR inhibitor rapamycin. Human HCC cells, a murine hepatoma cell line, endothelial cells (ECs), and vascular smooth muscle cells (VSMC) were employed in experiments. Results show that dual inhibition of mTOR and Hsp90 leads to effective disruption of oncogenic signaling cascades and substantially improves growth-inhibitory effects in vivo. Importantly, blocking Hsp90 abrogated the rapamycin-induced activation of Akt and of the downstream effector nuclear factor kappa-B (NF-kappaB) in HCC tumors. Furthermore, Hsp90 inhibition reduced the expression of platelet-derived growth factor-receptor-beta (PDGF-Rbeta) on VSMCs, and diminished vascular endothelial growth factor-receptor 2 (VEGFR-2) expression on ECs, which further improves the anti-angiogenic capacity of this regimen.. Blocking Hsp90 disrupts rapamycin-induced activation of alternative signaling pathways in HCCs and substantially improves the growth-inhibitory effects of mTOR inhibition in vivo. Hence, the concept of targeting tumor cells, ECs, and VSMCs by blocking Hsp90/mTOR could prove valuable for treatment of HCC.

Topics: Animals; Antibiotics, Antineoplastic; Carcinoma, Hepatocellular; Carrier Proteins; Cell Death; Cell Movement; Disease Models, Animal; DNA Fragmentation; DNA Primers; HSP90 Heat-Shock Proteins; Humans; Liver Neoplasms; Mice; Phosphotransferases (Alcohol Group Acceptor); RNA, Neoplasm; Sirolimus; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor A

Phosphorylation of proteins on serine or threonine residues that immediately precede proline (pSer/Thr-Pro) is specifically catalyzed by the peptidyl-prolyl cis-trans isomerase Pin1 and is a central signaling mechanism in cell proliferation and transformation. Although Pin1 is frequently overexpressed in hepatocellular carcinoma (HCC), the molecular mechanism of Pin1 in HCC has not been completely elucidated. Here, we show that Pin1 interacts with p70S6K in vitro and ex vivo. Overexpression of Pin1 resulted in enhanced p70S6K phosphorylation induced by insulin in SK-HEP-1 cells. In contrast, Pin1(-/-) mouse embryonic fibroblasts (MEFs) exhibited significantly decreased insulin-induced p70S6K phosphorylation compared with Pin1(+/+) MEFs. Furthermore, Pin1 enhanced the insulin-induced extracellular signal-regulated protein kinase (ERK)1/2 phosphorylation through its interaction with p70S6K, whereas the inhibition of p70S6K activity by rapamycin suppressed insulin-induced ERK1/2 phosphorylation in SK-HEP-1 cells. Hence, Pin1 affected activator protein-1 activity through p70S6K-ERK1/2 signaling in SK-HEP-1 cells. Most importantly, Pin1-overexpressing JB6 Cl41 cells enhanced neoplastic cell transformation promoted by insulin much more than green fluorescent protein-overexpressing JB6 Cl41 control cells. These results imply that Pin1 amplifies insulin signaling in hepatocarcinoma cells through its interaction with p70S6K, suggesting that Pin1 plays an important role in insulin-induced tumorigenesis and is a potential therapeutic target in hepatocarcinoma.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Cells, Cultured; Drug Synergism; Embryo, Mammalian; Fibroblasts; Gene Expression Regulation, Neoplastic; Green Fluorescent Proteins; Humans; Hypoglycemic Agents; Immunoblotting; Immunosuppressive Agents; Insulin; Liver Neoplasms, Experimental; Mice; Mice, Knockout; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Naphthoquinones; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Phosphorylation; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Small Interfering; Signal Transduction; Sirolimus; Transcription Factor AP-1; Two-Hybrid System Techniques

To test the effect of rapamycin (RAPA) on hepatic tumor growth and metastasis in Sprague-Dawley (SD) rat model and explore the possible mechanism.. SD rat hepatocellular carcinoma (HCC) model with metastatic potential was induced by diethylnitrosamine (DEN) and N-nitrosomorpholine (NMOR). 120 SD rats were randomized into four groups 16 weeks after DEN and NMOR treatment, and received 4-week intraperitoneal injection of RAPA (1.5 or 4.5 mg x kg(-1) x d(-1)), CsA (25 mg x kg(-1) x d(-1)) or equal volume of 0.9% saline, respectively. Tumor growth and metastasis were checked after the 4-week treatment. Serum vascular endothelial growth factor (VEGF) was determined by enzyme-linked immunosorbent assay (ELISA). Antiangiogenetic effects were assessed by CD34 immunostaining. The levels of hypoxia-inducible factor 1 alpha (HIF-1 alpha) and VEGF proteins and mRNAs were detected by immunohistochemistry, western blot and reverse transcriptase-polymerase chain reaction (RT-PCR).. The mean liver weight (5.58% +/- 0.42% and 5.69% +/- 0.74%), the metastatic liver nodules (5.12 +/- 0.68 and 5.67 +/-1.12), the metastasis lung nodules (0.43 +/- 0.11 and 0.45 +/- 0.83), and the lung metastasis rate (17.2% and 14.8%) were lower in rats treated with RAPA 1.5 mg x kg(-1) x d(-1) or 4.5 mg x kg(-1) x d(-1) than those in rats treated with saline, which were 10.42% +/- 1.86%, 12.36 +/- 3.45, 1.81 +/- 0.3 and 50.0% respectively (P < 0.01 or P < 0.05). The intratumoral microvessel density (MVD), serum VEGF, and the levels of HIF-1 alpha and VEGF were lower in RAPA-treated rats than those in control rats. However, CsA-treated rats showed an opposite trend compared with the RAPA-treated rats.. RAPA can repress the expression of angiogenesis-promoting factors HIF-1 alpha and VEGF, and significantly inhibits the growth and metastasis of HCC.

Topics: Animals; Carcinoma, Hepatocellular; Cyclosporine; Disease Models, Animal; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Immunosuppressive Agents; Liver Neoplasms, Experimental; Male; Microvessels; Neoplasm Metastasis; Neovascularization, Pathologic; Random Allocation; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sirolimus; Vascular Endothelial Growth Factors

The mammalian target of rapamycin inhibitors are immunosuppressive agents with antiproliferative effects and consequent potential application as anticancer agents. The safety and tolerance of calcineurin inhibitor (CNI)-free sirolimus-based immunosuppressant protocols in liver transplant recipients with malignancies or high risk of tumor recurrence has been scarcely evaluated.. Fourteen liver transplant recipients, including 12 men, of overall mean age of 57.4 +/- 12.4 years were distributed into two groups: group I, corresponding to 11 patients with malignant neoplasia, eight de novo neoplasia, and three recurrent hepatocarcinoma and; group II, three patients with high risk of tumor recurrence due to cholangiocarcinoma. Sirolimus was initiated at 2 mg od, with target levels of 3 to 9 ng/mL. Withdrawal of CNI was performed after reaching target levels of sirolimus. Periodic examinations of weight, arterial pressure, liver function tests, serum creatinine, triglycerides, cholesterol, sirolimus blood levels, and creatinine clearance were performed at 30, 60, 90, 180, and 360 days.. After a median follow-up of 221.5 days, eight group I patients (72.7%) were alive, including six with stable disease. All group II patients were alive without evidence of tumor recurrence after a median follow-up of 560 days. CNI was withdrawn in 11 patients (78.6%). Sirolimus was withdrawn in only one case due to severe symptomatic oral ulcers. No vascular complications or rejection episodes were observed.. A sirolimus-based immunosuppressant protocol was well tolerated and safe in liver transplant recipients with malignancies or a high risk of recurrence of neoplastic disease.

Topics: Adult; Calcineurin Inhibitors; Carcinoma, Hepatocellular; Drug Tolerance; Follow-Up Studies; Humans; Immunosuppressive Agents; Liver Neoplasms; Middle Aged; Recurrence; Safety; Sirolimus; Survival Rate; Survivors

Hepatocellular carcinoma (HCC) represents one of the most common malignancies globally, accounting for nearly one million new cases per year. Although the treatment of extrahepatic metastases from primary liver tumors is essentially palliative, a solitary metastasis from such tumors offers a possibility of cure by surgical resection. The adrenal gland is an uncommon site for metastasis from primary liver tumors.. We report a liver transplantation case of HCC and hepatitis B virus in a 23-year-old man with an excellent postoperative result. However, because an increased alpha-fetoprotein was evident and complete radiologic and blood tests were performed, all of which were normal. Three years posttransplantation, a right adrenal mass was identified by CT. PAAF was performed as well as adrenalectomy for a solitary adrenal metastasis from hepatocellular carcinoma.. The patient underwent adrenalectomy for the right adrenal metastasis at 3 years following liver transplantation for HCC. He is presently alive and disease-free 24 months after adrenalectomy.. Carefully selected patients with solitary metastasis from HCC may be considered for resection.

Topics: Adrenal Gland Neoplasms; Adrenalectomy; Adult; Carcinoma, Hepatocellular; Everolimus; Follow-Up Studies; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Neoplasm Metastasis; Sirolimus; Survival Analysis; Time Factors; Tomography, X-Ray Computed; Treatment Outcome; Young Adult

Topics: Carcinoma, Hepatocellular; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Neoplasm Recurrence, Local; Sirolimus

The aim of the present work was to evaluate the influence of cyclosporine (CsA) and sirolimus (SRL) on fatty acid (FA) desaturase activities. These enzymes (named Delta9, Delta6, and Delta5 desaturases) catalyze reactions leading to the biosynthesis of n-9, n-6, and n-3 FA families. n-3 FA family, derived from alpha-linolenic acid, is involved in the prevention of vascular events, which appear after successful kidney transplantation. Five groups of HepG(2) cells in culture were treated with either CsA (1 microg/microL and 2 microg/microL) or SRL (10 ng/mL and 20 ng/mL) for 3 days, including a control group without immunosuppressive treatment. We studied the incorporation and metabolic conversion of radioactive [1-(14)C]palmitic, linoleic, and eicosatrienoic acids. We also analyzed fatty acid composition. The distribution of radioactive metabolic products after incubation of these cells with [1-(14)C]palmitic acid revealed a decrease in Delta9 desaturase activity in the presence of each immunosuppressive drug: CsA = 0.61 +/- 0.01; SRL = 0.59 +/- 0.04 versus control = 0.79 +/- 0.05 (P < .01). We observed a significant increase in Delta6 and Delta5 desaturase activities under the influence of the immunosuppressive drugs: radiolabeled linoleic acid (CsA: 0.93 +/- 0.04; SRL: 1.02 +/- 0.03 vs control 0.60 +/- 0.03; P < .01) and eicosatrienoic acid (CsA: 1.12 +/- 0.02; SRL: 1.07 +/- 0.01 vs control 0.75 +/- 0.01; P < .01). In conclusion, CsA and SRL modulated the biosynthesis of polyunsaturated FAs, decreasing Delta9 desaturase and increasing Delta6 and Delta5 desaturase activities.

