sirolimus and Colonic-Neoplasms

Colorectal cancer is the second leading cause of cancer death in Switzerland. The nihilism that dominated the treatment of these patients for decades has been replaced by a measure of enthusiasm, given recent therapeutic advances. New anticancer drugs such as irinotecan and oxaliplatin have changed the standard chemotherapy treatment of metastatic colorectal cancer. However, the real hype has come from molecular targeted therapy. Identification of cellular processes characteristic of colon cancer has permitted therapeutic targeting with favorable therapeutic index. Inhibition of the epidermal growth factor receptor in the clinic has provided proof of principle that interruption of signal transduction cascades in patients has therapeutic potential. Angiogenesis, especially the vascular endothelial growth factor pathway, has been proven to be another highly successful molecular target. In this article, we will review molecular targets, which are under active clinical investigation in colon cancer.

Topics: Angiogenesis Inhibitors; Antibiotics, Antineoplastic; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzamides; Camptothecin; Cetuximab; Clinical Trials as Topic; Clinical Trials, Phase II as Topic; Colonic Neoplasms; Drug Delivery Systems; ErbB Receptors; Gefitinib; Humans; Imatinib Mesylate; Indoles; Irinotecan; Organoplatinum Compounds; Oxaliplatin; Phthalazines; Piperazines; Platelet-Derived Growth Factor; Protein Kinase Inhibitors; Pyridines; Pyrimidines; Pyrroles; Quinazolines; Quinolones; Receptors, Platelet-Derived Growth Factor; Receptors, Somatomedin; Signal Transduction; Sirolimus; Sunitinib

The importance of the network defined by phosphatidylinositol-3-kinase (PI3K), AKT and mammalian target of rapamycin (mTOR) downstream of Receptor Tyrosine Kinase (RTK) has been recognized for many years. However, the central role of RICTOR (rapamycin-insensitive companion of mTOR) in this pathway has only recently come to light. The function of RICTOR in pan-cancer still needs to be systematically elucidated. In this study, we examined RICTOR's molecular characteristics and clinical prognostic value by pan-cancer analysis. Our findings indicate that RICTOR was overexpressed in twelve cancer types, and a high RICTOR expression was linked to poor overall survival. Moreover, the CRISPR Achilles' knockout analysis revealed that RICTOR was a critical gene for the survival of many tumor cells. Function analysis revealed that RICTOR-related genes were mainly involved in TOR signaling and cell growth. We further demonstrated that the RICTOR expression was significantly influenced by genetic alteration and DNA-methylation in multiple cancer types. Additionally, we found a positive relationship between RICTOR expression and the immune infiltration of macrophages and cancer-associated fibroblasts in Colon adenocarcinoma and Head and Neck squamous cell carcinoma. Finally, we validated the ability of RICTOR in sustaining tumor growth and invasion in the Hela cell line using cell-cycle analysis, the cell proliferation assay, and wound-healing assay. Our pan-cancer analysis highlights the critical role of RICTOR in tumor progression and its potential as a prognostic marker for various cancer types.

Topics: Adenocarcinoma; Colonic Neoplasms; HeLa Cells; Humans; Prognosis; Rapamycin-Insensitive Companion of mTOR Protein; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors

We previously showed that lifelong rapamycin treatment of short-lived Apc. We asked, what effect would pretreatment of Apc. These data indicate that enteric rapamycin prevents or delays colon neoplasia in Apc

Topics: Adenomatous Polyposis Coli Protein; Animals; Carcinogenesis; Colon; Colonic Neoplasms; Disease Models, Animal; Female; Heterozygote; Humans; Intestinal Mucosa; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Transgenic; Sirolimus; Survival Analysis; Time Factors

Colorectal cancer is the third most frequently diagnosed malignancies among both men and women, which has an increased mortality but a poor prognosis. Targeting mTOR becomes an effective approach that shows promising antitumor activities in various cancers including colonic carcinoma. However, the potential mechanism against colon cancer remains incompletely understood. Here, we demonstrated that the anti-cancer effect of AZD8055 and OSI-027 is at least in part modulated by the gradual process of apoptosis initiation, progressing from mTOR suppression, 4EBP1 dephosphorylation, or EZH2 suppression, thereby leading to PUMA-dependent apoptosis via the intrinsic mitochondrial pathway. Furthermore, AZD8055 inhibited colorectal cancer tumor growth in mice significantly. PUMA deletion caused resistance of dual mTOR inhibitors, suggesting PUMA mediated carcinogenesis in vitro and in vivo. Collectively, these findings established a vital status of PUMA in driving the antineoplastic efficacy of targeting mTOR by AZD8055 and OSI-027 and offered the rationales for the current clinical assessment.

Topics: Animals; Apoptosis Regulatory Proteins; Colonic Neoplasms; DNA-Binding Proteins; Female; HCT116 Cells; Humans; Imidazoles; Mice; Mice, Inbred BALB C; Mice, Nude; Molecular Targeted Therapy; Morpholines; Proto-Oncogene Proteins; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Triazines; Xenograft Model Antitumor Assays

Colorectal cancer (CRC) is one of the most common cancers that is characterized by a high mortality due to the strong metastatic potential of the primary tumor and the high rate of therapy resistance. Hereby, evasion of apoptosis is the primary underlying cause of reduced sensitivity of tumor cells to chemo- and radiotherapy. Using RNA affinity chromatography, we identified the tripartite motif-containing protein 25 (TRIM25) as a bona fide caspase-2 mRNA-binding protein in colon carcinoma cells. Loss-of-function and gain-of-function approaches revealed that TRIM25 attenuates the protein levels of caspase-2 without significantly affecting caspase-2 mRNA levels. In addition, experiments with cycloheximide revealed that TRIM25 does not affect the protein stability of caspase-2. Furthermore, silencing of TRIM25 induced a significant redistribution of caspase-2 transcripts from RNP particles to translational active polysomes, indicating that TRIM25 negatively interferes with caspase-2 translation. Functionally, the elevation in caspase-2 upon TRIM25 depletion significantly increased the sensitivity of colorectal cells to drug-induced intrinsic apoptosis as implicated by increased caspase-3 cleavage and cytochrome c release. Importantly, the apoptosis-sensitizing effects by transient TRIM25 knockdown were rescued by concomitant silencing of caspase-2, demonstrating a critical role of caspase-2. Inhibition of caspase-2 by TRIM25 implies a survival mechanism that critically contributes to chemotherapeutic drug resistance in CRC.

Topics: Antineoplastic Agents; Caspase 2; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Cycloheximide; Cysteine Endopeptidases; Dactinomycin; Doxorubicin; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; HEK293 Cells; Humans; Loss of Function Mutation; Protein Stability; Sirolimus; Transcription Factors; Tripartite Motif Proteins; Ubiquitin-Protein Ligases

Relapse, metastasis, and chemo-resistance are the main factors responsible for the failure of surgical treatment of malignant tumors, and typically are the main obstacles to effective cancer treatment. Although significant advances have been made in the field of cancer chemotherapy, many patients still receive inadequate treatment due to the severe adverse effects of these drugs, resulting in an inability to reach therapeutic concentrations at the tumor site with systemic chemotherapy. Thus, a biological patch loaded with chemotherapeutic drugs could be an ideal strategy for the treatment of cancer at the tumor site.. We developed an acellular matrix using the submucosa of porcine jejunum, then loaded this matrix with different amounts of 5-fluorouracil (5-FU) and rapamycin nanoparticles. Cell proliferation and apoptosis were analyzed by flow cytometry and related markers were evaluated using real-time PCR and western blotting. The patches were evaluated in vitro to characterize their release kinetics and therapeutic feasibility. We then analyzed the therapeutic efficacy and systemic toxicity of these patches in vivo by using them in a mouse model of colon cancer.. The patches delivered 5-FU and rapamycin in a controlled manner for more than 8 weeks, arrested the cell cycle of LoVo cells and sw480 cells at G2/M phase, and induced apoptosis in vitro. The patches also suppressed the growth of xenografted tumors in vivo with lower adverse effects than typically observed with systemic administration of these drugs.. We demonstrated that patches loaded with 5-FU-RAPA-PLA-NP significantly inhibited the growth of colon cancer in vitro and in vivo. These results demonstrated the feasibility of the use of a multi-effect biological patch for cancer treatment.

