sirolimus and Uterine-Cervical-Neoplasms

The mechanistic (mammalian) targets of rapamycin (mTOR) inhibitors with known growth Inhibitory effect are currently in clinical trial for treatment of human cancer. The aim of this review is to present current incorporating these new drugs as single agents or in combination with other therapeutic modalities for treatment of gynecologic cancer.. A PubMed search was conducted on "mTOR inhibitors" and "human cancer". The relevant studies published between the year 2000 to present were reviewed. Those related to gynecologic cancer (cervical, endometrial and ovarian) were selected for this manuscript. The result of published data and their clinical application in gynecologic malignancies are presented.. mTOR is directly involved in many cell signaling pathways, and mTOR inhibitors have demonstrated anti-tumor activity against a variety of human malignancies, including gynecologic cancers. Combinations of mTOR inhibitors with other treatment modalities, e.g. cytotoxic chemotherapy, hormonal therapies, and other targeted molecular agents, have shown encouraging results particularly in endometrial and ovarian cancer.. Patients with advanced or recurrent gynecologic cancers who have failed initial treatment are need of new treatment modalities. There is strong evidence that mTOR inhibitors limit tumor proliferation and progression. The PI3k/AKT/mTOR pathway is often deregulated in gynecologic cancer. Patients with PIK3CA mutations are more responsive to PI3K/AKT/mTOR inhibitors than patients without these mutations. Routine screening for PIK3CA mutations warrants further investigation when PI3K/AKT/mTOR inhibitors are considered in treatment of patients with gynecologic cancer.

Topics: Antineoplastic Agents; Endometrial Neoplasms; Everolimus; Female; Humans; Ovarian Neoplasms; Sirolimus; TOR Serine-Threonine Kinases; Uterine Cervical Neoplasms

Bevacizumab and temsirolimus are active agents in gynecologic tumors. Temsirolimus attenuates upregulation of HIF-1α levels, a resistance mechanism for antiangiogenics, and targets the PI3-kinase/AKT/mTOR axis, commonly aberrant in these tumors.. We analyzed safety and responses in 41 patients with gynecologic cancers treated as part of a Phase I study of bevacizumab and temsirolimus.. Median age of the 41 women was 60 years (range, 33-80 years); median number of prior systemic therapies was 4 (1-11). Grade 3 or 4 treatment-related toxicities included: thrombocytopenia (10%), mucositis (2%), hypertension (2%), hypercholesterolemia (2%), fatigue (7%), elevated aspartate aminotransferase (2%), and neutropenia (2%). Twenty-nine patients (71%) experienced no treatment-related toxicity greater than grade 2. Full FDA-approved doses of both drugs (bevacizumab 15mg/kg IV Q3weeks and temsirolimus 25mg IV weekly) were administered without dose-limiting toxicity. Eight patients (20%) achieved stable disease (SD) > 6 months and 7 patients (17%), a partial response (PR) [total = 15/41 patients (37%)]. Eight of 13 patients (62%) with high-grade serous histology (ovarian or primary peritoneal) achieved SD > 6 months/PR.. Bevacizumab and temsirolimus was well tolerated. Thirty-seven percent of heavily-pretreated patients achieved SD > 6 months/PR, suggesting that this combination warrants further study.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Aspartate Aminotransferases; Bevacizumab; Carcinoma; Fatigue; Female; Humans; Hypercholesterolemia; Hypertension; Male; Middle Aged; Mucositis; Neutropenia; Ovarian Neoplasms; Sirolimus; Thrombocytopenia; Uterine Cervical Neoplasms

HPV infection has been associated with deregulation of the PI3K-Akt-mTOR pathway in invasive cervical carcinomas. This 2-stage phase II study assessed the activity of the mTOR inhibitor, temsirolimus, in patients with measurable metastatic and/or locally advanced, recurrent carcinoma of the cervix.. Temsirolimus 25mg i.v. was administered weekly in 4 week cycles. One response among the first 18 patients was required to proceed to the second stage of accrual. Correlative molecular studies were performed on archival tumor tissue.. Thirty-eight patients were enrolled. Thirty-seven patients were evaluable for toxicity and 33 for response. One patient experienced a partial response (3.0%). Nineteen patients had stable disease (57.6%) [median duration 6.5 months (range 2.4-12.0mo)]. The 6-month progression free survival rate was 28% (95% CI: 14-43%). The median progression free survival was 3.52 months [95% CI (1.81-4.70)]. Adverse effects were mild-moderate in most cases and similar to other temsirolimus studies. No toxicity>grade 3 was observed. Assessment of PTEN and PIK3CA by IHC, copy number analyses and PTEN promoter methylation status did not reveal subsets associated with disease stability.. Single agent temsirolimus has modest activity in cervical carcinoma with about two-thirds of patients exhibiting stable disease. Molecular markers for treatment benefit remain to be identified.