Topics: Arachidonic Acids; Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclosporine; Fatty Acid Desaturases; Fatty Acids; Humans; Immunosuppressive Agents; Kinetics; Linoleic Acid; Liver Neoplasms; Palmitic Acid; Sirolimus

To study the effects of sirolimus (SRL) on the growth of transplanted human hepatocellular carcinoma (HCC) in nude mice.. HepG2 cells were Implanted into the liver of nude mice. The implanted mice were then treated with SRL and tacrolimus (FK506). The expression of vascular endothelial growth factor (VEGF) and proliferating cell nuclear antigen (PCNA) was detected by immunohistology, microvessel density (MVD) was counted by immunostaining with anti-CD34 antibody for endothelial cells. Tumor apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay.. The tumor weight was (352+/-38) mg, (683+/-53) mg and (675+/-45) mg in SRL, FK506 and control group respectively. The tumor weight was significantly decreased in SRL group (P < 0.01), and there was no difference between FK506 group and control group. The expression of VEGF and PCNA protein was remarkably down-regulated in SRL group compared to control group (P < 0.05), and it was not significantly different between FK506 group and control group (P > 0.05). Compared to the control group, MVD was significanly decreased in SRL group, and the apoptosis index of tumor cell was significantly higher in SRL group (P < 0.01).. SRL inhibits transplanted HCC tumor growth by reducing tumor angiogenesis, inhibiting tumor proliferation and inducing tumor apoptosis.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Hep G2 Cells; Humans; Immunohistochemistry; Liver; Liver Neoplasms, Experimental; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Neovascularization, Pathologic; Proliferating Cell Nuclear Antigen; Sirolimus; Tacrolimus; Treatment Outcome; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

The success of sorafenib in the treatment of advanced hepatocellular carcinoma (HCC) has focused interest on the role of Ras signaling in this malignancy. We investigated the molecular alterations of the Ras pathway in HCC and the antineoplastic effects of sorafenib in combination with rapamycin, an inhibitor of mTOR pathway, in experimental models.. Gene expression (qRT-PCR, oligonucleotide microarray), DNA copy number changes (SNP-array), methylation of tumor suppressor genes (methylation-specific PCR) and protein activation (immunohistochemistry) were analysed in 351 samples. Anti-tumoral effects of combined therapy targeting the Ras and mTOR pathways were evaluated in cell lines and HCC xenografts.. Different mechanisms accounted for Ras pathway activation in HCC. H-ras was up-regulated during different steps of hepatocarcinogenesis. B-raf was overexpressed in advanced tumors and its expression was associated with genomic amplification. Partial methylation of RASSF1A and NORE1A was detected in 89% and 44% of tumors respectively, and complete methylation was found in 11 and 4% of HCCs. Activation of the pathway (pERK immunostaining) was identified in 10.3% of HCC. Blockade of Ras and mTOR pathways with sorafenib and rapamycin reduced cell proliferation and induced apoptosis in cell lines. In vivo, the combination of both compounds enhanced tumor necrosis and ulceration when compared with sorafenib alone.. Ras activation results from several molecular alterations, such as methylation of tumor suppressors and amplification of oncogenes (B-raf). Sorafenib blocks signaling and synergizes with rapamycin in vivo, preventing tumor progression. These data provide the rationale for testing this combination in clinical studies.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Benzenesulfonates; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; DNA Methylation; Drug Synergism; Female; Gene Dosage; Genes, ras; Humans; Liver Neoplasms; Liver Neoplasms, Experimental; Mice; Mice, Nude; Neoplasm Transplantation; Niacinamide; Phenylurea Compounds; Promoter Regions, Genetic; Protein Kinases; Pyridines; ras Proteins; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Sirolimus; Sorafenib; TOR Serine-Threonine Kinases; Transplantation, Heterologous

The insulin-like growth factor (IGF) signaling axis is frequently dysregulated in hepatocellular carcinoma (HCC). Therefore, we investigated whether the specific targeting of the IGF type 1 receptor (IGF-1R) might represent a new therapeutic approach for this tumor.. Total and phosphorylated levels of IGF-1R were measured in 21 paired samples of human HCCs and adjacent nontumoral livers using ELISA. The antineoplastic potency of a novel anti-IGF-1R antibody, AVE1642, was examined in five human hepatoma cell lines.. Overexpression of IGF-1R was detected in 33% of HCCs and increased activation of IGF-1R was observed in 52% of tumors. AVE1642 alone had moderate inhibitory effects on cell viability. However, its combination with gefitinib, an epidermal growth factor receptor (EGFR) inhibitor, induced supra-additive effects in all cell lines that were associated with cell cycle blockage and inhibition of AKT phosphorylation. The combination of AVE1642 with rapamycin also induced a synergistic reduction of viability and of AKT phosphorylation. Of marked interest, AVE1642 alone up-regulated the phosphorylated and total levels of HER3, the main partner of EGFR, and AVE1642-induced phosphorylation of HER3 was prevented by gefitinib. Moreover, the down-regulation of HER3 expression with siRNA reduced AKT phosphorylation and increased cell sensitivity to AVE1642.. These findings indicate that hepatoma cells overcome IGF-1R inhibition through HER3 activation in an EGFR-dependent mechanism, and that HER3 represents a critical mediator in acquired resistance to anti-IGF-1R therapy. These results provide a strong rational for targeting simultaneously EGFR and IGF-1R in clinical trials for HCC].

Topics: Antibiotics, Antineoplastic; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Drug Synergism; ErbB Receptors; Gefitinib; Humans; Insulin; Liver Neoplasms; Phosphorylation; Proto-Oncogene Proteins c-akt; Quinazolines; Receptor, ErbB-3; Receptor, IGF Type 1; RNA, Small Interfering; Signal Transduction; Sirolimus

Tumor recurrence after liver transplantation for hepatocellular carcinoma is associated with a poor prognosis. Because immunosuppression is a well-known risk factor for tumor growth, it is surprising that its possible role in the outcome of liver transplantation has been poorly evaluated. We performed a case-control review of prospectively collected data and compared 2 groups of patients according to the type of immunosuppression after liver transplantation for hepatocellular carcinoma at a single center. One hundred six patients received tacrolimus and mycophenolate mofetil, and 121 received sirolimus. Patients in the sirolimus group had significantly higher recurrence-free survival rates than patients in the tacrolimus group (P = 0.0003). The sirolimus group also had significantly higher patient survival rates than the tacrolimus group at 1 year (94% versus 79%), 3 years (85% versus 66%), and 5 years (80% versus 59%; P = 0.001). Sirolimus was well tolerated, and the patients in this study did not have the increase in surgical complications noted by other investigators. Leukopenia was the most common side effect, but it typically resolved with dose reduction. Dyslipidemia and mouth ulcers were common but were easily controlled. In summary, the data suggest a beneficial effect of sirolimus immunosuppression on recurrence-free survival, which translates into patient survival benefits.

Topics: Carcinoma, Hepatocellular; Case-Control Studies; Chemotherapy, Adjuvant; Drug Therapy, Combination; Female; Humans; Immunosuppressive Agents; Kaplan-Meier Estimate; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Mycophenolic Acid; Proportional Hazards Models; Recurrence; Retrospective Studies; Risk Assessment; Risk Factors; Sirolimus; Tacrolimus; Time Factors; Treatment Outcome

Liver transplantation is considered the best treatment of hepatocellular carcinoma but its efficacy depends on the risk of tumour recurrence. The risk of recurrence depends on tumour characteristics but is also influenced by immunosuppressive regimens. Immunosuppressants of the mTOR inhibitors family share anti-tumour properties which are already taken into account in the treatment of renal carcinoma and advanced hepatocellular carcinoma. These features make interesting their use in liver transplantation for hepatocellular carcinoma, with the following goals: to reduce the risk of post-transplant tumour recurrence, to expand the indications of liver transplantation for hepatocellular carcinoma and eventually to slow down tumour growth during the waiting period. However, to date, the potential role of mTOR inhibitors after liver transplantation for hepatocellular carcinoma is only based on small encouraging proof of concept studies. Large randomized studies are therefore required to further define the specific indications of these compounds. The demonstration of a beneficial impact of mTOR inhibitors in the setting of liver transplantation for hepatocellular carcinoma would be a major therapeutical advance.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Humans; Immunosuppressive Agents; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Liver Transplantation; Neoplasm Recurrence, Local; Protein Serine-Threonine Kinases; Sirolimus; TOR Serine-Threonine Kinases

To study the effects of Rapamycin (RPM) on angiogenesis and tumor progression in human hepatocellular carcinoma (HCC) implantation mice.. Tumor tissues of HCC were implanted into the liver of nude mice. Then, nude mice were treated with RPM and cyclosporine A (CsA). Real-time PCR was used to detect the mRNA expression of vascular endothelial growth factor (VEGF). Immunohistochemical stain and image analysis were used to detect the protein expression of VEGF and proliferating cell nuclear antigen (PCNA) and microvessel density (MVD) was counted by endothelial cells immunostained by anti-CD34 antibody. The concentration of VEGF in the peripheral blood was detected by ELISA.. (1) The tumor weights were (372 +/- 35) mg, (769 +/- 39) mg and (751 +/- 42) mg in RPM, CsA and control group respectively. The tumor weight was significantly decreased in RPM group and no difference in CsA group compared with control group. (2) The expression of VEGF mRNA, VEGF and PCNA protein in tumor tissues and concentration of VEGF in the peripheral blood were remarkably down-regulated in RPM group compared with control group (P < 0.05) and were not remarkably different in CsA group from in control (P > 0.05).(3) Comparing with the control, the tumor MVD was remarkably decreased in RPM group (P < 0.05), and no difference in CsA group (P > 0.05).. RPM can inhibit angiogenesis and tumor progression of HCC by down-regulated the gene and protein expression of VEGF and inhibited the growth of tumor.