Topics: Acellular Dermis; Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Drug Carriers; Drug Synergism; Fluorouracil; Humans; Male; Mice, Inbred BALB C; Mice, Nude; Sirolimus

Colorectal cancer (CRC) occurs with more aggressiveness in kidney transplant recipients compared to the general population. Immunosuppressive therapy plays a crucial role in the development of post-transplant malignancy. Concretely, cyclosporine A (CsA) has intrinsic pro-oncologic properties, while several studies report a regression of cancer after the introduction of rapamycin (RAPA). However, their effect on the extracellular vesicle (EV) content from CRC cell lines and their relevance in the pre-metastatic niche have not yet been studied. Here, we investigated the effect of RAPA and CsA in EV-miRNAs from metastatic and non-metastatic CRC cell lines and the role of relevant miRNAs transferred into a pre-metastatic niche model. EV-miRNA profiles showed a significant upregulation of miR-6127, miR-6746-5p, and miR-6787-5p under RAPA treatment compared to CsA and untreated conditions in metastatic cell lines that were not observed in non-metastatic cells. From gene expression analysis of transfected lung fibroblasts, we identified 22 shared downregulated genes mostly represented by the histone family involved in chromatin organization, DNA packaging, and cell cycle. These results suggest that EV-miR-6127, miR-6746-5p and miR-6787-5p could be a potential epigenetic mechanism induced by RAPA therapy in the regulation of the pre-metastatic niche of post-transplant colorectal cancer.

Topics: Cell Line, Tumor; Colonic Neoplasms; Cyclosporine; Epigenesis, Genetic; Extracellular Vesicles; Gene Expression Profiling; Humans; Immunosuppression Therapy; MicroRNAs; Sirolimus; Transcription, Genetic

Inflammation contributes to colon cancer initiation. The disease along with allergy and autoimmunity has been on the rise in Western and more recently in developing countries. This shared rise may imply a shared cause. Streptomycetes are known as soil residents and produce numerous antiproliferative, anti-inflammatory/immunosuppressive compounds, e.g. rapamycin and tacrolimus. Recently, Streptomyces has been shown in gut microbiome with a lower prevalence in humans than nonhumans whose microbiomes might be more representative of past humans' in a hunter-gatherer and farming environment. It was previously suggested that Streptomyces producing antiproliferatives/immunosuppressants would be 'old friends' against allergy and autoimmunity as well as inflammatory bowel diseases. Here, it is suggested that these streptomycetes within gut microbiome have also been evolved as 'old friends' to suppress colon tumorigenesis through their numerous antiproliferatives/immunosuppressants. Subsequently, the shortage of exposure to nature in our current lifestyle has cost us the shortage of these friends and vulnerability to colon cancer. An attractive research area in the future would be whether the shortage of Streptomyces exposure can be the underlying reason for colon cancer, allergy and autoimmunity rise, and if the restoration of these 'old friends' through probiotics or more exposure to nature can prevent colon cancer.

Topics: Anti-Inflammatory Agents; Carcinogenesis; Colonic Neoplasms; Friends; Gastrointestinal Microbiome; Humans; Life Style; Microbiota; Probiotics; Sirolimus; Soil; Soil Microbiology; Streptomyces; Tacrolimus

The natural plant-derived product S-allylmercapto-cysteine (SAMC) has been studied in cancer therapy as a single and combination chemotherapeutic agent. The present study was employed to verify the combination use of SAMC and rapamycin that is the mTOR inhibitor with anticancer ability but has limited efficacy due to drug resistance, and to explore the underlying mechanisms. We combined rapamycin and SAMC for colorectal cancer treatment in the HCT‑116 cancer cells and a xenograft murine model. The in vivo study was established by xenografting HCT‑116 cells in BALB/c nude mice. It was found that the combination therapy had enhanced tumor-suppressing ability with the upregulation of the Bax/Bcl-2 ratio as a consequence of activated apoptosis, inhibition of autophagic activity and prevention of Akt phosphorylation. The rapamycin and SAMC combination activated antioxidant transcription expressions of Nrf2 and downstream gene NQO1. Concomitantly, autophagosome cargo p62 was downregulated, indicating that the p62 played a negative-regulatory role between Nrf2 and autophagy. Our results show that the combination of SAMC and rapamycin enhanced the anticancer ability, which could be used for the treatment of colorectal cancer. The underling mechanism of autophagy/p62/Nrf2 pathway discovered may provide a new direction for drug development, especially for traditional Chinese medicines.

Topics: Animals; Antineoplastic Agents; Antioxidants; Apoptosis; Autophagy; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Cysteine; Down-Regulation; Garlic; HCT116 Cells; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; NF-E2-Related Factor 2; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; RNA-Binding Proteins; Signal Transduction; Sirolimus; Transplantation, Heterologous; Xenograft Model Antitumor Assays

We estimated the effect of various immunosuppressants (ISs) and metformin (M) to provide theoretical background of optimal therapeutic strategy for de novo colon cancer after liver transplantation (LT). Three colon cancer cell lines (HT29, SW620, and HCT116) were used in in vitro studies. HT29 was also used in BALB/c-nude mice animal models. Following groups were used in both in vitro and in vivo studies: sirolimus (S), tacrolimus (T), cyclosporin A (CsA), M, metformin/sirolimus (Met/S), metformin/tacrolimus (Met/T), and metformin/cyclosporin A (Met/CsA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed and western blot analyses were performed for mTOR pathway proteins, apoptosis proteins, and epithelial-mesenchymal-transition (EMT) proteins. Tumor volume was measured for 4 weeks after inoculation. MTT-assay revealed significant cell viability inhibition in all 3 colon cancer cell lines in groups of S, M, and Met/S. Of note, group Met/S showed synergistic effect compare to M or S group. Western blot analysis showed significant low levels of all investigated proteins in groups of S and Met/S in both in vitro and in vivo experiment. Tumor growth was significantly inhibited only in the Met/S group. Combination of Met and S showed the most potent inhibition in all colon cancer cell lines. This finding might have application for de novo colon cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Colonic Neoplasms; Cyclosporine; Disease Models, Animal; Drug Synergism; Drug Therapy, Combination; HCT116 Cells; HT29 Cells; Humans; Metformin; Mice; Mice, Inbred BALB C; Mice, Nude; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Smad3 Protein; Tacrolimus; Transplantation, Heterologous