Topics: Adult; Aged; Antineoplastic Agents; Class I Phosphatidylinositol 3-Kinases; Disease-Free Survival; Female; Humans; Immunohistochemistry; Middle Aged; Neoplasm Recurrence, Local; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; PTEN Phosphohydrolase; Sirolimus; TOR Serine-Threonine Kinases; Uterine Cervical Neoplasms

This study was carried out to determine the effect of autophagy modulation in radiation treatment of cervical cancer cells. HeLa and CaSki cells were irradiated with γ-rays (2 Gy/min) after treatment with an autophagy inducer (rapamycin) and inhibitor (3-MA). Expression of LC3 and cell death in two cell preparations were examined. In addition, expression of Caspase-3 and PARP were examined after radiation alone and with autophagy inhibitor treatment. A notable increment of LC3 expression was detected after radiation in both cell lines. Cell viability was observed to decrease in 3-MA-treated cells compared to radiation alone, and even further in rapamycin-treated cells. Apoptosis was confirmed to occur later than autophagy in radiation treatment, and inhibition of autophagy derived a decrease in apoptosis. In conclusion, radiation-induced autophagy may be regulated by modulators, and autophagy augmentation yields an increase in cervical cancer cell death under radiation.Impact statement

Topics: Apoptosis; Autophagy; Cell Line, Tumor; Female; Humans; Sirolimus; Uterine Cervical Neoplasms

Cervical cancer (CC) is a common gynecological malignant tumor, causing poor survival rate. Circular RNAs (circRNAs) are abundantly expressed in CC with their stable loop structure. However, the underlying mechanism and biological function of circRNAs remained unclear. Using quantitative real-time polymerase chain reaction (qRT-PCR) or western blot assay, we measured the expression of hsa_circ_0001495, miR-526b-3p, and transmembrane Bax inhibitor motif containing 6 (TMBIM6) in CC tissues and cells. The relationship between miR-526b-3p and hsa_circ_0001495 or TMBIM6 was investigated by bioinformatics analysis, dual-luciferase and RIP analysis. Enzyme linked immunosorbent assay (ELISA) was conducted to evaluate glucose consumption and lactate production. 5-ethynyl-2'-deoxyuridine (EDU) assay were used to test cell proliferation. Cell apoptosis was analyzed by using flow cytometry assay. Transwell and wound-healing assays were used to measure cell invasion and migration. The expression of proteins was examined by western blot. Xenograft assay was applied to detect the effect of hsa_circ_0001495 in vivo. Our finding showed that hsa_circ_0001495 and TMBIM6 expression were upregulated, while miR-526b-3p was downregulated in CC tissues and cell lines. Hsa_circ_0001495 knockdown or TMBIM6 knockdown suppressed cell proliferation, migration, glycolysis, while promoted cell apoptosis in vitro, and hsa_circ_0001495 silence curbed tumor growth in vivo. Beside, hsa_circ_0001495 exerted its function in CC by positively regulating TMBIM6. Furthermore, hsa_circ_0001495 acted as a sponge for miR-526b-3p to regulate TMBIM6 expression. Hsa_circ_0001495/miR-526b-3p/TMBIM6 axis also regulated the phosphorylation of mammalian target of rapamycin (mTOR) in CC cells. In summary, hsa_circ_0001495 regulated the progression of CC by regulating miR-526b-3p/TMBIM6/mTOR pathway.