Topics: Animals; Carcinoma, Hepatocellular; Humans; Liver Neoplasms, Experimental; Mice; Mice, Nude; Neovascularization, Pathologic; Proliferating Cell Nuclear Antigen; RNA, Messenger; Sirolimus; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and highly resistant to available chemotherapies. Mammalian target of rapamycin (mTOR) functions to regulate protein translation, angiogenesis and cell cycle progression in many cancers including HCC. In the present study, subcutaneous patient-derived HCC xenografts were used to study the effects of an mTOR inhibitor, RAD001 (everolimus), on tumour growth, apoptosis and angiogenesis. We report that oral administration of RAD001 to mice bearing patient-derived HCC xenografts resulted in a dose-dependent inhibition of tumour growth. RAD001-induced growth suppression was associated with inactivation of downstream targets of mTOR, reduction in VEGF expression and microvessel density, inhibition of cell proliferation, up-regulation of p27(Kip1) and down-regulation of p21(Cip1/Waf1), Cdk-6, Cdk-2, Cdk-4, cdc-25C, cyclin B1 and c-Myc. Our data indicate that the mTOR pathway plays an important role in angiogenesis, cell cycle progression and proliferation of liver cancer cells. Our study provides a strong rationale for clinical investigation of mTOR inhibitor RAD001 in patients with HCC.

Topics: Animals; Apoptosis; Body Weight; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Proliferation; Everolimus; Humans; Liver Neoplasms; Male; Mice; Mice, SCID; Microvessels; Phosphorylation; Protein Kinases; Sirolimus; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

The mammalian target of rapamycin (mTOR) inhibitors play a key role in regulating signal transduction by blocking the mTOR pathway and combining anticancer and immunosuppressive properties. This study was undertaken to determine the prevalence and clinicopathological relevance of phospho-p70S6 (p-p70S6) kinase in hepatocellular carcinoma (HCC) and to investigate the effects of rapamycin on HCC in vitro.. A total of 196 patients with HCCs were treated either with surgical resection (n=106) or liver transplantation (n=90). Tumour tissue was investigated for p-p70S6, phospho-AKT, Ki-67, Cyclin-D1 and apoptosis, and staining results were correlated with clinicopathologically relevant parameters.. Overall, p-p70S6 was detected in 24.5% (48/196) of HCCs. In the resection group, 26.4% (28/106) of HCC were positive and 22.2% (20/90) in the transplant group. p-p70S6 was significantly associated with elevated Cyclin-D1 immunoexpression and was correlated with decreased overall survival (P=0.011) in patients resected with a clear margin. In multivariate COX regression analysis, p-p70S6 was identified as an independent prognostic parameter in patients resected with a clear margin. Rapamycin induced apoptosis and growth inhibition by G0/G1 cell cycle arrest in vitro. However, in HCC patients p-p70S6 kinase was not associated with proliferation or apoptosis.. Activation of p70S6 kinase indicates aggressive tumour behaviour in patients with clear margin-resected HCC. Identification of p-p70S6 kinase in HCC selects high-risk patients who may benefit from drugs targeting the mTOR pathway.

Topics: Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cyclin D1; Enzyme Activation; Female; Humans; Immunohistochemistry; Immunosuppressive Agents; In Situ Nick-End Labeling; Male; Microarray Analysis; Phosphorylation; Prognosis; Regression Analysis; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Survival Analysis

Novel therapeutic strategies are needed to prevent the tumor recurrence or metastasis after liver transplantation for hepatocellular carcinoma (HCC). This study was to investigate the effect of rapamycin, alone and in combination with sorafenib, on HCC in vivo.. Xenograft of a highly metastatic human HCC tumor (LCI-D20) was used to evaluate primary tumor growth and lung metastasis after treatment with rapamycin alone or in combination with sorafenib. Tumor cell proliferation was determined by Ki-67 immunostaining. To detect tumor cell apoptosis, the terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling assay was used. Tumor angiogenesis was investigated by using a monoclonal anti-CD31 antibody. A vascular endothelial growth factor ELISA kit was used to measure vascular endothelial growth factor protein levels in the mice serum.. Rapamycin, alone and in combination with sorafenib, strongly inhibited primary tumor growth and lung metastases in LCI-D20 model. Furthermore, the combination therapy significantly enhanced the effect of antitumor on primary tumor growth compared with single treatment with either rapamycin (P < 0.001) or sorafenib (P < 0.001). Rapamycin alone inhibited HCC cell proliferation, induced apoptosis, and decreased tumor angiogenesis. Nevertheless, the combination therapy showed a significant inhibition of tumor cell proliferation (P < 0.05). Additionally, the combination therapy also further enhanced suppression of tumor cell angiogenesis compared with rapamycin treatment (P < 0.01). However, the induction of apoptosis in combination therapy group was not significantly higher than in the rapamycin-treated group (P > 0.05).. The combination therapy of rapamycin and sorafenib could be a new and promising therapeutic approach to the treatment of HCC and prevention of HCC recurrence after liver transplantation.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzenesulfonates; Blotting, Western; Carcinoma, Hepatocellular; Cell Proliferation; Enzyme-Linked Immunosorbent Assay; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Liver Neoplasms; Male; Mice; Mice, Nude; Neovascularization, Pathologic; Niacinamide; Phenylurea Compounds; Platelet Endothelial Cell Adhesion Molecule-1; Pyridines; Sirolimus; Sorafenib; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Receptor tyrosine kinase inhibitors (RTKIs) and mTOR inhibitors are potential novel anticancer therapies for HCC. We hypothesized that combination targeted on distinctive signal pathways would provide synergistic therapeutics.. ABT-869, a novel RTKI, and rapamycin were investigated in HCC pre-clinical models.. Rapamycin, but not ABT-869, inhibited in vitro growth of Huh7 and SK-HEP-1 HCC cells in a dose dependant manner. However, in subcutaneous Huh7 and SK-HEP-1 xenograft models, either ABT-869 or rapamycin can significantly reduce tumor burden. Combination treatment reduced the tumors to the lowest volume (95+/-20mm(3)), and was significantly better than single agent treatment (p<0.05). Immunohistochemical staining of tumor shows that ABT-869 potently inhibits VEGF in HCC in vivo. In addition, the MAPK signaling pathway has been inhibited by significant inhibition of phosphorylation of p44/42 MAP kinase by ABT-869 in vivo. Rapamycin inhibits phosphorylation of p70 S6 kinase and 4E-BP-1, downstream targets of mTOR, and decreases VEGF. Combination treatment showed synergistic effect on expression levels of p27 in vivo. Dramatic inhibition of neo-angiogenesis by ABT-869 was also demonstrated.. HCC could potentially be treated with the combination treatment of ABT-869 and rapamycin. Clinical trials on combination therapy are warranted.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Movement; Cell Survival; Drug Therapy, Combination; Humans; Immunosuppressive Agents; Indazoles; Liver Neoplasms; Mice; Mice, SCID; Neovascularization, Pathologic; Phenylurea Compounds; Platelet-Derived Growth Factor; Protein Kinases; Receptor Protein-Tyrosine Kinases; Sirolimus; Subcutaneous Fat; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Sirolimus (SRL) acts as a primary immunosuppressant or antitumor agent. The aim of the present study was to evaluate the influence of SRL on the recurrence rate and survival of patients after orthotopic liver transplantation (OLT) for hepatocellular carcinoma (HCC) exceeding the Milan criteria.. We retrospectively examined 73 consecutive patients who underwent OLT for HCC exceeding the Milan criteria from March 2004 through December 2005. Among them, 27 patients were treated with SRL-based immunosuppressive protocols after OLT, and 46 patients by an FK506-based protocol. Statistical analysis was based on the intent-to-treat method.. The 2 groups were comparable in all clinicopathologic parameters. The mean overall survival was 594 +/- 35 days in the SRL group and 480 +/- 42 days in the FK506 group (P = .011); the mean disease-free survival period was 519 +/- 43 days in the SRL group and 477 +/- 48 days in the FK506 group (P = .234). Multivariate analysis revealed Child's status (P = .004) and immunosuppressive protocol (P = .015) were the significant factors affecting overall survival. Only microvascular invasion (P = .004) was significantly associated with disease-free survival. Among 24 surviving patient in the SRL group, 2 patients had SRL discontinued for toxicity; 10 had SRL monotherapy immunosuppression.. The SRL-based immunosuppressive protocol improved the overall survival of patients after OLT for HCC exceeding the Milan criteria, probably by postponing recurrence and with better tolerability.