The mammalian target of rapamycin (mTOR) is commonly activated in colon cancer. mTOR complex 1 (mTORC1) is a major downstream target of the PI3K/ATK pathway and activates protein synthesis by phosphorylating key regulators of messenger RNA translation and ribosome synthesis. Rapamycin analogs Everolimus and Temsirolimus are non-ATP-competitive mTORC1 inhibitors, and suppress proliferation and tumor angiogenesis and invasion. We now show that apoptosis plays a key role in their anti-tumor activities in colon cancer cells and xenografts through the DR5, FADD and caspase-8 axis, and is strongly enhanced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and 5-fluorouracil. The induction of DR5 by rapalogs is mediated by the ER stress regulator and transcription factor CHOP, but not the tumor suppressor p53, on rapid and sustained inhibition of 4E-BP1 phosphorylation, and attenuated by eIF4E expression. ATP-competitive mTOR/PI3K inhibitors also promote DR5 induction and FADD-dependent apoptosis in colon cancer cells. These results establish activation of ER stress and the death receptor pathway as a novel anticancer mechanism of mTOR inhibitors.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antineoplastic Agents; Apoptosis; Cell Cycle Proteins; Cell Line, Tumor; Colonic Neoplasms; Everolimus; Female; Humans; Mice, Nude; Phosphoproteins; Phosphorylation; Protein Kinase Inhibitors; Receptors, TNF-Related Apoptosis-Inducing Ligand; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factor CHOP; Xenograft Model Antitumor Assays

Recent studies have correlated physical activity with a better prognosis in cachectic patients, although the underlying mechanisms are not yet understood. In order to identify the pathways involved in the physical activity-mediated rescue of skeletal muscle mass and function, we investigated the effects of voluntary exercise on cachexia in colon carcinoma (C26)-bearing mice. Voluntary exercise prevented loss of muscle mass and function, ultimately increasing survival of C26-bearing mice. We found that the autophagic flux is overloaded in skeletal muscle of both colon carcinoma murine models and patients, but not in running C26-bearing mice, thus suggesting that exercise may release the autophagic flux and ultimately rescue muscle homeostasis. Treatment of C26-bearing mice with either AICAR or rapamycin, two drugs that trigger the autophagic flux, also rescued muscle mass and prevented atrogene induction. Similar effects were reproduced on myotubes in vitro, which displayed atrophy following exposure to C26-conditioned medium, a phenomenon that was rescued by AICAR or rapamycin treatment and relies on autophagosome-lysosome fusion (inhibited by chloroquine). Since AICAR, rapamycin and exercise equally affect the autophagic system and counteract cachexia, we believe autophagy-triggering drugs may be exploited to treat cachexia in conditions in which exercise cannot be prescribed.

Topics: Aminoimidazole Carboxamide; Animals; Autophagosomes; Autophagy; Cachexia; Cell Line, Tumor; Colonic Neoplasms; Female; Humans; Lysosomes; Mice; Mice, Inbred BALB C; Muscle Fibers, Skeletal; Muscle Weakness; Muscle, Skeletal; Neoplasm Transplantation; Physical Conditioning, Animal; Ribonucleotides; Sirolimus; Survival Analysis

Resistance to microtubule-stabilizing agents is a major hurdle for successful cancer therapy. We investigated combined treatment of microtubule-stabilizing agents (MSAs) with inhibitors of angiogenesis to overcome MSA resistance.. Treatment regimens of clinically relevant MSAs (patupilone and paclitaxel) and antiangiogenic agents (everolimus and bevacizumab) were investigated in genetically defined MSA-resistant lung (A549EpoB40) and colon adenocarcinoma (SW480) tumor xenografts in nude mice (CD1-Foxn1, ICRnu; 5-14 per group). Tumor growth delays were calculated by Kaplan-Meier analysis with Holm-Sidak tests. All statistical tests were two-sided.. Inhibition of mTOR-kinase by everolimus only minimally reduced the proliferative activity of β tubulin-mutated lung adenocarcinoma cells alone and in combination with the MSA patupilone, but everolimus inhibited expression and secretion of vascular endothelial growth factor (VEGF) from these cells. mTOR-kinase inhibition strongly sensitized tumor xenografts derived from these otherwise MSA-resistant tumor cells to patupilone. Tumors treated with the combined modality of everolimus and patupilone had statistically significantly reduced tumor volume and stronger tumor growth delay (16.2 ± 1.01 days) than control- (7.7 ± 0.3 days, P = .004), patupilone- (10 ± 0.97 days, P = .009), and everolimus-treated (10.6 ± 1.4 days, P = .014) tumors. A combined treatment modality with bevacizumab also resensitized this MSA-refractory tumor model to patupilone. Treatment combination also strongly reduced microvessel density, corroborating the relevance of VEGF targeting for the known antivasculature-directed potency of MSA alone in MSA-sensitive tumor models. Resensitization to MSAs was also probed in P glycoprotein-overexpressing SW480-derived tumor xenografts. Different bevacizumab regimens also sensitized this otherwise-resistant tumor model to clinically relevant MSA paclitaxel.. A treatment combination of MSAs with antiangiogenic agents is potent to overcome tumor cell-linked MSA resistance and should be considered as strategy for MSA-refractory tumor entities.

Topics: Adenocarcinoma; Angiogenesis Inhibitors; Animals; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Bevacizumab; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Drug Resistance, Neoplasm; Epothilones; Everolimus; Humans; Kaplan-Meier Estimate; Lung Neoplasms; Mice; Mice, Nude; Microtubules; Paclitaxel; Real-Time Polymerase Chain Reaction; RNA, Neoplasm; Sirolimus; TOR Serine-Threonine Kinases; Tubulin Modulators; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

The mTORC1 inhibitors, rapamycin and its analogs, are known to show only modest antitumor activity in clinic, but the underlying mechanisms remain largely elusive. Here, we found that activated AKT signaling is associated with rapamycin resistance in breast and colon cancers by sustained phosphorylation of the translational repressor 4E-BP1. Treatment of tumor cells with rapamycin or the AKT inhibitor MK2206 showed a limited activity in inhibiting 4E-BP1 phosphorylation, cap-dependent translation, cell growth and motility. However, treatment with both drugs resulted in profound effects in vitro and in vivo. Mechanistic investigation demonstrated that the combination treatment was required to effectively inhibit PRAS40 phosphorylation on both Ser183 and Thr246 mediated by mTORC1 and AKT respectively, and with the combined treatment, dephosphorylated PRAS40 binding to the raptor/mTOR complex was enhanced, leading to dramatic repression of mTORC1-regulated 4E-BP1 phosphorylation and translation. Knockdown of PRAS40 or 4E-BP1 expression markedly reduced the dependence of tumor cells on AKT/mTORC1 signaling for translation and survival. Together, these findings reveal a critical role of PRAS40 as an integrator of mTORC1 and AKT signaling for 4E-BP1-mediated translational regulation of tumor cell growth and motility, and highlight PRAS40 phosphorylation as a potential biomarker to evaluate the therapeutic response to mTOR/AKT inhibitors.