Topics: Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Cell Movement; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Membrane Proteins; MicroRNAs; RNA, Circular; Sirolimus; TOR Serine-Threonine Kinases; Uterine Cervical Neoplasms

In contrast to normal cells, cancer cells typically undergo metabolic reprogramming. Studies have shown that oncogenes play an important role in this metabolic reprogramming. CD38 is a multifunctional transmembrane protein that is expressed abnormally in a variety of tumor types. To investigate the effect and possible mechanism of CD38 in cervical cancer cells and to provide a new therapeutic target for the treatment of cervical cancer, the present study identified that CD38 is involved in regulating cell metabolism in cervical cancer cells. Liquid chromatography‑tandem mass spectrometry and bioinformatic analyses revealed that differentially abundant proteins in CD38‑overexpressed cervical cancer cells (CaSki‑CD38 and HeLa‑CD38) are predominantly involved in glycolytic pathways, oxidative phosphorylation and the NAD/NADH metabolic process. Further experiments using an ATP test kit and lactate test kit revealed that CD38 promotes glucose consumption, increases lactate accumulation and increases ATP production. In addition, CD38 increases the phosphorylation of phosphatidylserine/threonine kinase (AKT), mechanistic target of rapamycin (mTOR) and phosphatidylinositol‑4,5‑bisphosphate 3‑kinase (PI3K), which play a key role in tumor metabolism. Furthermore, it was found that the energy metabolism of cervical cancer cells was inhibited following treatment with the mTOR inhibitor rapamycin. In conclusion, the results of the present study suggested that CD38 regulates the metabolism of cervical cancer cells by regulating the PI3K/AKT/mTOR pathway, which may be a candidate target for the treatment of cervical cancer.

Topics: ADP-ribosyl Cyclase 1; Energy Metabolism; Female; HeLa Cells; Humans; Membrane Glycoproteins; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Uterine Cervical Neoplasms

BNIP1, a member of the BH3-only protein family, plays essential roles in a variety of biological processes. However, the mechanism and function of BNIP1 are still unknown in cervical cancer. We aim to explore the roles of BNIP1 on cervical cancer cell proliferation, apoptosis, migration, and invasion abilities by mTOR signaling pathway.. qRT-PCR and Western blot assays were performed to assess BNIP, mTOR, and p70S6K1 expressions. CCK-8, transwell and flow cytometry assays were used to measure the representative proliferation, migration, invasion, and apoptosis abilities.. Our findings indicated that BNIP1 is down-expressed in cervical cancer tissues and cells, and was negatively associated with lymphatic metastasis. Overexpression of BNIP1 suppressed proliferation, migration and invasion, and promoted apoptosis of cervical cancer cells. Silence of BNIP1 by siRNAs accelerated proliferation, migration and invasion, and inhibited apoptosis of cervical cancer cells. In addition, we found that BNIP1 significantly inhibited mTOR, p70S6K1, and p-p70S6K1 expressions; BNIP1 affected the proliferation, apoptosis, migration, and invasion abilities of cervical cancer cells by regulating mTOR expression.. BNIP1 can be considered a marker for cervical carcinoma therapy.

Topics: Apoptosis; Cell Movement; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Neoplasm Staging; Proto-Oncogene Proteins c-bcl-2; Ribosomal Protein S6 Kinases, 70-kDa; RNA Interference; RNA, Small Interfering; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Uterine Cervical Neoplasms

In cervical cancer, the association between HPV infection and dysregulation of phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway (PI3K/AKT/mTOR pathway) places mTOR as an attractive therapeutic target. The failure of current treatment modalities in advanced stages of this cancer and drawbacks of already available mTOR inhibitors demand for novel drug candidates. In the present study we identified the presence of a mTOR inhibitor in an active fraction of the ethyl acetate extract of Streptomyces sp OA293. The metabolites(s) in the active fraction completely inhibited mTORC1 and thereby suppressed activation of both of its downstream targets, 4E-BP1 and P70S6k, in cervical cancer cells. In addition, it also stalled Akt activation via inhibition of mTORC2. The mechanism of mTOR inhibition detailed in our study overcomes significant drawbacks of well known mTOR inhibitors such as rapamycin and rapalogs. The active fraction induced autophagy and Bax mediated apoptosis suggesting that mTOR inhibition resulted in programmed cell death of cancer cells. The molecular weight determination of the components in active fraction confirmed the absence of any previously known natural mTOR inhibitor. This is the first report of complete mTOR complex inhibition by a product derived from microbial source.