Topics: Adolescent; Adult; Carcinoma, Hepatocellular; Disease-Free Survival; Female; Follow-Up Studies; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Male; Neoplasm Invasiveness; Neoplasm Metastasis; Patient Selection; Retrospective Studies; Sirolimus; Survival Analysis; Survivors; Tacrolimus

Although continuous improvements have been made in fighting rejection with immunosuppressive drugs in transplant recipients, this success story has been tempered by an associated high incidence of cancer. The latest projections are that cancer might exceed cardiovascular disease as the leading cause of death among transplant recipients. Indeed, immunosuppression reduces our natural ability to destroy cancer cells and to inhibit viral infections potentially linked to cancer development. Therefore, a strategy to counter the problem of cancer in transplantation is needed. Recently, mammalian target of rapamycin (mTOR) inhibitors have demonstrated potential as both immunosuppressive and anticancer agents. Although mTOR inhibitors prevent organ transplant rejection, this class of drugs has potential anticancer properties that may be useful in the "balance of effects" toward cancer-free survival in transplant recipients. Mechanisms of mTOR inhibitors' anticancer effects are multiple, affecting processes including angiogenesis, cell proliferation, cell survival, and molecular oncogenic signaling. Importantly, experimental work supports the view that tumor inhibition can be accomplished with mTOR inhibitors while protecting allografts against rejection. Most recently, prospective randomized clinical studies have been initiated to test the concept that mTOR inhibitors reduce cancer while simultaneously inhibiting allograft rejection. One such study, the SiLVER trial, examines hepatocellular carcinoma recurrence in mTOR inhibitor-treated liver transplant patients. More robust evidence as to whether cancer risk can be reduced in transplant recipients with mTOR inhibitors may come from such clinical trials.

Topics: Angiogenesis Inhibitors; Antineoplastic Agents; Carcinoma, Hepatocellular; Graft Rejection; Humans; Immunosuppression Therapy; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Neoplasm Recurrence, Local; Protein Kinases; Randomized Controlled Trials as Topic; Sirolimus; TOR Serine-Threonine Kinases

Topics: Carcinoma, Hepatocellular; Follow-Up Studies; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Neoplasm Recurrence, Local; Sirolimus; Treatment Outcome

The proinflammatory cytokine interleukin (IL)-6 has been proposed to be one of the mediators that link obesity-derived chronic inflammation with insulin resistance. Signaling through the mammalian target of rapamycin (mTOR) has been found to impact insulin sensitivity under various pathological conditions, through serine phosphorylation and inhibition of insulin receptor substrate by the downstream effector of mTOR, ribosomal S6 kinase 1 (S6K1). However, an involvement of mTOR in IL-6-induced insulin resistance has not yet been reported. Here we show that rapamycin, the inhibitor of mTOR signaling, rescues insulin signaling and glycogen synthesis from IL-6 inhibition in HepG2 hepatocarcinoma cells as well as in mouse primary hepatocytes. IL-6 activates S6K1 in these cells, but unexpectedly, S6K1 is not involved in IL-6 inhibition of insulin signaling, since the effect of IL-6 persists in cells with drastically reduced S6K1 levels induced by RNA interference, suggesting that the function of mTOR signaling is through a mechanism different from the prevailing model of S6K1 phosphorylation of insulin receptor substrate-1. Interestingly, we find that the phosphorylation of STAT3 on Ser(727) and STAT3 transcriptional activity are regulated by mTOR upon IL-6 stimulation and that STAT3 is required for IL-6 inhibition of insulin signaling. Furthermore, IL-6-induced SOCS3 expression is inhibited by rapamycin, and ectopic expression of SOCS3 blocks the ability of rapamycin to enhance insulin sensitivity in the presence of IL-6. Taken together, we propose that mTOR plays a key role in IL-6-induced hepatic insulin resistance by regulating STAT3 activation and subsequent SOCS3 expression.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; DNA Primers; Genes, Reporter; Homeostasis; Humans; Insulin; Insulin Resistance; Interleukin-6; Lentivirus; Liver Neoplasms; Luciferases; Protein Kinases; Recombinant Proteins; Ribosomal Protein S6 Kinases; RNA Interference; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with an annual occurrence of one million new cases. At present there is no effective treatment for HCC individuals that not amenable to curative therapies. Recent studies show the PI3K/Akt/mTOR signal pathway is involved in multiple cellular functions including proliferation, differentiation, tumorigenesis, and apoptosis. Rapamycin (a specific Mtor inhibitor) could lead to G(1) arrest of many malignant cell lines, and currently analogs of rapamycin are being investigated as a cancer chemotherapeutic adjuvant. This study investigated rapamycin and chemotherapeutic agent 5-fluorouracil (5-Fu) in combination treatment induced apoptosis and cell senescence in hepatocarcinoma cell line SMMC-7721 cells. Treating SMMC-7721 cells with rapamycin plus 5-Fu led to not only apoptosis but also cell senescence, and the senescent cells exhibited significantly less clonogenic potential than 5-Fu individually treated cells. Further study showed rapamycin plus 5-Fu-induced senescence-like growth arrest was accompanied by down-regulation of AP-1 and NF kappa B transcription activity. These results suggest that inhibitors of mTOR may have anticancer potential when used together with some other chemotherapeutic agents, and that down-regulation of AP-1 and NF kappa B transcription activity might take part in a senescence-like growth arrest program induced by rapamycin plus 5-Fu.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cellular Senescence; Fluorouracil; Humans; Liver Neoplasms; Protein Kinases; Sirolimus; TOR Serine-Threonine Kinases

Rapamycin is a potent new immunosuppressant with a mechanism of action that is distinct from that of calcineurin inhibitors, but few clinical data on rapamycin in liver transplantation are available. Hence it is necessary to evaluate the efficacy and side-effects of rapamycin-based immunosuppression in liver transplant patients.. We retrospectively analysed 39 liver transplantation patients who took rapamycin as an immunosuppressant. This series consisted of 28 patients with hepatocellular carcinoma, 9 patients with chronic fulminant hepatitis, and 2 patients with end-stage liver cirrhosis. Eight patients used rapamycin for monotherapy, and 31 used rapamycin-based immunosuppression. In the 31 patients, 7 patients used rapamycin instead of mycophenolate mofetil to treat acute rejection.. In the 28 patients with hepatocellular carcinoma, the one-year survival rate was 67% without any tumor recurrence. The acute rejection in 7 patients was relieved in 1-2 weeks after the administration of rapamycin. All the 8 patients who received rapamycin monotherapy survived for at least 6 months and liver function tests and biopsy showed nothing abnormal. Jaundice in 8 patients with chronic rejection was reduced sharply after use of rapamycin.. Rapamycin given alone or in conjunction with calcineurin inhibitors appears to be an effective primary immunosuppressant regimen for orthotopic liver transplantation patients. Further studies are warranted to evaluate the efficacy and side-effect profile of rapamycin in liver transplant patients.

Topics: Acute Disease; Adult; Carcinoma, Hepatocellular; Female; Graft Rejection; Humans; Immunosuppressive Agents; Liver Failure, Acute; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Retrospective Studies; Sirolimus; Treatment Outcome

Experience with sirolimus (SRL)-based immunosuppression following orthotopic liver transplantation (OLT) is rapidly accumulating. In combination with calcineurin inhibitors (CNIs), SRL may reduce the incidence of acute rejection and lower overall required drug levels. This study sought to quantify long-term outcome following OLT in patients with cirrhosis and concomitant hepatocellular carcinoma (HCC) who were treated with an SRL-based regimen as a primary therapy. From January 2000 to June 2007, 97 patients underwent OLT for end-stage liver disease and HCC at the University of Colorado Health Sciences Center. Of those, 45 patients received SRL, in addition to CNIs, as a component of their primary immunosuppression regimen post-OLT. Conversely, 52 patients received the standard immunosuppression regimen including CNIs, mycophenolate mofetil, and corticosteroids. The 2 treatment groups were compared with respect to the following variables: age, gender, tumor stage by explant, grade, size, presence of vascular invasion, focality, Child's class, baseline creatinine, and warm and cold ischemic times. The 2 groups were comparable by all factors save for cold ischemic time, which was significantly longer in the CNI-treated group. Overall survival at 1 and 5 years post-OLT for patients treated with SRL was 95.5% and 78.8%, respectively. Conversely, survival in patients treated with CNIs exclusively at the same time intervals was 83% and 62%. Although there was no difference in the incidence of major complications, the SRL group experienced a modest improvement in renal function. Cumulatively, these data suggest a potential survival benefit with SRL-based therapy in patients undergoing OLT for end-stage liver disease and concomitant malignancy.

Topics: Adrenal Cortex Hormones; Calcineurin Inhibitors; Carcinoma, Hepatocellular; Cyclosporine; Drug Therapy, Combination; Female; Graft Rejection; Humans; Immunosuppressive Agents; Kaplan-Meier Estimate; Kidney Diseases; Liver Failure; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Mycophenolic Acid; Neoplasm Recurrence, Local; Proportional Hazards Models; Retrospective Studies; Risk Assessment; Sirolimus; Tacrolimus; Time Factors; Treatment Outcome

Topics: Antibiotics, Antineoplastic; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Division; Cellular Senescence; Fluorouracil; Liver Neoplasms; Protein Kinases; Sirolimus; TOR Serine-Threonine Kinases

Hepatocellular carcinoma (HCC) is resistant to chemotherapy. We reported that sirolimus, an mTOR inhibitor, has antiangiogenic properties in HCC. Since antiangiogenic therapy may enhance chemotherapy effects, we tested the antitumorigenic properties of sirolimus combined with doxorubicin in experimental HCC.. Morris Hepatoma (MH) cells were implanted into livers of syngeneic rats. Animals were assigned to sirolimus, pegylated liposomal doxorubicin, both combined or control groups. Tumoral growth was followed by MRI. Antiangiogenic effects were assessed by CD31 immunostaining and capillary tube formation assays. Cell proliferation was monitored in vitro by thymidine incorporation. Expression of p21 and phosphorylated MAPKAP kinase-2 was quantified by immunoblotting.. Animals treated with the combination developed smaller tumors with decreased tumor microvessel density compared to animals that received monotherapies. In vitro, inhibition of mTOR further impaired capillary formation in the presence of doxorubicin. Doxorubicin reduced endothelial cell proliferation; inhibition of mTOR accentuated this effect. Doxorubicin stimulated p21 expression and the phosphorylation of MAPKAP kinase-2 in endothelial cells. Addition of mTOR inhibitor down-regulated p21, but did not decrease MAPKAP kinase-2 phosphorylation.. Sirolimus has additive antitumoral and antiangiogenic effects when administered with doxorubicin. These findings offer a rationale for combining mTOR inhibitors with chemotherapy in HCC treatment.

Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Aorta; Apoptosis; Carcinoma, Hepatocellular; Cells, Cultured; Disease Models, Animal; Doxorubicin; Endothelial Cells; Intracellular Signaling Peptides and Proteins; Liver Neoplasms, Experimental; Male; Neoplasm Transplantation; Oncogene Protein p21(ras); Phosphorylation; Polyethylene Glycols; Protein Serine-Threonine Kinases; Rats; Rats, Inbred ACI; Sirolimus; Transcription Factors

Hepatocellular carcinoma is a leading cause of global cancer mortality, with standard chemotherapy being minimally effective in prolonging survival. We investigated if combined targeting of vascular endothelial growth factor protein and expression might affect hepatocellular carcinoma growth and angiogenesis.. We treated patient-derived hepatocellular carcinoma xenografts with (i) bevacizumab; (ii) rapamycin; and (iii) bevacizumab plus rapamycin. Western blotting was employed to determine changes in the proteins. Apoptosis, vascular endothelial growth factor expression, microvessel density, and cell proliferation were analyzed by immunohistochemistry.. Hepatocellular carcinoma growth was inhibited by bevacizumab plus rapamycin treatment to a significantly greater degree than bevacizumab or rapamycin monotherapy. Reductions in tumor growth by bevacizumab plus rapamycin were associated with inhibition of downstream targets of the mammalian target-of-rapamycin pathway, reductions in vascular endothelial growth factor expression, and tumor microvessel density. Potentially additive effects of bevacizumab plus rapamycin included reductions in vascular endothelial growth factor expression, cyclin D1, and cyclin B1. In an intra-peritoneal model of hepatocellular carcinoma, bevacizumab plus rapamycin potently inhibited both intra-liver and intra-abdominal tumor growth, reduced ascites levels, and significantly prolonged mouse survival.. Bevacizumab and rapamycin, which are both clinically approved drugs, may represent a novel molecularly-targeted combination treatment for hepatocellular carcinoma.

Topics: Angiogenesis Inhibitors; Animals; Antibiotics, Antineoplastic; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Bevacizumab; Carcinoma, Hepatocellular; Disease Models, Animal; Drug Therapy, Combination; Liver Neoplasms; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, SCID; Multiprotein Complexes; Proteins; Signal Transduction; Sirolimus; Survival Rate; TOR Serine-Threonine Kinases; Transcription Factors; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

This paper illustrates the use of the chick embryo chorioallantoic membrane (CAM) assay to determine the single and combined antiangiogenic effects of very low doses of vinblastine (VBL) and rapamycin (RAP) in human hepatocellular carcinoma (HCC). The angiogenic response induced by human HCC biopsy specimens was inhibited by each drug and sinergistically by their combination. Morever, immunohistochemical detection of microvessels with anti-CD31 mAB showed that their area was significantly lower in specimens treated with VBL and RAP in combination. Sinergy on the part of these well-known drugs when used in combination as antiangiogenics at very low doses may be of significance in the designing of new ways of treating HCC.

Topics: Angiogenesis Inhibitors; Animals; Carcinoma, Hepatocellular; Chick Embryo; Chorioallantoic Membrane; Fibroblast Growth Factor 2; Humans; Image Processing, Computer-Assisted; Liver Neoplasms; Microcirculation; Neovascularization, Pathologic; Sirolimus; Vinblastine

Cellular mechanisms that regulate the replication of hepatitis C virus (HCV) RNA are poorly understood. p21-activated kinase 1 (PAK1) is a serine/threonine kinase that has been suggested to participate in antiviral signaling. We studied its role in the cellular control of HCV replication. Transfection of PAK1-specific small interfering RNA enhanced viral RNA and protein abundance in established replicon cell lines as well as cells infected with chimeric genotype 1a/2a HCV, despite reducing cellular proliferation, suggesting specific regulation of HCV replication. PAK1 knockdown did not reduce interferon regulatory factor 3-dependent gene expression, indicating that this regulation is independent of the retinoic acid-inducible gene I/interferon regulatory factor 3 pathway. On the other hand, LY294002 and rapamycin abolished PAK1 phosphorylation and enhanced HCV abundance, suggesting that the mammalian target of rapamycin (mTOR) is involved in PAK1 regulation of HCV. Small interfering RNA knockdown of the mTOR substrate p70 S6 kinase abrogated PAK1 phosphorylation and enhanced HCV RNA abundance, whereas overexpression of a constitutively active alternate substrate, eukaryotic translation initiation factor 4E-binding protein 1, increased cap-independent viral translation and viral RNA abundance without influencing PAK1 phosphorylation. Similar data indicated that mTOR is regulated by both phosphatidylinositol 3-kinase/Akt and ERK. Taken together, the data indicate that p70 S6 kinase activates PAK1 and contributes to phosphatidylinositol 3-kinase- and ERK-mediated regulation of HCV RNA replication.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Chromones; Extracellular Signal-Regulated MAP Kinases; Hepacivirus; Humans; Models, Biological; Morpholines; p21-Activated Kinases; Phosphatidylinositol 3-Kinases; Promoter Regions, Genetic; Protein Kinases; Protein Serine-Threonine Kinases; Ribosomal Protein S6 Kinases; Sirolimus; TOR Serine-Threonine Kinases; Virus Replication

Hepatocellular carcinoma (HCC) is amenable to only few treatments. Inhibitors of the kinase mTOR are a new class of immunosuppressors already in use after liver transplantation. Their antiproliferative and antiangiogenic properties suggest that these drugs could be considered to treat HCC. We investigated the antitumoral effects of mTOR inhibition in a HCC model.. Hepatoma cells were implanted into livers of syngeneic rats. Animals were treated with the mTOR inhibitor sirolimus for 4 weeks. Tumor growth was monitored by MR imaging. Antiangiogenic effects were assessed in vivo by microvessel density and corrosion casts and in vitro by cell proliferation, tube formation and aortic ring assays.. Treated rats had significantly longer survival and developed smaller tumors, fewer extrahepatic metastases and less ascites than controls. Sirolimus decreased intratumoral microvessel density resulting in extensive necrosis. Endothelial cell proliferation was inhibited at lower drug concentrations than hepatoma cells. Tube formation and vascular sprouting of aortic rings were significantly impaired by mTOR inhibition. Casts revealed that in tumors treated with sirolimus vascular sprouting was absent, whereas intussusception was observed.. mTOR inhibition significantly reduces HCC growth and improves survival primarily via antiangiogenic effects. Inhibitors of mTOR may have a role in HCC treatment.

Topics: Angiogenesis Inhibitors; Animals; Antibiotics, Antineoplastic; Capillaries; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Enzyme Inhibitors; Liver Neoplasms, Experimental; Neoplasm Transplantation; Neovascularization, Pathologic; Phosphorylation; Protein Kinases; Rats; Regional Blood Flow; Sirolimus; TOR Serine-Threonine Kinases

Rapamycin, isolated from Streptomyces hygroscopicus, is recently reported to have immunosuppressant and anti-tumor effects on a large variety of cancers. To date, no detailed data are available about the effects of rapamycin on hepatocellular carcinoma cells.. In this study, the anti-proliferation effects of rapamycin on hepatocellular carcinoma cells BEL-7402 and HepG-2 in vitro were studied.. Cell viability was assessed by MTT assay and [3H]-thymidine uptake, cell apoptosis was observed by Hoechst 33258 staining and flow cytometry (FCM). The variation of caspase-3 and apoptotic related genes was assayed by Western blotting, cell mitochondrial membrane potential was also investigated by using standard methods.. Rapamycin could inhibit the growth of hepatocellular carcinoma cells and cause apoptosis significantly; the suppression was both in time- and dose-dependent manner, marked morphological changes of cell apoptosis were observed very clearly by Hoechst 33258 staining. Rapamycin exhibits induction apoptosis by activation of caspase-3 and disruption of the mitochondrial membrane potential on hepatocellular carcinoma cells in vitro. Western blotting analysis demonstrated that anti-apoptotic protein Bcl-2 was down-regulated while pro-apoptotic protein Bcl-xl up-regulated remarkably in a time-dependent manner when apoptosis occurred.. Rapamycin has significant anti-proliferation effect by induction of apoptosis via activation of caspase-3 and disruption of mitochondrial membrane potential, as well as by down-regulation of anti-apoptotic protein Bcl-2 and up-regulation of pro-apoptotic protein Bcl-xl on hepatocellular carcinoma cells. The data provide a potential mechanism for rapamycin-induced apoptosis in hepatocellular carcinoma cells, suggesting that rapamycin may serve as both an effective adjunctive reagent for the treatment of residual cancer cells and immunosuppressant after liver transplantation of hepatocellular carcinoma, and that in vivo anti-cancer effects as well as its potential clinical effectiveness need further investigation.

Topics: Apoptosis; bcl-X Protein; Blotting, Western; Carcinoma, Hepatocellular; Caspase 3; Cell Line, Tumor; Cell Proliferation; Flow Cytometry; Humans; Immunosuppressive Agents; Liver Neoplasms; Membrane Potential, Mitochondrial; Proto-Oncogene Proteins c-bcl-2; Sirolimus

The mammalian target of rapamycin (mTOR) pathway, a major regulator of translation, is frequently activated in hepatocellular carcinomas. We investigated the effects of mTOR activation in the human HepaRG cells, which possess potent hepatocytic differentiation capability. Differentiation of HepaRG cells into functional and polarized hepatocyte-like cells correlated with a decrease in mTOR and Akt activities. Stable cell lines expressing an activated mutant of mTOR were generated. Sustained activation of mTOR impaired the hepatocytic differentiation capability of these cells as shown by impaired formation of bile canaliculi, absence of polarity, and reduced secretion of alpha1-antitrypsin. An inhibitor of mTOR, rapamycin, was able to revert this phenotype. Furthermore, increased mTOR activity in HepaRG cells resulted in their resistance to the antiproliferative effects of transforming growth factor-beta1. Profiling of polysome-bound transcripts indicated that activated mTOR specifically targeted genes posttranscriptionally regulated on hepatocytic differentiation. Three major biological networks targeted by activated mTOR were identified: (a) cell death associated with tumor necrosis factor superfamily members, IFNs and caspases; (b) lipid homeostasis associated with the transcription factors PPARalpha, PPARdelta, and retinoid X receptor beta; and (c) liver development associated with CCAAT/enhancer binding protein alpha and hepatic mitogens. In conclusion, increased mTOR activity conferred a preneoplastic phenotype to the HepaRG cells by altering the translation of genes vital for establishing normal hepatic energy homeostasis and moderating hepatocellular growth.