Topics: Adaptor Proteins, Signal Transducing; Animals; Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Drug Synergism; Female; Heterocyclic Compounds, 3-Ring; Humans; MCF-7 Cells; Mechanistic Target of Rapamycin Complex 1; Mice, Nude; Multiprotein Complexes; Phosphoproteins; Proto-Oncogene Proteins c-akt; Random Allocation; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Xenograft Model Antitumor Assays

With this study we aimed to research the effects of immunosuppressive drugs, their cumulative doses, and viral infections on development of malign tumors in patients who have undergone treatment for 5 years.. We examined 100 patients who underwent renal transplantation from 2004 to 2009. Patients had mycophenolate mofetil and steroid in addition to cyclosporine, sirolimus, or tacrolimus as immunosuppressive treatment. For malignancy screening, physical examination, radiologic and endoscopic screening were done, and immunosuppressive drugs and their cumulative doses, age, sex, body mass index (BMI), dialysis history, and viral infection history were investigated.. The mean age of patients was 42.03 ± 11.30 years. There were 1 colon cancer patient, 1 retroperitoneal liposarcoma, 1 renal oncocytoma, 3 Kaposi sarcoma patients treated with cyclosporine; in those treated with Tac there were 1 basal cell carcinoma, 1 Kaposi sarcoma, 2 thyroid carcinoma, 1 breast carcinoma, 1 bladder carcinoma, 1 renal cell carcinoma, and 1 colon carcinoma patients. The mean age of patients having carcinoma was statistically significant compared with those without cancer (P < .01). The prednisolone cumulative dose was significantly higher in carcinoma patients than in patients without carcinoma (P < .01).. The use of long-term chronic immunosuppressive therapy may increase the development of cancer. The risk of carcinoma increases with increasing drug dose and time period of the immunosuppressive drug. There was not a negative effect on cancer prevalence in patients with cyclosporine or tacrolimus. But the cumulative dose of steroids significantly increased malignancy occurence.

Topics: Adult; Breast Neoplasms; Carcinoma; Colonic Neoplasms; Cyclosporine; Early Detection of Cancer; Female; Humans; Immunosuppressive Agents; Kidney Transplantation; Male; Middle Aged; Mycophenolic Acid; Neoplasms; Retroperitoneal Neoplasms; Sarcoma, Kaposi; Sirolimus; Steroids; Tacrolimus; Thyroid Neoplasms; Time Factors; Urologic Neoplasms

To detect the cellular sensitivity to adriamycin (ADR) by assessing autophagy, apoptosis, and multidrug resistance gene 1 (mdr1) expression in LoVo/Adr cells.. LoVo/Adr cells were designated accordingly into ADR group, Rapamycin (RAPA) group, ADR plus RAPA, and control group in main observations. Autophage, cell death and mdr1 were examined.. IC50 value of ADR in LoVo/Adr was significantly decreased in response to RAPA (P < 0.05). Autophagy rate of LoVo/Adr cells was higher in the ADR or RAPA-alone group than in control (p < 0.05), while ADR/RAPA combination has significantly increased autophagy rate compared to ADR or RAPA alone (p < 0.05). Compared with controls, apoptosis rate in the RAPA group had no difference (p > 0.05); whereas there was significant difference in ADR group (p < 0.05). Furthermore, apoptosis rate was significantly different in combined RAPA/ADR compared to ADR (p < 0.05). Expression of mRNA and protein P-gp level of mdr 1 gene in LoVo/Adr cells were significantly decreased under RAPA-treated groups at 25 µmol/L and 50 µmol/L (p < 0.05).. This study has indicated that the inhibition of the mTOR pathway reverses multidrug resistance in colorectal cancer cells, which is associated with increased autophagy, apoptosis and reduced mdr1 gene expression in drug-resistant cells treated with Adriamycin.

Topics: Adenocarcinoma; Antibiotics, Antineoplastic; Apoptosis; ATP Binding Cassette Transporter, Subfamily B; Autophagy; Cell Line, Tumor; Colonic Neoplasms; Doxorubicin; Drug Resistance, Neoplasm; Humans; RNA, Messenger; Sirolimus; TOR Serine-Threonine Kinases

A dozen clinical trials examining a combination of temsirolimus and cetuximab in treating metastatic colon cancer are currently underway. We investigated the role of cancerous inhibitor of protein phosphatase 2A (CIP2A) in the synergism between temsirolimus and cetuximab in colon cancer.. Five colon cancer cell lines were used for in vitro studies. Signal transduction pathways were assessed by immunoblotting. The synergism between studied drugs was analyzed with combination indexes. Gene silencing was performed using small interfering RNAs. The efficacies of temsirolimus and cetuximab were tested in nude mice with colon cancer xenografts. Transcriptional activity was assessed using a reporter assay. The inhibitors leupeptin, chloroquine, and MG132 were used to assess protein degradation. The association between CIP2A, clinicopathological parameters, and survival was examined by immunohistochemical staining using a tumor tissue microarray.. Temsirolimus decreased the resistance of cells to cetuximab by both inhibiting transcription of CIP2A and increasing degradation of CIP2A through the lysosomal-autophagy pathway. The mammalian target of rapamycin (mTOR) protein immunoprecipitated along with CIP2A. Temsirolimus decreased expression of phosphorylated extracellular regulated protein kinase (pErk) and phosphorylated v-akt murine thymoma viral oncogene (pAKT) and decreased the interaction of CIP2A and mTOR in cell lines without the K-ras codon 12 mutation. CIP2A was a prognostic marker only in colon cancer patients with weak expression of pErk or pAKT.. Temsirolimus decreases cellular resistance to cetuximab by regulating CIP2A expression in colon cancer cells. Potential biomarkers for CIP2A inhibitors include pErk and pAKT.

Topics: Animals; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autoantigens; Biomarkers, Tumor; Blotting, Western; Cell Proliferation; Cetuximab; Colonic Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Immunoprecipitation; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Nude; Phosphorylation; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Sirolimus

Intracellular Mg(2+) levels are strictly regulated; however, the biological importance of intracellular Mg(2+) levels and the pathways that regulate them remain poorly understood. Here, we determined that intracellular Mg(2+) is important in regulating both energy metabolism and tumor progression. We determined that CNNM4, a membrane protein that stimulates Mg(2+) efflux, binds phosphatase of regenerating liver (PRL), which is frequently overexpressed in malignant human cancers. Biochemical analyses of cultured cells revealed that PRL prevents CNNM4-dependent Mg(2+) efflux and that regulation of intracellular Mg(2+) levels by PRL and CNNM4 is linked to energy metabolism and AMPK/mTOR signaling. Indeed, treatment with the clinically available mTOR inhibitor rapamycin suppressed the growth of cancer cells in which PRL was overexpressed. In ApcΔ(14/+) mice, which spontaneously form benign polyps in the intestine, deletion of Cnnm4 promoted malignant progression of intestinal polyps to adenocarcinomas. IHC analyses of tissues from patients with colon cancer demonstrated an inverse relationship between CNNM4 expression and colon cancer malignancy. Together, these results indicate that CNNM4-dependent Mg(2+) efflux suppresses tumor progression by regulating energy metabolism.