Topics: Apoptosis; Autophagy; bcl-2-Associated X Protein; Biological Products; Cell Line, Tumor; Cell Proliferation; Female; HEK293 Cells; Humans; Mechanistic Target of Rapamycin Complex 1; Phosphatidylinositol 3-Kinase; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; Streptomyces; TOR Serine-Threonine Kinases; Uterine Cervical Neoplasms

BACKGROUND Epithelial-mesenchymal transition (EMT) plays an important role in cancer tumorigenesis. Transforming growth factor β1 (TGF-β1) can induced EMT, which could increase tumor migration and invasion. Moreover, recent studies have been proven that mammalian target of rapamycin (mTOR) is a critical regulator of EMT. We investigated the mechanisms of mTOR in transforming growth factor β1 (TGF-β1)-induced EMT in cervical cancer cells. MATERIAL AND METHODS HeLa and SiHa cells were treated with 10 ng/ml TGF-β1 to induce EMT. Then, they were treated with or without rapamycin. CCK8 assay was performed to determine cell proliferation. Cell migration was detected by wound-healing assay; apoptosis was analyzed by flow cytometry; mTOR inhibitors inhibited mTOR pathway to assess the expression of E-cadherin, Vimentin STAT3, Snail2, p-p70s6k, and PKM2 expression. RESULTS TGF-β1 promoted proliferation and migration, and attenuated apoptosis in cervical carcinoma cells. Rapamycin abolished TGF-β1-induced EMT cell proliferation and migration and reversed TGF-β1-induced EMT. E-cadherin were suppressed, whereas Vimentin and PKM2 were increased in HeLa and SiHa cells after stimulation with TGF-β1. Moreover, mTOR was activated in the process of TGF-β1-induced EMT. Rapamycin inhibited the phosphorylation of p70s6k. Furthermore, inhibition of the mTOR pathway decreased PKM2 expression. CONCLUSIONS Inhibition of the mTOR pathway abolished TGF-β1-induced EMT and reduced mTOR/p70s6k signaling, which downregulated PKM2 expression. Our results provide novel mechanistic insight into the anti-tumor effects of inhibition of mTOR.

Topics: Apoptosis; Cadherins; Carrier Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Female; HeLa Cells; Humans; Membrane Proteins; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Thyroid Hormone-Binding Proteins; Thyroid Hormones; TOR Serine-Threonine Kinases; Transforming Growth Factor beta1; Uterine Cervical Neoplasms

6-Gingerol, a potent nutraceutical, has been shown to have antitumor activity in different tumors, although its mechanism of action is not well understood. In this study, we evaluated antitumor activities of 6-gingerol on human oral (SCC4, KB) and cervical cancer (HeLa) cell lines with or without wortmannin, rapamycin, and cisplatin. Tumor cell proliferation was observed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium, inner salt assay, cell cycle analysis by propidium iodide labeling and flow cytometry, apoptosis by Annexin-V binding assay, and caspase activity by chemiluminescence assay. 6-Gingerol showed dose-dependent cytotoxicity in all three cell lines. Combinations of 6-gingerol with wortmannin and cisplatin showed additive effects, while with rapamycin, it showed 50% cytotoxicity that was equivalent to IC50 of 6-gingerol alone. Treatment with 6-gingerol resulted in G2-phase arrest in KB and HeLa cells and S-phase arrest in SCC4 cells. 6-Gingerol, wortmannin, and rapamycin treatment showed almost two-fold higher expression of caspase 3 in all cell lines. The results imply that 6-gingerol either alone or in combination with PI-3 K inhibitor and cisplatin may provide better therapeutic effects in oral and cervical carcinoma. Thus, 6-gingerol appears to be a safe and potent chemotherapeutic/chemopreventive compound acting through cell cycle arrest and induction of apoptosis in human oral and cervical tumor cells.

Topics: Androstadienes; Apoptosis; Caspase 3; Catechols; Cell Cycle Checkpoints; Cell Division; Cell Line, Tumor; Cell Proliferation; Cisplatin; Fatty Alcohols; Female; HeLa Cells; Humans; Mouth Neoplasms; Sirolimus; Uterine Cervical Neoplasms; Wortmannin