Topics: Carcinoma, Hepatocellular; Cell Differentiation; Cell Growth Processes; Cell Line; Down-Regulation; Hepatocytes; Homeostasis; Humans; Lipid Metabolism; Liver Neoplasms; Polyribosomes; Protein Kinases; Proto-Oncogene Proteins c-akt; RNA; Sirolimus; TOR Serine-Threonine Kinases; Transfection

We report long-term outcomes and side effects after transplantation for hepatocellular carcinoma (HCC) using de novo, sirolimus-based immunosuppression (IS).. A total of 70 patients with HCC (mean age: 54.4+/-7 years, female/male: 12/58) were transplanted and included in the study. Immunosuppression included de novo sirolimus, low-dose calcineurin inhibitor for 6 to 12 months, with short-course (3 months) or no steroids.. After 49 months-median follow-up, eight patients have experienced an HCC recurrence, 2 of 34 when Milan criteria were respected (6%) and 6 of 36 when beyond Milan criteria (17%). One- and 4-year tumor-free survivals were 85 and 73%, when Milan criteria were respected and 82% and 75% when they were not, respectively. (P=0.9). After recurrence, mean survival was 23+/-28 months. Half (35 of 70) of the patients experienced a rejection. Incisional hernia (24 of 70, 34%), wound infection (12 of 70, 17%), anemia (39 of 70, 56%), leucopenia (39 of 70, 56%), high triglyceride (43 of 70, 61%), and cholesterol (28 of 70, 40%) levels and mouth ulcers (20 of 70, 29%) were among the most frequent complications. No hepatic artery thrombosis was observed.. These data suggest that de novo sirolimus-based immunosuppression is associated with satisfactory outcomes after transplantation, even in selected patients beyond Milan criteria. The protocol has proven safe, with an acceptable side-effect profile. This study supports the conduct of larger randomized trials investigating sirolimus after transplantation for HCC.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Dose-Response Relationship, Drug; Female; Graft Rejection; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Longitudinal Studies; Male; Middle Aged; Neoplasm Recurrence, Local; Patient Selection; Pilot Projects; Sirolimus; Survival Analysis; Treatment Outcome

Orthotopic liver transplantation (OLT) is increasingly being applied for cure in patients with cirrhosis and concomitant hepatocellular carcinoma (HCC). In recipients with limited tumor burden, OLT achieves reasonable long-term outcome. This study sought to identify clinical and pathologic variables predictive of long-term disease-free survival and the presence of vascular invasion. From 1992 to 2006, 130 patients underwent OLT for cirrhosis and HCC. Malignancy was diagnosed in 107 patients prior to OLT and in 23 patients on pathologic examination of the explant. Nine clinical and pathologic variables were considered including: TNM stage, nodularity, vascular invasion, Milan criteria, incidental lesion, differentiation, tumor size, preOLT transarterial chemoembolization (TACE), and administration of sirolimus-based immunosuppression. The overall incidence of HCC recurrence was 17% with the majority (82%) being stage III. Cumulatively, tumor recurrence-free survival (RFS) is 84, 74, and 67% at 1, 3, and 5 years respectively. Independent predictors of RFS included stage III and poorly differentiated lesions (P<0.05). Furthermore, stage III tumors and those >3.5 cm in size were predictive of vascular invasion. Importantly, preOLT, TACE and postOLT sirolimus had no influence on survival. Pathologic variables including tumor stage and grade have a significant impact on outcome. Importantly, it seems that TACE and sirolimus had no beneficial effect.

Topics: Adult; Aged; Blood Vessels; Carcinoma, Hepatocellular; Chemoembolization, Therapeutic; Female; Humans; Immunosuppressive Agents; Incidence; Liver; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Neoplasm Staging; Preoperative Care; Sirolimus; Survival Analysis; Treatment Outcome

The phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway is one of the major oncogenic pathways and is activated in many types of human cancers, including hepatocellular carcinoma. It can also be activated by the hepatitis C virus (HCV) nonstructural 5A (NS5A) protein. In the present study, we set out to determine the regulatory effects of this pathway on the replication of hepatitis B virus (HBV). Our results demonstrate that the expression of a constitutively active Akt1 profoundly inhibited HBV RNA transcription and consequently reduced HBV DNA replication in HepG2 cells. This suppression of HBV gene transcription was apparently mediated by the activation of mTOR, as it was abolished by the mTOR inhibitor rapamycin. Moreover, treatment of HBV-expressing HepG2.2.15 cells with inhibitors of PI3K, Akt, and mTOR increased the transcription of 3.5-kb and 2.4-kb viral RNA as well as the replication of HBV DNA. This observation implies that the basal level activation of this pathway in HepG2 cells regulated HBV replication. Consistent with previous reports showing that the HCV NS5A protein could bind to the p85 subunit of PI3K and activate the PI3K-Akt signal transduction pathway, our results showed that expression of this protein could inhibit HBV RNA transcription and reduce HBV DNA replication in HepG2 cells. Taken together, our results suggest that the activation of the PI3K-Akt pathway during liver oncogenesis may be at least partially responsible for the elimination of HBV replication from tumor cells and may also provide an explanation for the observed suppression of HBV replication by HCV coinfection.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Transformation, Viral; DNA Replication; DNA, Viral; Hepacivirus; Hepatitis B; Hepatitis B virus; Hepatitis C; Humans; Immunosuppressive Agents; Phosphatidylinositol 3-Kinases; Protein Kinases; Proto-Oncogene Proteins c-akt; RNA, Viral; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription, Genetic; Viral Nonstructural Proteins; Virus Replication

In this study, we demonstrate that interleukin-4 (IL-4) protects human hepatocellular carcinoma (HCC) cell line Hep3B from apoptosis induced by transforming growth factor-beta (TGF-beta). Further investigation of IL-4-transduced signaling pathways revealed that both insulin response substrate 1 and 2 (IRS-1/-2) and extracellular signal-regulated kinase (ERK) pathways were activated after IL-4 stimulation. The IRS-1/-2 activation was accompanied by the activation of phosphotidylinositol-3-kinase (PI3K), leading to Akt and p70 ribosomal protein S6 kinase (p70S6K). Interestingly, a protein kinase C (PKC) inhibitor, Gö6976, inhibited the phosphorylation of Akt, suggesting that the Akt activation was PKC-dependent. Using specific inhibitors for PI3K or ERK, we demonstrated that the PI3K pathway, but not the ERK pathway, was required for protection. The constitutively active form of PI3K almost completely rescued TGF-beta-induced apoptosis, further supporting the importance of the PI3K pathway in the protective effect of IL-4. Furthermore, a dominant negative Akt and/or Gö6976 only partially blocked the anti-apoptotic effect of IL-4. Similarly, rapamycin, which interrupted the activation of p70S6K, also only partially blocked the protective effect of IL-4. However, in the presence of both rapamycin and dominant negative Akt with or without Gö6976, IL-4 almost completely lost the anti-apoptotic effect, suggesting that both Akt and p70S6K pathways were required for the protective effect of IL-4 against TGF-beta-induced apoptosis.

Topics: Apoptosis; bcl-Associated Death Protein; Carbazoles; Carcinoma, Hepatocellular; Cell Line, Tumor; Humans; Indoles; Insulin Receptor Substrate Proteins; Interleukin-4; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Phosphatidylinositol 3-Kinases; Phosphoproteins; Protein Kinase C; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Transforming Growth Factor beta

Hepatocellular carcinoma (HCC) is the most invasive and frequently diagnosed malignancy and the second leading cause of cancer death in many regions of Asia. The PI3K/Akt/mTOR signal pathway is involved in multiple cellular functions including proliferation, differentiation, tumorigenesis, and apoptosis. Up-regulation of telomerase activity is thought to be a critical step leading to cell transformation.. This study investigated changes in mTOR pathway and telomerase activity in hepatocarcinoma cell line SMMC-7721 treated with chemotherapeutic agent 5-fluorouracil (5-Fu). We detected apoptosis of hepatocarcinoma cells by TUNEL assay. Telomerase activity, hTERT transcription level and p- p70 S6k was demonstrated by the telomeric repeat amplification protocol and silver staining assay, Dual-Luciferase Reporter Assay and Western blot analysis respectively.. Treating SMMC-7721 cells with 5-Fu leads to apoptosis of the cells, and reduction in telomerase activity, as well as a dramatic reduction in the activated form of p70 S6 kinase, a mTOR substrate. The 5-Fu treatment nearly abolishes transcription of hTERT (the major component of telomerase) mRNA. Treating SMMC-7721 cells with Rapamycin, a specific mTOR inhibitor, significantly reduce hTERT protein level but did not affect hTERT transcription. 5-Fu and rapamycin were synergistic in regards to down-regulation of telomerase activity in hepatocarcinoma cells.. These results suggest that chemotherapeutic agent 5-Fu may down-regulate telomerase activity at both transcriptional level and PI3K/Akt/mTOR pathway-dependent post-transcriptional level to facilitate hepatocellular carcinoma cell apoptosis.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Down-Regulation; Fluorouracil; Gene Expression Regulation, Neoplastic; Humans; In Situ Nick-End Labeling; Liver Neoplasms; Protein Kinases; Signal Transduction; Sirolimus; Telomerase; TOR Serine-Threonine Kinases; Transcription, Genetic

To report a retrospective analysis of preliminary results of 36 patients who received sirolimus (SRL, Rapamune, rapamycin) in a consecutive cohort of 248 liver allograft recipients.. Thirty-six liver transplant patients with hepatocellular carcinoma (HCC) who were switched to SRL-based immunosuppression therapy from tacrolimus were enrolled in this study. The patients who were diagnosed as advanced HCC before orthotopic liver transplantation (OLT) were divided into group A (n = 11), those who were found to have HCC recurrence and/or metastasis after OLT were assigned to group B (n = 18), and those who developed renal insufficiency caused by calcineurin inhibitor (CNI) were assigned to group C (n = 7) after OLT.. The patients were followed up for a median of 10.4 mo (range, 3.8-19.1 mo) after conversion to SRL therapy and 12.3 mo (range, 5.1-34.4 mo) after OLT. Three patients developed mild acute cellular rejection 2 wk after initiating SRL therapy, which was fully reversed after prednisolone pulse therapy. In group A, only 1 patient was found to have HCC recurrence and metastasis 12 mo after OLT. In group B, 66.7% (12/18) patients (2 with progressive tumor, 7 with stable tumor and 3 without tumor) were still alive due to conversing to SRL and/or resection for HCC recurrence at the end of a median follow-up of 6.8 mo post conversion and 10.7 mo posttransplant. In group C, no HCC recurrence was demonstrated in 7 patients, and renal function became normal after SRL therapy. Thrombocytopenia (n = 2), anemia (n = 8), and oral aphthous ulcers (n = 7) found in our cohort were easily manageable.. The conversion to SRL-based immunosuppression may inhibit the recurrence and metastasis of HCC and improve CNI-induced renal insufficiency in OLT patients with HCC.