Topics: Adenocarcinoma; AMP-Activated Protein Kinases; Animals; Antibiotics, Antineoplastic; Cation Transport Proteins; Colonic Neoplasms; Colonic Polyps; Energy Metabolism; Gene Expression Regulation, Neoplastic; Humans; Ion Transport; Magnesium; Mice; Mice, Knockout; Neoplasm Proteins; Neoplasms, Experimental; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Unresectable colorectal liver metastases remain a major unresolved issue and more effective novel regimens are urgently needed. While screening synergistic drug combinations for colon cancer therapy, we identified a novel multidrug treatment for colon cancer: chemotherapeutic agent melphalan in combination with proteasome inhibitor bortezomib and mTOR (mammalian target of rapamycin) inhibitor rapamycin. We investigated the mechanisms of synergistic antitumor efficacy during the multidrug treatment. All experiments were performed with highly metastatic human colon cancer CX-1 and HCT116 cells, and selected critical experiments were repeated with human colon cancer stem Tu-22 cells and mouse embryo fibroblast (MEF) cells. We used immunochemical techniques to investigate a cross-talk between apoptosis and autophagy during the multidrug treatment. We observed that melphalan triggered apoptosis, bortezomib induced apoptosis and autophagy, rapamycin caused autophagy and the combinatorial treatment-induced synergistic apoptosis, which was mediated through an increase in caspase activation. We also observed that mitochondrial dysfunction induced by the combination was linked with altered cellular metabolism, which induced adenosine monophosphate-activated protein kinase (AMPK) activation, resulting in Beclin-1 phosphorylated at Ser 93/96. Interestingly, Beclin-1 phosphorylated at Ser 93/96 is sufficient to induce Beclin-1 cleavage by caspase-8, which switches off autophagy to achieve the synergistic induction of apoptosis. Similar results were observed with the essential autophagy gene, autophagy-related protein 7, -deficient MEF cells. The multidrug treatment-induced Beclin-1 cleavage was abolished in Beclin-1 double-mutant (D133A/D146A) knock-in HCT116 cells, restoring the autophagy-promoting function of Beclin-1 and suppressing the apoptosis induced by the combination therapy. These observations identify a novel mechanism for AMPK-induced apoptosis through interplay between autophagy and apoptosis.

Topics: AMP-Activated Protein Kinases; Animals; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Boronic Acids; Bortezomib; Cell Line, Tumor; Colonic Neoplasms; Drug Synergism; Enzyme Activation; Humans; Kinetics; Melphalan; Membrane Proteins; Mice; Mitochondria; Phosphorylation; Phosphoserine; Pyrazines; Sirolimus

The PI3K/AKT/mTOR pathway is frequently dysregulated in cancers and inhibition of mTOR has demonstrated the ability to modulate pro-survival pathways. As such, we sought to determine the ability of the mTOR inhibitor everolimus to potentiate the antitumor effects of irinotecan in colorectal cancer (CRC).. The combinatorial effects of everolimus and irinotecan were evaluated in vitro and in vivo in CRC cell lines harboring commonly found mutations in PIK3CA, KRAS and/or BRAF. Pharmacokinetically-directed dosing protocols of everolimus and irinotecan were established and used to assess the in vivo antitumor effects of the agents. At the end of treatment, 3-6 tumors per treatment arm were harvested for biomarker analysis by NMR metabolomics.. Everolimus and irinotecan/SN38 demonstrated synergistic anti-proliferative effects in multiple CRC cell lines in vitro. Combination effects of everolimus and irinotecan were determined in CRC xenograft models using clinically-relevant dosing protocols. Everolimus demonstrated significant tumor growth inhibition alone and when combined with irinotecan in HT29 and HCT116 tumor xenografts. Metabolomic analysis showed that HT29 tumors were more metabolically responsive than HCT116 tumors. Everolimus caused a decrease in glycolysis in both tumor types whilst irinotecan treatment resulted in a profound accumulation of lipids in HT29 tumors indicating a cytotoxic effect.. Quantitative analysis of tumor growth and metabolomic data showed that the combination of everolimus and irinotecan was more beneficial in the BRAF/PIK3CA mutant HT29 tumor xenografts, which had an additive effect, than the KRAS/PIK3CA mutant HCT116 tumor xenografts, which had a less than additive effect.

Topics: Animals; Antineoplastic Agents, Phytogenic; Camptothecin; Cell Line, Tumor; Class I Phosphatidylinositol 3-Kinases; Colonic Neoplasms; Drug Synergism; Everolimus; Female; Humans; Immunosuppressive Agents; Irinotecan; Metabolome; Mice; Mice, Nude; Mutation; Neoplasm Transplantation; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); ras Proteins; Sirolimus; Transplantation, Heterologous; Xenograft Model Antitumor Assays

A 23-year-old male presented with a three-week-history of crampy abdominal pain and melaena. Colonoscopy revealed a friable mass filling the entire lumen of the cecum; histologically, it was classified as perivascular epithelioid cell tumor (PEComa). An magnetic resonance imaging scan showed, in addition to the primary tumor, two large mesenteric lymph node metastases and four metastatic lesions in the liver. The patient underwent right hemicolectomy and left hemihepatectomy combined with wedge resections of metastases in the right lobe of the liver, the resection status was R0. Subsequently, the patient was treated with sirolimus. After 4 mo of adjuvant mammalian target of rapamycin inhibition he developed two new liver metastases and a local pelvic recurrence. The visible tumor formations were again excised surgically, this time the resection status was R2 with regard to the pelvic recurrence. The patient was treated with 12 cycles of doxorubicin and ifosfamide under which the disease was stable for 9 mo. The clinical course was then determined by rapid tumor growth in the pelvic cavity. Second line chemotherapy with gemcitabine and docetaxel was ineffective, and the patient died 23 mo after the onset of disease. This case report adds evidence that, in malignant PEComa, the mainstay of treatment is curative surgery. If not achievable, the effects of adjuvant or palliative chemotherapy are unpredictable.

Topics: Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Biopsy; Chemotherapy, Adjuvant; Colectomy; Colonic Neoplasms; Colonoscopy; Doxorubicin; Drug Resistance, Neoplasm; Fatal Outcome; Hepatectomy; Humans; Ifosfamide; Liver Neoplasms; Magnetic Resonance Imaging; Male; Pelvic Neoplasms; Perivascular Epithelioid Cell Neoplasms; Predictive Value of Tests; Reoperation; Sirolimus; Time Factors; Treatment Outcome; Young Adult

The phosphatidylinositide-3-kinase (PI3K) signaling pathway is critical for multiple cellular functions including metabolism, proliferation, angiogenesis, and apoptosis, and is the most commonly altered pathway in human cancers. Recently, we developed a novel mouse model of colon cancer in which tumors are initiated by a dominant active PI3K (FC PIK3ca). The cancers in these mice are moderately differentiated invasive mucinous adenocarcinomas of the proximal colon that develop by 50 days of age. Interestingly, these cancers form without a benign intermediary or aberrant WNT signaling, indicating a non-canonical mechanism of tumorigenesis. Since these tumors are dependent upon the PI3K pathway, we investigated the potential for tumor response by the targeting of this pathway with rapamycin, an mTOR inhibitor. A cohort of FC PIK3ca mice were treated with rapamycin at a dose of 6 mg/kg/day or placebo for 14 days. FDG dual hybrid PET/CT imaging demonstrated a dramatic tumor response in the rapamycin arm and this was confirmed on necropsy. The tumor tissue remaining after treatment with rapamycin demonstrated increased pERK1/2 or persistent phosphorylated ribosomal protein S6 (pS6), indicating potential resistance mechanisms. This unique model will further our understanding of human disease and facilitate the development of therapeutics through pharmacologic screening and biomarker identification.