Protein arginine methylation is a common posttranslational modification resulting in the generation of asymmetric dimethylarginine (aDMA) and symmetric dimethylarginine (sDMA). Currently, the regulation of aDMA or sDMA by hypoxia, nutrient stavation or cytokines in the tumor microenvironment remains largely unknown. Here we show that p90aDMA, p70aDMA and p90sDMA, endogenous proteins containing aDMA or sDMA with mass 70 or 90 kDa, were widely and dominantly expressed in breast cancer cell lines. Notably, it was p90aDMA rather than p90sDMA that accumulated in the nucleus upon stimulation of cancer cells with interleukin (IL)-2, IL-4, IL-6 but not IL-8. In addition, the p90aDMA accumulation could be inhibited after treatment with a global methyltrasferase inhibitor, adenosine-2',3'-dialdehyde (AdOx). It seemed that some endogenous proteins in cancer cells were asymmetrically arginine-methylated upon exposure to some cytokines.. Furthermore, endogenous proteins of aDMA, such as p90aDMA and p70aDMA, were degraded in response to hypoxia, nutrient starvation and rapamycin treatment in breast and cervical cancer cells. IL-2/4/6 slightly increased basal autophagy but slightly decreased the rapamycin‑induced autophagy in cancer cells, suggesting that IL-2/4/6 and autophagy inducers play distinct roles in the regulation of aDMA of proteins. Conversely, rapamycin accumulated p90sDMA in MDA-MB‑231 and MCF-7 cells. Taken together, our results add a new dimension to the complexity of arginine methylated regulation in response to various stimuli and provide the first evidence that aDMA serves as one specific degradation signal of selective autophagy.

Topics: Arginine; Autophagy; Breast Neoplasms; Cell Line, Tumor; Female; Humans; Interleukin-2; Interleukin-4; Interleukin-6; MCF-7 Cells; Molecular Weight; Nuclear Proteins; Protein Processing, Post-Translational; Sirolimus; Tumor Microenvironment; Uterine Cervical Neoplasms

Progranulin (PGRN) is an autocrine growth factor with tumorigenic roles in various tumors including cervical cancer. In this study, we investigated mammalian target of rapamycin (mTOR) signaling in response to PGRN induction and the contribution of the PGRN-stimulated PI3K/Akt/mTOR signaling pathway in the transformation and progression of cervical cancer. Here we identified a strong linkage between PGRN and phosphorylated-mTOR in cervical cancer tissues. PGRN promoted the phosphorylation of mTOR and activated mTOR signaling in human cervical mucosa epithelial cells and cervical cancer cells, and TNFR2 was needed for PGRN-stimulated mTOR signaling. Inhibition of mTOR signaling with rapamycin decreased PGRN-stimulated protein synthesis, transformation and proliferation of cervical cells in vitro, and tumor formation and growth in vivo. Thus, our findings update the signal transduction pathways of PGRN by suggesting that mTOR signaling contributes to PGRN-stimulated carcinogenesis of cervical cancer. Inhibition of PGRN/PI3K/Akt/mTOR signaling may be targeted in treatment of cervical cancer.

Topics: Animals; Anoikis; Carcinogenesis; Cell Movement; Cell Proliferation; Cervix Uteri; Female; HeLa Cells; Humans; Intercellular Signaling Peptides and Proteins; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Phosphatidylinositol 3-Kinases; Phosphorylation; Progranulins; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Uterine Cervical Neoplasms

We previously reported that microRNA-218 was frequently lost in cervical cancer and restoration of microRNA-218 increased cellular radio-sensitivity via inhibiting. Herein, we aim to investigate the effects of microRNA-218 on cellular response to mTOR inhibition. The expression of microRNA-218 and Rictor were measured by Taqman PCR and real time PCR in a panel of 15 cervical cancer tissues. MicroRNA-218 was stably overexpressed in four cervical cancer cell lines and a series of in vitro and in vivo experiments were performed to investigate cellular sensitivity to Rapamycin. In primary cultured cervical cancer cells, the expression of microRNA-218 was negatively correlated with the mRNA level of Rictor, which predicted cellular sensitivity to Rapamycin (p = 0.002, R(2)  = 0.6810). In vitro, overexpression of microRNA-218 significantly reduced the level of Rictor and enhanced the growth-inhibition, cell cycle arrest, and apoptosis induced by Rapamycin. In vivo, overexpression of microRNA-218 further enhanced the suppressive effects of Rapamycin on tumor growth. In conclusion, we demonstrated that microRNA-218 could re-sensitize cervical cancer to Rapamycin through targeting Rictor. Moreover, patients with loss of microRNA-218 presented notable resistance to Rapamycin, indicating that microRNA-218 might be a valid marker for patients-stratification in future clinical trials.