Topics: Adaptor Proteins, Signal Transducing; Adult; Calcineurin; Carcinoma, Hepatocellular; Female; Humans; Immunosuppressive Agents; Incidence; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Neoplasm Metastasis; Neoplasm Recurrence, Local; Phosphoproteins; Renal Insufficiency; Retrospective Studies; Sirolimus; Tacrolimus; Transplantation; Transplantation, Homologous

Green tea extract and its major component (-)-epigallocatechin-3-gallate (EGCG) exhibit antiangiogenic activities in various experimental tumor models. A growing body of evidence has established that hypoxia-inducible factor-1alpha (HIF-1alpha) and its downstream target, vascular endothelial growth factor (VEGF), play a critical role in tumor angiogenesis. In this study, we investigated the effect of green tea extract and EGCG on HIF-1alpha and VEGF expression in human cervical carcinoma (HeLa) and hepatoma (HepG2) cells. Our results showed that green tea extract and EGCG significantly inhibited hypoxia- and serum-induced HIF-1alpha protein accumulation in these cancer cells but had no effects on HIF-1alpha mRNA expression. Suppression of HIF-1alpha protein by green tea extract and EGCG also resulted in a drastic decrease in VEGF expression at both mRNA and protein levels. The mechanisms of green tea extract and EGCG inhibition of hypoxia-induced HIF-1alpha protein accumulation seem to involve the blocking of both phosphatidylinositol 3-kinase/Akt and extracellular signal-regulated kinase 1/2 signaling pathways and the enhancing of HIF-1alpha protein degradation through the proteasome system. In addition, green tea extract and EGCG inhibited serum-induced HIF-1alpha protein and VEGF expression by interfering with the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling pathways, which play a crucial role in the protein translational machinery cascade. Functionally, green tea extract and EGCG abolished both chemoattractant- and hypoxia-stimulated HeLa cell migration. Our data suggested that HIF-1alpha/VEGF function as therapeutic target for green tea extract and EGCG in the context of cancer chemoprevention and anticancer therapy.

Topics: Angiogenesis Inhibitors; Anticarcinogenic Agents; Carcinoma; Carcinoma, Hepatocellular; Catechin; Cell Hypoxia; Culture Media; Extracellular Signal-Regulated MAP Kinases; Female; HeLa Cells; Humans; Hypoxia-Inducible Factor 1; Liver Neoplasms; Phosphatidylinositol 3-Kinases; Plant Extracts; RNA, Messenger; Sirolimus; Uterine Cervical Neoplasms; Vascular Endothelial Growth Factor A

To investigate the effects of rapamycin (RPM) in inhibiting the growth and metastasis of hepatocellular carcinoma (HCC).. Human HCC cells of the line MHCC97H with a high potential of metastasis were divided into 3 groups to be cultured with cyclosporine A (CsA) 100 ng/ml, RPM 10 ng/ml, or CsA + RPM for 48 hours. Flow cytometry was used to examine the apoptosis and cell cycle MTT method was used to examine the effect of RMP on the proliferation of the MHCC97H cells. RT-PCR was used to detect the mRNA expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hypoxia-inducible factor-1alpha (HIF-1alpha), and transforming growth factor b (TGFb). Another MHCC97H cells were cultured in complete medium without RPM for 48 hours, then the protein expression of VEGF in the supernatant was detected by ELISA. Twenty-eight nude LCI-D20 mice were inoculated with human HCC cells and then divided into 4 groups to be fed with CsA (25 mg/kg), RPM (2 mg/kg), CsA + RPM, and normal saline (0.2 ml, as control group) for 35 days. Then the mice were killed to take the weight of inoculated tumor, measure the blood drug concentration, calculate the lung metastasis rate and number of metastatic foci, and observe pathology of the lung.. CsA showed no effect on the cycle of the MHCC97H cells. The MHCC97H cells of the RPM and CsA + RPM groups arrested at the stage G(0)/G(1) (both P = 0.000). MMT method also showed that the proliferation of the MHCC97H cells in the RPM and CsA + RPM groups were blocked (P = 0.003 and P = 0.002). However, CsA did not influence the proliferation of the MHCC97H cells. Flow cytometry showed that RPM did not promote the apoptosis of the MHCC97H cells. RT-PCR showed that RPM down-regulated the mRNA expression of VEGF and HIF-1alpha (both P < 0.05), however, did not influence the mRNA expression of bFGF, TGFb, and TGFb. The VEGF protein level in the supernatant of the culture fluid of MHCC97H cells of the RPM group was (890.3 +/- 25.1) pg/ml, significantly lower than that of the control group, (1583.7 +/- 17.3) pg/ml (P = 0.000). The tumor inhibiting rate of the RPM group was 63.7%, not significantly different from that of the RPM + CsA group (80.9%, P = 1.000). The metastatic rate of the CsA and control groups were both 100% with a higher number of metastatic tumors in the CsA group (P = 0.046).. RPM significantly inhibits the growth and metastasis of HCC. RPM-based immunosuppressive regimen may be of value in HCC patients receiving liver transplantation.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Enzyme-Linked Immunosorbent Assay; Fibroblast Growth Factor 2; Flow Cytometry; Humans; Immunosuppressive Agents; Liver Neoplasms, Experimental; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sirolimus; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Rapamycin, isolated from Streptomyces hygroscopicus, is recently reported to have immunosuppressive and antitumor effects on a large variety of cancers. This study was to investigate the role of Caspase-3 in rapamycin-induced apoptosis of hepatocellular carcinoma BEL-7402 cells.. BEL-7402 cells were treated with different concentrations (5, 10, 20, 30, 40, 50 nmol/L) of rapamycin. Cell viability was detected by MTT assay; cell apoptosis was observed by flow cytometry (FCM) and Hoechst 33258 staining. The activity of Caspase-3 was determined by Caspase colorimetric assay kit and Western blot.. Rapamycin inhibited the growth of BEL-7402 cells and induced apoptosis significantly in time- and dose-dependent manners. Marked morphologic changes of cell apoptosis, such as chromatin condensation and nuclear fragmentation, were observed clearly at 48 h after exposion to rapamycin; Caspase-3 was activated by the loss of Caspase-3 proenzyme (32-ku) and its 20-ku subunit appeared at 24 h after incubation. Caspase-3 inhibitor z-DEVD-FMK could block the apoptosis induced by rapamycin.. Rapamycin can inhibit growth and induce apoptosis of BEL-7402 cells. The activation of Caspase-3 may play an important role in cell apoptosis.

Topics: Antibiotics, Antineoplastic; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Caspase Inhibitors; Cell Line, Tumor; Cell Proliferation; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Humans; Oligopeptides; Sirolimus

Standard immunosuppression after organ transplantation stimulates tumor growth. Sirolimus has a strong antiproliferative and a tumor inhibiting effect. The purpose is to assess the effect on tumor growth of the immuno-suppressive compounds sirolimus and tacrolimus alone and in combination on cells of human hepatocellular carcinoma.. We used the human cell lines SK-Hep 1 and Hep 3B derived from hepatocellular carcinoma. Proliferation analyses after treatment with sirolimus, tacrolimus, or the combination of both were performed. FACS analyses were done to reveal cell cycle changes and apoptotic cell death. The expression of apoptosis-related proteins was estimated by Western blots.. Sirolimus alone or combined with tacrolimus inhibited the growth of both cell lines after 5 d by up to 35% in SK-Hep 1 cells, and by up to 68% in Hep 3B cells at 25 ng/mL. Tacrolimus alone stimulated the growth by 12% after 5 ng/mL and by 25% after 25 ng/mL in Hep 3B cells. We found an increase of apoptotic Hep 3B cells from 6 to 16%, and a G1-arrest in SK-Hep 1 cells with an increase of cells from 61 to 82%, when sirolimus and tacrolimus were combined. Bcl-2 was down-regulated in Hep 3B, but not in SK-Hep 1 cells after combined treatment.. Sirolimus appears to inhibit the growth of hepatocellular carcinoma cells alone and in combination with tacrolimus. Sirolimus seems to inhibit the growth stimulation of tacrolimus.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Division; Cell Line, Tumor; Drug Combinations; G1 Phase; Humans; Immunosuppressive Agents; Liver Neoplasms; Sirolimus; Tacrolimus