Topics: Animals; Apoptosis; Cell Proliferation; Class I Phosphatidylinositol 3-Kinases; Colon; Colonic Neoplasms; Fluorodeoxyglucose F18; Humans; Male; MAP Kinase Signaling System; Mice; Multimodal Imaging; Necrosis; Phosphatidylinositol 3-Kinases; Positron-Emission Tomography; Protein Kinase Inhibitors; Signal Transduction; Sirolimus; Tomography, X-Ray Computed; TOR Serine-Threonine Kinases

Anorexia-cachexia syndrome (ACS) is a major determinant of cancer-related death that causes progressive body weight loss due to depletion of skeletal muscle mass and body fat. Here, we report the development of a novel preclinical murine model of ACS in which lymphomas harbor elevated Myc and activated mTOR signaling. The ACS phenotype in this model correlated with deregulated expression of a number of cytokines, including elevated levels of interleukin-10 which was under the direct translational control of mTOR. Notably, pharmacologic intervention to impair protein synthesis restored cytokine production to near-normal levels, delayed ACS progression, and extended host survival. Together, our findings suggest a new paradigm to treat ACS by strategies which target protein synthesis to block the production of procachexic factors.

Topics: Animals; Anorexia; Antineoplastic Agents; Body Weight; Cachexia; Cell Line, Tumor; Colonic Neoplasms; Disease Models, Animal; Female; Harringtonines; Homoharringtonine; Humans; Interleukin-10; Kaplan-Meier Estimate; Lymphoma; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Protein Biosynthesis; Proto-Oncogene Proteins c-myc; Signal Transduction; Sirolimus; Syndrome; TOR Serine-Threonine Kinases

The mammalian target of rapamycin (mTOR) is frequently activated in colon cancers due to mutations in the phosphatidylinositol 3-kinase (PI3K) pathway. Targeting mTOR with allosteric inhibitors of mTOR such as rapamycin reduces colon cancer progression in several experimental models. Recently, a new class of mTOR inhibitors that act as ATP-competitive inhibitors of mTOR, has been developed. The effectiveness of these drugs in colon cancer cells has however not been fully characterized.. LS174T, SW480 and DLD-1 colon cancer cell lines were treated with PP242 an ATP-competitive inhibitor of mTOR, NVP-BEZ235, a dual PI3K/mTOR inhibitor or rapamycin. Tumor cell growth, proliferation and survival were assessed by MTS assay, 5-bromo-2'-deoxyuridine (BrDU) incorporation or by quantification of DNA fragmentation respectively. In vivo, the anticancer activity of mTOR inhibitors was evaluated on nude mice bearing colon cancer xenografts.. PP242 and NVP-BEZ235 reduced the growth, proliferation and survival of LS174T and DLD-1 colon cancer cells more efficiently than rapamycin. Similarly, PP242 and NVP-BEZ235 also decreased significantly the proliferation and survival of SW480 cells which were resistant to the effects of rapamycin. In vivo, PP242 and NVP-BEZ235 reduced the growth of xenografts generated from LS174T and SW480 cells. Finally, we also observed that the efficacy of ATP-competitive inhibitors of mTOR was enhanced by U0126, a MEK inhibitor.. Taken together, these results show that ATP-competitive inhibitors of mTOR are effective in blocking colon cancer cell growth in vitro and in vivo and thus represent a therapeutic option in colon cancer either alone or in combination with MEK inhibitors.

Topics: Animals; Antibiotics, Antineoplastic; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colonic Neoplasms; Female; Humans; Imidazoles; Immunohistochemistry; Indoles; Male; Mice; Protein Kinase Inhibitors; Purines; Quinolines; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

The incidence of Kaposi sarcoma (KS) has substantially increased among immunocompromised patients, suggesting a role for immunosuppressive drugs. The aim of this study was to evaluate the incidence, features, and outcome of KS among 307 kidney transplantation patients at our center between January 1994 and June 2010. During the study period, the 10 patients who developed KS (3.25%) showed a mean age at transplantation of 35.8 ± 8.7 years (range, 22 to 49 years). The mean interval between transplantation and occurrence of KS was 24.7 ± 21.36 months (range, 6 to 64 months). The mean time of antithymocyte globulin induction was 9.5 days (range, 6 to 13 days). KS was restricted to the skin in 7 cases, among which, one presented with associated Hodgkin lymphoma. Visceral involvement (one lung and one colon) was observed in two cases. One patient presented with a gastric KS without skin lesions. Immunosuppressive treatment was reduced, then withdrawn in three cases, resulting in regression of KS a few weeks later, but with graft loss requiring hemodialysis at 1, 3 and 4 months. Among the remaining 7 cases, we stopped mycophenalate mofetil (MMF) and switched from calcineurin inhibitors to sirolimus. Allograft function remained stable after the switch. Only one patient who already had allograft dysfunction due to biopsy-proven chronic allograft nephropathy. Deteriorated progressively, undergoing hemodialysis at 2 years after KS diagnosis. In conclusion, we observed a relatively high incidence of KS among our cases. The introduction of sirolimus resulted in complete regression of KS lesions with preserved graft function.

Topics: Adult; Calcineurin Inhibitors; Colonic Neoplasms; Drug Substitution; Drug Therapy, Combination; Female; Graft Rejection; Graft Survival; Humans; Immunosuppressive Agents; Incidence; Kidney Transplantation; Lung Neoplasms; Male; Middle Aged; Renal Dialysis; Sarcoma, Kaposi; Sirolimus; Skin Neoplasms; Stomach Neoplasms; Time Factors; Treatment Outcome; Tunisia

PHLPP belongs to a novel family of protein phosphatases that serve as negative regulators of Akt. There are two isoforms, PHLPP1 and PHLPP2, identified in this family. Our previous studies indicated a tumor suppressor role of both PHLPP isoforms in colon cancer. Here we report that the expression of PHLPP is controlled by mTOR-dependent protein translation in colon and breast cancer cells. Treating cells with rapamycin or knockdown of mTOR using RNAi results in a marked decrease of PHLPP protein expression. In contrast, stable knockdown of TSC2, a negative regulator of mTOR activity, increases PHLPP expression. The rapamycin-mediated down-regulation of PHLPP is blocked by expression of a rapamycin-insensitive mutant of p70S6K. In addition, depletion of 4E-BP1 expression by RNAi results in an increase of PHLPP expression and resistance to rapamycin-induced down-regulation. Moreover, inhibition of mTOR activity by amino acid or glucose starvation reduces PHLPP expression in cells. Functionally, we show that rapamycin-mediated inhibition of PHLPP expression contributes to rapamycin resistance in colon cancer cells. Thus, our studies identify a compensatory feedback regulation in which the activation of Akt is inhibited by up-regulation of PHLPP through mTOR, and this mTOR-dependent expression of PHLPP subsequently determines the rapamycin sensitivity of cancer cells.

Topics: Adaptor Proteins, Signal Transducing; Antibiotics, Antineoplastic; Cell Cycle Proteins; Cell Line, Tumor; Colonic Neoplasms; Down-Regulation; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Nuclear Proteins; Phosphoprotein Phosphatases; Phosphoproteins; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

Targeting the mTOR signaling pathway with rapamycin in cancer therapy has been less successful than expected due in part to the removal of a negative feedback loop resulting in the over-activation of the PI3K/Akt signaling pathway. As the c-Jun N-terminal kinase (JNK) signaling pathway has been found to be a functional target of PI3K, we investigate the role of JNK in the anticancer efficacy of rapamycin.. The colon cancer cell line LS174T was treated with rapamycin and JNK phosphorylation was analyzed by Western Blot. Overexpression of a constitutively negative mutant of JNK in LS174T cells or treatment of LS174T cells with the JNK inhibitor SP600125 were used to determine the role of JNK in rapamycin-mediated tumor growth inhibition.. Treatment of LS174T cells with rapamycin resulted in the phosphorylation of JNK as observed by Western Blot. The expression of a negative mutant of JNK in LS174T cells or treatment of LS174T cells with SP600125 enhanced the antiproliferative effects of rapamycin. In addition, in vivo, the antitumor activity of rapamycin was potentiated on LS174T tumor xenografts that expressed the dominant negative mutant of JNK.. Taken together, these results show that rapamycin-induced JNK phosphorylation and activation reduces the antitumor efficacy of rapamycin in LS174T cells.