Topics: Adult; Animals; Apoptosis; Carrier Proteins; Cell Cycle Checkpoints; Cell Line, Tumor; Female; G1 Phase; HeLa Cells; Humans; Mice; Mice, Inbred BALB C; MicroRNAs; Middle Aged; Rapamycin-Insensitive Companion of mTOR Protein; Sirolimus; Uterine Cervical Neoplasms; Xenograft Model Antitumor Assays

Cisplatin is used as a cytotoxic agent for the management of cervical cancer. However, the severity of the side-effects limits the use of this drug, particularly at high doses. Resistance to cisplatin is often attributed to a disruption in the normal apoptotic response via aberrant activation of pathways such as the mTOR pathway. Here we assess the role of mTOR and its effect on cell death sensitization and autophagy in response to a low concentration of cisplatin in cervical cancer cells. Additionally we measured the expression profile of mTOR in normal, low- and high-grade squamous intraepithelial (LSIL and HSIL) lesions and cancerous tissue. An in vitro model of cervical cancer was established using HeLa and CaSki cells. mTOR protein expression as well as autophagy-related proteins were evaluated through Western blotting. Inhibition of mTOR was achieved with the use of rapamycin and RNA silencing. A low concentration of cisplatin administered as a single agent induces autophagy, but not apoptosis. Cisplatin cytotoxicity was greatly enhanced in cancer cells when mTOR had been inhibited prior to cisplatin treatment which was likely due to autophagy being increased above cisplatin-induced levels, thereby inducing apoptosis. Cervical tissue samples revealed an increase in mTOR protein expression in LSIL and carcinoma tissue which suggests a change in autophagy control. Our data suggest that utilising a lower dose of cisplatin combined with mTOR inhibition is a viable treatment option and addresses the challenge of cisplatin dose-dependent toxicity, however future studies are required to confirm this in a clinical setting.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autophagy; Cell Cycle; Cisplatin; Dose-Response Relationship, Drug; Female; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Protein Kinase Inhibitors; RNA Interference; Signal Transduction; Sirolimus; Squamous Intraepithelial Lesions of the Cervix; TOR Serine-Threonine Kinases; Transfection; Uterine Cervical Neoplasms

Radiotherapy is widely used to treat cancer because it has the advantage of physically and functionally conserving the affected organ. To improve radiotherapy and investigate the molecular mechanisms of cellular radioresistance, we established a clinically relevant radioresistant (CRR) cell line, SAS-R, from SAS cells. SAS-R cells continue to proliferate when exposed to fractionated radiation (FR) of 2 Gy/day for more than 30 days in vitro. A xenograft tumor model of SAS-R was also resistant to 2 Gy/day of X-rays for 30 days. The density of blood vessels in SAS-R tumors was higher than in SAS tumors. Everolimus, a mammalian target of rapamycin (mTOR) inhibitor, sensitized microvascular endothelial cells to radiation, but failed to radiosensitize SAS and SAS-R cells in vitro. Everolimus with FR markedly reduced SAS and SAS-R tumor volumes. Additionally, the apoptosis of endothelial cells (ECs) increased in SAS-R tumor tissues when both Everolimus and radiation were administered. Both CD34-positive and tomato lectin-positive blood vessel densities in SAS-R tumor tissues decreased remarkably after the Everolimus and radiation treatment. Everolimus-induced apoptosis of vascular ECs in response to radiation was also followed by thrombus formation that leads to tumor necrosis. We conclude that FR combined with Everolimus may be an effective modality to overcome radioresistant tumors via targeting tumor ECs.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Endothelial Cells; Everolimus; Female; Head and Neck Neoplasms; HeLa Cells; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Radiation-Sensitizing Agents; Sirolimus; Squamous Cell Carcinoma of Head and Neck; Uterine Cervical Neoplasms; Xenograft Model Antitumor Assays