Immunosuppressive therapies associated with organ transplantation produce an increased risk of cancer development. Malignancies are increased in transplant recipients because of the impaired immune system. Moreover, experimental data point to a tumor-promoting activity of various immunosuppressive agents. In this study, we compared the effects of 4 immunosuppressive agents with different mechanisms of action (cyclosporine, rapamycin, mycophenolic acid, and leflunomide) on the in vitro growth of various tumor cell lines and umbilical vein endothelial cells. To varying degrees rapamycin (10 ng/mL), mycophenolic acid (300 nmol/L), and leflunomide (30 micromol/L) highly inhibited the growth of human rhabdomyosarcoma, hepatocellular carcinoma, colorectal carcinoma, and endothelial cells. In contrast, cyclosporine (100 ng/mL) did not affect their growth. Our data suggest that regimens containing rapamycin, mycophenolic acid, or leflunomide, which have both immunosuppressive and antitumor activities, should be preferred in transplant recipients to minimize the risk of tumors.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Cell Survival; Colorectal Neoplasms; Cyclosporine; Humans; Immunosuppression Therapy; Immunosuppressive Agents; Isoxazoles; Jurkat Cells; Leflunomide; Liver Neoplasms; Mycophenolic Acid; Rhabdomyosarcoma; Sirolimus

This 44-year-old woman developed multifocal hepatocellular carcinoma (HCC) within hepatitis B-induced liver cirrhosis. At the time of listing for transplantation the HCC had progressed beyond the Milan criteria. Due to her young age, high grade of histological differentiation according to biopsy, and lack of therapeutic alternatives, she was listed for transplantation. She received an organ from the Eurotransplant marginal liver list. Immunosuppression was reduced to tacrolimus monotherapy within 4 months. Five months after transplantation bilateral bulky ovarian metastases were seen on computed tomography (CT) scan. A bilateral salphingo-oophorectomy was performed and immunosuppression switched to sirolimus monotherapy. Fourteen months after this procedure and 19 months after transplantation, the patient is asymptomatic with stable liver function. She is free of recurrence as judged by CT scan, bone scan, and alpha-fetoprotein. In conclusion, radical surgical treatment and immunosuppression using sirolimus may achieve tumor-free survival in selected patients with advanced or recurrent HCC.

Topics: Adult; Antibiotics, Antineoplastic; Carcinoma, Hepatocellular; Female; Hepatitis B; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Ovarian Neoplasms; Radiography; Sirolimus; Treatment Outcome

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Humans; Immunosuppressive Agents; Liver Neoplasms; Male; Middle Aged; Neoplasm Regression, Spontaneous; Sirolimus

An increasing number of patients with hepatocellular carcinoma (HCC) are undergoing evaluation for listing for liver transplantation. Criteria for selection require ongoing review for suitability. A consecutive series of 40 patients with HCC within the standard Milan criteria (single tumors n = 19 < 5 cm, or up to 3 tumors < 3 cm) and beyond (Extended Criteria; single tumors n = 21 < 7.5 cm, multiple tumors < 5 cm) underwent liver transplant with a sirolimus-based immunosuppressive protocol designed to minimize exposure to calcineurin inhibitors and steroids. At 44.3 +/- 19.3 months (mean +/- standard deviation) follow-up, 1- and 4-year survivals (Kaplan-Meier) are 94.1 +/- 5.7% and 87.4 +/- 9.3%, in the Milan group, respectively, and 90.5 +/- 6.4% and 82.9 +/- 9.3% in the Extended Criteria group, respectively. Five patients died during follow-up, only 1 from recurrent HCC. Five tumor recurrences have occurred at median 17 (mean 22 +/- 17) months posttransplant, 1 in the Milan group and 4 in the Extended Criteria group. Median survival in the patients with recurrent tumor is 42 months (mean 45 +/- 25), and the median postrecurrence survival is 15.5 months (mean 23 +/- 16). The rate of patients who were alive and free of tumor at 1 and 4 years is 94.1 +/- 5.7% and 81.1 +/- 9.9%, respectively, in the Milan group and is 90.5 +/- 6.4% and 76.8 +/- 10.5%, respectively, in the Extended Criteria group. Five patients had sirolimus discontinued for toxicity, while 24 of 35 surviving patients have sirolimus monotherapy immunosuppression. In conclusion, the Milan criteria for liver transplantation in the presence of HCC can be carefully extended without compromising outcomes. This sirolimus based immunosuppression protocol appears to have beneficial effects on tumor recurrence and survival with an acceptable rate of rejection and toxicity.

Topics: Carcinoma, Hepatocellular; Disease-Free Survival; Female; Follow-Up Studies; Graft Rejection; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Male; Middle Aged; Neoplasm Recurrence, Local; Sirolimus

Topics: Antibiotics, Antineoplastic; Carcinoma, Hepatocellular; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation; Sirolimus

mTOR and P70 S6 kinase (S6K) play a key role in regulating protein translation. The role of mTOR and S6K in hepatocellular carcinoma has not been investigated, but this pathway is of particular interest because an effective inhibitor, rapamycin, is available. This study was undertaken to determine the prevalence and clinicopathological correlates of mTOR pathway activation in hepatocellular carcinoma and to determine whether rapamycin inhibits the pathway in cell culture.. Total and phosphorylated mTOR and S6K protein expression were studied by immunohistochemistry in hepatocellular carcinomas (n = 73), fibrolamellar carcinomas (n = 13), and hepatic adenomas (n = 15). Results were correlated with tumor growth pattern as defined by the WHO (trabecular, pseudoglandular/acinar, compact, and scirrhous), tumor size, Ki-67 proliferation index, and the modified Edmondson nuclear grade, which has a scale of 1 to 4. HepG2 and Hep3B cell lines were treated with rapamycin to see the effect on proliferation and S6K phosphorylation.. Increased expression of total mTOR was seen in 5% of hepatocellular carcinoma, whereas overexpression of phospho-mTOR was evident in 15% of hepatocellular carcinoma. Phospho-mTOR positivity correlated with increased expression of total S6K, which was found in 45% of cases. Total S6K overexpression was positively correlated with tumor nuclear grade, inversely with tumor size, and was unassociated with the proliferation index or WHO growth pattern. Rapamycin treatment of HepG2 and Hep3B cell lines markedly inhibited cell proliferation and reduced S6K phosphorylation in both cell lines.. The mTOR pathway is activated in a subset of hepatocellular carcinoma. Rapamycin can inhibit proliferation of neoplastic hepatocytes in cell culture.

Topics: Adenoma; Antibiotics, Antineoplastic; Carcinoma, Hepatocellular; Cell Proliferation; Female; Genetic Heterogeneity; Hepatitis; Humans; Immunoenzyme Techniques; Ki-67 Antigen; Liver Neoplasms; Male; Middle Aged; Phosphorylation; Protein Kinases; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

A membrane protein trafficking mutant (Trf1) of HuH-7 alters the asialoglycoprotein (ASGPR) and transferrin receptor subcellular distribution. Expression cloning of a cDNA complementing the trf1 mutation led to the discovery of a novel casein Kinase 2 catalytic subunit (CK2alpha"). To purify potential CK2alpha" phosphorylation-dependent sorting proteins from cytosol, the ASGPR cytoplasmic domain was expressed as a GST fusion protein and immobilized on glutathione-agarose. In the absence of phosphorylation, only trace amounts of cytosol protein were bound and eluted. When the fusion protein was phosphorylated, a heterocomplex of potential sorting proteins was recovered. Mass spectrometer and immunoblot analysis identified five of these proteins as gp96, HSP70, HSP90, cyclophilin-A, and FKBP18. Treatment of HuH-7 with rapamycin to disrupt the heterocomplex reduced surface ASGPR binding activity by 65 +/- 5.7%. In Trf1 cells, surface-binding activity was 48 +/- 7% of that in HuH-7 and was not further reduced by rapamycin treatment. Immunoanalysis showed significantly fewer surface receptors on rapamycin-treated HuH7 cells than on nontreated cells, with no affect on the level of surface receptors in Trf1 cells. The data presented provide evidence that phosphorylation of the ASGPR cytoplasmic domain is required for the binding of specific molecular chaperones with the potential to regulate receptor trafficking.

Topics: Amino Acid Sequence; Asialoglycoprotein Receptor; Binding Sites; Carcinoma, Hepatocellular; Catalytic Domain; Cloning, Molecular; DNA, Complementary; Humans; Kinetics; Liver Neoplasms; Molecular Chaperones; Molecular Sequence Data; Mutagenesis; Phosphorylation; Protein Subunits; Recombinant Fusion Proteins; Sirolimus; Telomeric Repeat Binding Protein 1; Tumor Cells, Cultured

Topics: Carcinoma, Hepatocellular; Cell Division; Humans; Immunosuppressive Agents; Liver Neoplasms; Sirolimus; Tacrolimus; Tumor Cells, Cultured

As resolved by electrophoresis in non-reducing SDS gels, transferrin newly made in Hep G2 cells migrates as a very diffuse set of species. During a subsequent 1-h chase all transferrin polypeptides are converted to a single, rapidly migrating species. These changes in gel mobility are due to alterations in the pattern of disulfide bonding, are not caused by carbohydrate processing, and occur while the protein is in the rough endoplasmic reticulum. Cyclosporin A causes an approximately 10-min lag in transferrin folding, after which folding resumes at the normal rate. Cyclosporin A also retards transferrin maturation from the endoplasmic reticulum and its secretion, at concentrations that do not affect secretion of other hepatoma proteins. Neither FK506 nor rapamycin affect transferrin folding. We conclude that an initial stage in transferrin folding is accelerated by an endoplasmic reticulum peptidyl-proline isomerase that is inhibited by cyclosporin A.

Topics: Animals; Anti-Bacterial Agents; Carcinoma, Hepatocellular; Cyclosporins; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum; Humans; Kinetics; Liver Neoplasms; Mice; Polyenes; Precipitin Tests; Protein Conformation; Sirolimus; Tacrolimus; Transferrin; Tumor Cells, Cultured

Topics: Amino Acid Isomerases; Animals; Carcinoma, Hepatocellular; Carrier Proteins; Cell Line; Cyclosporins; DNA Replication; Gene Expression; Humans; Immunosuppressive Agents; Liver; Liver Diseases; Liver Neoplasms; Liver Neoplasms, Experimental; Organ Specificity; Peptidylprolyl Isomerase; Polyenes; Rats; Sirolimus; Tacrolimus; Tacrolimus Binding Proteins; Transcription, Genetic