Topics: Adenocarcinoma; Animals; Anthracenes; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Enzyme Inhibitors; Female; Humans; JNK Mitogen-Activated Protein Kinases; Mice; Mice, Nude; Models, Animal; Phosphorylation; Signal Transduction; Sirolimus; Xenograft Model Antitumor Assays

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Benzenesulfonates; Bevacizumab; Cetuximab; Colonic Neoplasms; Diagnosis, Differential; Dose-Response Relationship, Drug; Drug Delivery Systems; Drug Eruptions; ErbB Receptors; Erlotinib Hydrochloride; Everolimus; Facial Dermatoses; Humans; Indoles; Liver Neoplasms; Male; Middle Aged; Neoplasms; Niacinamide; Phenylurea Compounds; Pyridines; Pyrroles; Quinazolines; Sirolimus; Sorafenib; Sunitinib; Trastuzumab

Most genetically engineered mouse (GEM) models for colon cancer are based on tissuewide or germline gene modification, resulting in tumors predominantly of the small intestine. Several of these models involve modification of the adenomatous polyposis coli (Apc) gene and are excellent models for familial cancer predisposition syndromes. We have developed a stochastic somatic mutation model for sporadic colon cancer that presents with isolated primary tumors in the distal colon and recapitulates the entire adenoma-carcinoma-metastasis axis seen in human colon cancer. Using this model, we have analyzed tumors that are either solely mutant in the Apc gene or in combination with another colon cancer-associated mutant gene, the Kras G12D allele. Because of the restricted location in the distal colon, the natural history of the tumors can be analyzed by serial colonoscopy. As the mammalian target of rapamycin (mTOR) pathway is a critical component of the complex signaling network in colon cancer, we used this model to assess the efficacy of mTOR blockade through rapamycin treatment of mice with established tumors. After treatment, Apc mutant tumors were more than 80% smaller than control tumors. However, tumors that possessed both Apc and Kras mutations did not respond to rapamycin treatment. These studies suggest that mTOR inhibitors should be further explored as potential colorectal cancer therapies in patients whose tumors do not have activating mutations in KRAS.

Topics: Animals; Antibiotics, Antineoplastic; Colonic Neoplasms; Disease Models, Animal; Disease Progression; Genes, APC; Intracellular Signaling Peptides and Proteins; Mice; Mice, Knockout; Mutation; Neoplasm Metastasis; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins p21(ras); Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Mammalian target of rapamycin (mTOR) inhibitors such as rapamycin have shown modest effects in cancer therapy due in part to the removal of a negative feedback loop leading to the activation of the phosphatidylinositol 3-kinase/Akt (PI3K/Akt) signaling pathway. In this report, we have investigated the role of FOXO1, a downstream substrate of the PI3K/Akt pathway in the anticancer efficacy of rapamycin.. Colon cancer cells were treated with rapamycin and FOXO1 phosphorylation was determined by Western blot. Colon cancer cells transfected with a constitutively active mutant of FOXO1 or a control plasmid were treated with rapamycin and the antiproliferative efficacy of rapamycin was monitored.. Rapamycin induced the phosphorylation of FOXO1 as well as its translocation from the nucleus to the cytoplasm, leading to FOXO1 inactivation. The expression of an active mutant of FOXO1 in colon cancer cells potentiated the antiproliferative efficacy of rapamycin in vitro and its antitumor efficacy in vivo.. Taken together these results show that rapamycin-induced FOXO1 inactivation reduces the antitumor efficacy of rapamycin.

Topics: Animals; Antibiotics, Antineoplastic; Cell Growth Processes; Cell Line, Tumor; Colonic Neoplasms; Female; Forkhead Box Protein O1; Forkhead Transcription Factors; HT29 Cells; Humans; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Nude; Multiprotein Complexes; Proteins; Proto-Oncogene Proteins c-akt; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Transfection; Xenograft Model Antitumor Assays

Malignancies are a well-known complication of immunosuppressive therapy among renal transplant recipients, representing an important cause of long-term morbidity and mortality. Rapamycin has been shown to limit the proliferation of a number of malignant cell lines in vivo and in vitro.. Fifteen patients developed the following malignancies at a mean of 90.3 months (range = 10-252) after kidney transplantation: metastatic gastric cancer (n = 1), metastatic colon cancer (n = 1), bilateral nephrourothelioma (n = 1), skin cancer (n = 2), Kaposi's sarcoma (n = 2), posttransplant lymphoproliferative disorder (PTLD; n = 4), renal cell carcinoma T1 (n = 1), MALT lymphoma (n = 1), intramucous colon carcinoma (n = 1), liposarcoma of the spermatic cord (n = 1). After the diagnosis of malignancy, the patients were switched from calcineurin inhibitor-based immunosuppression to rapamycin (monotherapy, n = 3), or associated with steroids (n = 6) or with mycophenolate mofetil (n = 6).. Both patients with metastatic cancer underwent chemotherapy but succumbed after 6 and 13 months. Two patients with PTLD who underwent chemotherapy died after 12 and 36 months. At a mean follow-up of 32.7 months (range = 7-56), the remaining 11 patients are cancer-free. Two patients lost their grafts after 24 and 36 months after the switch due to chronic rejection. Renal graft function remained stable in all other patients from diagnosis throughout follow-up.. Our observations suggested that rapamycin-based immunosuppression offers the possibility for regression of nonmetastatic tumors. Nevertheless, it is difficult to assess whether tumor regression was due to rapamycin treatment or to the reduced immunosuppression.

Topics: Cell Division; Cell Line, Tumor; Colonic Neoplasms; Genital Neoplasms, Male; Humans; Immunosuppression Therapy; Immunosuppressive Agents; Kidney Transplantation; Liposarcoma; Male; Neoplasm Metastasis; Neoplasms; Sirolimus; Skin Neoplasms; Stomach Neoplasms