The incidence of head and neck squamous cell carcinomas (HNSCC) associated with human papillomavirus (HPV) infection has increased over the past decades in the United States. We aimed at examining the global impact of HPV-associated HNSCC and whether the established key role of mTOR activation in HNSCC is also observed in HPV(+) HNSCC lesions, thereby providing novel treatment options for HPV-associated HNSCC patients.. An international HNSCC tissue microarray (TMA) was used to analyze the expression of p16(INK4A), a surrogate for HPV infection, and Akt-mTOR pathway activation. Results were confirmed in a large collection of HPV(-) and HPV(+) HNSCC cases and in a cervical cancer (CCSCC) TMA. Observations were validated in HNSCC and CCSCC-derived cell lines, which were xenografted into immunodeficient mice for tumorigenesis assays.. Approximately 20% of all HNSCC lesions could be classified as HPV(+), irrespective of their country of origin. mTOR pathway activation was observed in most HPV(+) HNSCC and CCSCC lesions and cell lines. The preclinical efficacy of mTOR inhibition by rapamycin and RAD001 was explored in HPV(+) HNSCC and CCSCC tumor xenografts. Both mTOR inhibitors effectively decreased mTOR activity in vivo and caused a remarkable decrease in tumor burden. These results emphasize the emerging global impact of HPV-related HNSCCs and indicate that the activation of the mTOR pathway is a widespread event in both HPV(-) and HPV-associated HNSCC and CCSCC lesions.. The emerging results may provide a rationale for the clinical evaluation of mTOR inhibitors as a molecular targeted approach for the treatment of HPV-associated malignancies.

Topics: Animals; Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Cervix Uteri; Cohort Studies; Cyclin-Dependent Kinase Inhibitor p16; DNA, Viral; Female; Humans; Immunoenzyme Techniques; Mice; Mice, Nude; Mouth Neoplasms; Neoplasm Proteins; Papillomaviridae; Papillomavirus Infections; Polymerase Chain Reaction; Proto-Oncogene Proteins c-akt; Sirolimus; Tissue Array Analysis; TOR Serine-Threonine Kinases; Uterine Cervical Neoplasms; Xenograft Model Antitumor Assays

The mammalian target of rapamycin (mTOR) pathway is activated in a range of malignant cancers, but its role in human cervical cancer has not been well defined. This study aims to investigate the activation of mTOR pathway in cervical carcinomas and whether inhibition of mTOR with rapamycin, as well as specific siRNA, could lead to decreased proliferation, induced cell cycle arrest and apoptosis.. A cervical cancer tissue microarray was tested for activation of the mTOR pathway. The effects on HeLa cell survival and downstream signaling were determined following mTOR inhibition by increasing doses of rapamycin, or silencing by siRNA.. The mTOR pathway is activated in cervical carcinomas. mTOR-specific siRNA effectively suppressed HeLa cell growth through mechanisms including inhibition of the cell cycle and increased apoptosis, which were similar to the mechanisms of rapamycin action.. The mTOR signaling pathway is activated in cervical carcinomas. Inhibition of mTOR represents a potential therapeutic strategy for cervical cancers.

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Survival; Cisplatin; Down-Regulation; Female; HeLa Cells; Humans; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Phosphatidylinositol 3-Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Uterine Cervical Neoplasms

Paclitaxel, a potent anti-neoplastic agent, has been found to be effective against several tumours, including cervical cancer. However, the exact mechanism underlying the cytotoxic effects of pacitaxel, especially in the survival-signalling pathway, is poorly understood. The aim of this study was to investigate the molecular pathway of the cytotoxic effect of paclitaxel in human cervical cancer cell lines. Four human cervical cancer cell lines were treated for 24 h with various concentration of paclitaxel, and the sensitivity was analysed by an MTT assay. The cell cycle progression and sub-G1 population were analysed by flow cytometry. Apoptosis was further measured by DNA fragmentation and microscope examination. The protein expression was determined by Western blot analysis. Our results showed that HeLa cells demonstrated the highest sensitivity to paclitaxel, whereas CaSki cells showed the lowest. In cervical cancer cells, paclitaxel induced apoptosis through an intrinsic pathway with prior G2/M arrest. In addition, we showed that paclitaxel downregulated the phosphorylation of Akt in both HeLa and CaSki cells. Interestingly, in CaSki cells, which were more suggestive of a resistant phenotype, paclitaxel induced the activation of mTOR as a downstream target of Akt. Pre-treatment with rapamycin inhibited activation of mTOR signalling and significantly enhanced the sensitivity of CaSki cells to paclitaxel by increasing apoptotic cell death. This effect was mediated, at least partly, through caspase activation. Overall, paclitaxel exerts its anti-tumour effects on cervical cancer cells by inducing apoptosis through intrinsic pathway, and rapamycin targeted to mTOR can sensitise paclitaxel-resistant cervical cancer cells.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Communication; Cell Cycle; Cell Line, Tumor; DNA, Neoplasm; Dose-Response Relationship, Drug; Down-Regulation; Drug Screening Assays, Antitumor; Female; HeLa Cells; Humans; Paclitaxel; Phosphatidylinositol 3-Kinases; Protein Kinases; Sirolimus; TOR Serine-Threonine Kinases; Uterine Cervical Neoplasms

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

Infections with high-risk human papillomaviruses (HPVs) are linked to more than 95% of cervical cancers. HPVs replicate exclusively in differentiated cells and the function of the HPV E7 oncoprotein is essential for viral replication. In this study, we investigated the mechanism that regulates E7 expression in differentiated cells. The level of E7 protein was strongly induced in HPV-containing Caski, HOK-16B, and BaP-T cells during growth in methylcellulose-containing medium, a condition that induces differentiation. Enhanced expression of E7 was observed between 4 and 8 h of culturing in methylcellulose and was maintained for up to 24 h. The increase was not due to altered stability of the E7 protein or an increase in the steady-state level of the E7 mRNA. Instead, the translation of the E7 mRNA was enhanced during differentiation. More than 70 to 80% of the E7 mRNA was found in the polysome fractions in the differentiated cells. Consistent with this observation, higher levels of the phosphorylated translator inhibitor 4E-BP1 were observed in differentiated HPV-containing cells but not in differentiated non-HPV tumor cells or primary keratinocytes. The mTOR kinase inhibitor rapamycin blocked phosphorylation of 4E-BP1 and significantly decreased the level of E7 protein in Caski cells, suggesting that phosphorylation of 4E-BP1 is linked to E7 expression. Prevailing models for the molecular mechanisms underlying E7 expression have focused largely on transcriptional regulation. The results presented in this study demonstrate a significant role of the cellular translation machinery to maintain a high level of E7 protein in differentiated cells.

Topics: Adaptor Proteins, Signal Transducing; Antibiotics, Antineoplastic; Cell Cycle Proteins; Cell Differentiation; Eukaryotic Initiation Factor-4E; Female; Gene Expression Regulation, Neoplastic; Gene Expression Regulation, Viral; HeLa Cells; Humans; Keratinocytes; Models, Genetic; Papillomaviridae; Papillomavirus E7 Proteins; Phosphoproteins; Phosphorylation; Protein Biosynthesis; Protein Processing, Post-Translational; RNA, Messenger; RNA, Viral; Sirolimus; Time Factors; Up-Regulation; Uterine Cervical Neoplasms; Virus Replication

Clinical trials using rapamycin analogues or HER1/epidermal growth factor receptor (EGFR) inhibitors show that each class of agent has activity against a range of human solid tumors. Because blockade of mitogen-activated protein kinase signaling occurs following HER1/EGFR inhibition in some cell types, we tested the combination of rapamycin and erlotinib in SiHa, Me180, and CaSki human cervical carcinomas xenografts in severe combined immunodeficient mice. In tissue culture, all three cell lines showed decreased phosphorylated S6 ribosomal protein and decreased phosphorylated extracellular signal-regulated kinase (ERK) following treatment with rapamycin and erlotinib, respectively. In SiHa tumors, suppression of phosphorylated S6 was induced by either drug alone, whereas phosphorylated ERK decreased with erlotinib, and enhancement of these effects was obtained with the combination. Continuous treatment of xenografts for 3 weeks led to significant tumor growth delay compared with vehicle control for rapamycin as single agent (P = 0.003) and greater for the combination (P = 0.04 versus rapamycin). Significant antiangiogenic effect was obtained in SiHa xenografts using the drugs together (measured by microvascular density and vascular endothelial growth factor plasma levels) but not for the single agents. Me180 and CaSki xenografts showed significant growth delay with rapamycin but not with erlotinib. Erlotinib treatment resulted in decreased phosphorylated ERK, associated with enhanced suppression of phosphorylated S6 and improved growth delay in Me180 but not in CaSki tumors. These results support the further clinical investigation of rapamycin and EGFR inhibitor combinations in anticancer therapy but highlight the problem of intertumoral heterogeneity in the prediction of in vivo response.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Cell Proliferation; Drug Interactions; Epidermal Growth Factor; Erlotinib Hydrochloride; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Mice; Mice, SCID; Neoplasm Transplantation; Neoplasms, Experimental; Phosphorylation; Quinazolines; Ribosomal Protein S6; Signal Transduction; Sirolimus; Transplantation, Heterologous; Uterine Cervical Neoplasms