Despite recent progress, colon cancer is often resistant to combination chemotherapy, highlighting the need for development of novel therapeutic approaches. An attractive target is hypoxia-inducible factor-1alpha (HIF-1alpha), a key transcription factor with a pivotal role in tumor cell metabolism. One potential class of therapeutic agents targeting HIF-1alpha are mammalian target of rapamycin inhibitors such as rapamycin. A second class are topoisomerase I inhibitors, such as irinotecan, which are able to inhibit the accumulation of HIF-1alpha. We here investigated whether combination of rapamycin and irinotecan was active in human colon cancer models.. Human metastatic tumors were xenografted in nude mice and treated with low doses of irinotecan alone, rapamycin alone, or combination of both drugs. The cellular effects of irinotecan and rapamycin were further characterized for HT-29 and HCT-116 colon cancer cells in vitro.. In contrast to single-agent therapy, xenografted tumors treated with combination of irinotecan and rapamycin showed potent inhibition of the mammalian target of rapamycin/HIF-1alpha axis, which was accompanied by a dramatic reduction in tumor volume. In vitro experiments showed that exposure to low concentrations of the two drugs resulted in massive HT-29 cell death under hypoxic, but not normoxic, conditions, in full agreement with a cytotoxic effect mediated through HIF-1alpha rather than through induction of genotoxic lesions. HCT-116 cells were less sensitive to the combined treatment due to constitutive activation of phosphatidylinositol 3-kinase/Akt and Ras/mitogen-activated protein kinase pathways.. These results identify HIF-1alpha as a promising target and provide a rationale for clinical trials of low-dose irinotecan and rapamycin combination toward metastatic colon cancer.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Camptothecin; Cell Line, Tumor; Colonic Neoplasms; Glycolysis; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Irinotecan; Male; MAP Kinase Signaling System; Mice; Phosphatidylinositol 3-Kinases; Protein Kinases; Proto-Oncogene Proteins c-akt; Sirolimus; TOR Serine-Threonine Kinases; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Toll-like receptor 4 (TLR4) signaling in tumor cells can promote tumor escape and tumor progression, for which TLR4-triggered resistance of tumor cells to apoptosis has been proposed as one of the mechanisms. Rapamycin is an immunosuppressant agent widely used for treatment of transplantation rejection and autoimmune diseases, and recently used for cancer therapy. However, the underlying mechanisms responsible for antitumor effects of rapamycin remain to be fully elucidated. Here we report that rapamycin can reverse TLR4-triggered resistance of colon cancer cells to oxaloplatin- or doxorubicin-induced apoptosis by disrupting Akt and subsequent NF-kappaB activation, suppressing upregulation of anti-apoptotic protein Bcl-xL. Furthermore, Akt/NF-kappaB inhibitors also reverses the apoptosis resistance, accordingly, Akt constitutive activation rescues NF-kappaB activation and Bcl-xL expression in rapamycin-pretreated colon cancer cells, suggesting Akt disruption is critical to the process. Therefore, rapamycin may abrogate TLR4-triggered tumor apoptosis resistance by inhibiting Akt/NF-kappaB pathways and Bcl-xL expression, providing experimental evidence for the anti-tumor effect of rapamycin.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; bcl-X Protein; Cell Line, Tumor; Colonic Neoplasms; Doxorubicin; Humans; Immunosuppressive Agents; Lipopolysaccharides; Mice; NF-kappa B; Proto-Oncogene Proteins c-akt; Sirolimus; Toll-Like Receptor 4; Up-Regulation

Radiotherapy exerts direct antivascular effects in tumors and also induces a proangiogenic stress response in tumor cells via the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (mTOR) pathway. Therefore, the combination of radiotherapy and antiangiogenic therapy with mTOR inhibitor RAD001 (Everolimus) might exert additive/synergistic effects on tumor growth.. Effects of radiation combined with mTOR inhibitor RAD001 were studied on proliferation of murine colon cancer CT-26, human pancreatic cancer L3.6pl, and human umbilical vascular endothelial cells in vitro. In vivo tumor growth of subcutaneous colon cancer CT 26 and orthotopic pancreatic cancer L3.6pl was assessed after fractionated radiotherapy (5 x 2 or 5 x 4 Gy) with or without the addition of the mTOR inhibitor RAD001. RAD001 (1.5 mg/kg/d) was administered until the end of experiments beginning before or after radiotherapy.. A single dose of 2 Gy reduced in vitro proliferation of L3.6pl (-16%), CT-26 (-70%), and human umbilical vascular endothelial cells (HUVEC; -72%). The mTOR inhibitor RAD001 (10 ng/mL) suppressed proliferation of HUVEC (-83%), L3.6pl (-8%), and CT-26 (-82%). Combination of even low concentrations of 0.01 ng/mL RAD001 and 0.25 Gy radiation significantly reduced proliferation of HUVECs (-57%), whereas additive effects of RAD001 and radiation on tumor cells were seen only at the highest concentrations tested. In vivo, RAD001 introduced before radiotherapy (5 x 2 Gy) improved tumor growth control in mice (L3.6pl: 326 mm(3) versus 1144 mm(3); CT-26: 210 mm(3) versus 636 mm(3); P < 0.05 versus control). RAD001 turned out to possess a dose-modifying effect on radiotherapy.. Endothelial cells seem to be most sensitive to combination of mTOR inhibition and radiotherapy. Additive tumor growth delay using the mTOR inhibitor RAD001 and radiotherapy in vivo therefore might rely on combined antiangiogenic and antivascular effects.

Topics: Angiogenesis Inhibitors; Animals; Cell Division; Cell Line, Tumor; Colonic Neoplasms; Endothelium, Vascular; Everolimus; Humans; Mice; Microcirculation; Pancreatic Neoplasms; Radiation-Sensitizing Agents; Sirolimus

Toll-like receptor 4 (TLR4) signaling in tumor cells can mediate tumor cell immune escape and tumor progression, being regarded as one of the mechanisms for chronic inflammation in tumorigenesis and progression. So, intervention of TLR4-mediated immune escape and metastasis has been proposed as one of the approaches to cancer prevention and treatment. Rapamycin, an immunosuppressant agent widely used for treatment of autoimmune diseases and transplantation rejection, is recently used for cancer therapy. However, the underlying mechanisms remain to be fully understood. In the present study, we demonstrate that rapamycin can significantly inhibit TLR4-triggered IL-6 and PGE(2) production and invasion of colon cancer cells. Suppression of TLR4-induced IL-6 and PGE(2) production is responsible for the rapamycin-mediated decrease of TLR4-evoked invasion of colon cancer cells. Furthermore, disruption of NF-kappaB pathway contributes to the inhibition of TLR4-induced IL-6, PGE(2) production and invasion by rapamycin in colon cancer cells. Rapamycin can also downregulate TLR4 expression. Therefore, we demonstrate that rapamycin may abrogate TLR4-triggered tumor cell immune escape and invasion by downregulating TLR4 expression and inhibiting TLR4-activated NF-kappaB pathway, thus providing new mechanistic explanation for the antitumor effect of rapamycin.

Topics: Animals; Autocrine Communication; Cell Line, Tumor; Cell Movement; Colonic Neoplasms; Dinoprostone; Down-Regulation; Enzyme Activation; Gene Expression Regulation, Neoplastic; Interleukin-6; Lipopolysaccharides; Mice; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; NF-kappa B; Sirolimus; Toll-Like Receptor 4

The mammalian target of rapamycin (mTOR) inhibitor CCI-779 (temsirolimus) is a recently Food and Drug Administration-approved anticancer drug with efficacy in certain solid tumors and hematologic malignancies. In cell culture studies, CCI-779 at the commonly used nanomolar concentrations generally confers a modest and selective antiproliferative activity. Here, we report that, at clinically relevant low micromolar concentrations, CCI-779 completely suppressed proliferation of a broad panel of tumor cells. This "high-dose" drug effect did not require FKBP12 and correlated with an FKBP12-independent suppression of mTOR signaling. An FKBP12-rapamycin binding domain (FRB) binding-deficient rapamycin analogue failed to elicit both the nanomolar and micromolar inhibitions of growth and mTOR signaling, implicating FRB binding in both actions. Biochemical assays indicated that CCI-779 and rapamycin directly inhibited mTOR kinase activity with IC(50) values of 1.76 +/- 0.15 and 1.74 +/- 0.34 micromol/L, respectively. Interestingly, a CCI-779-resistant mTOR mutant (mTOR-SI) displayed an 11-fold resistance to the micromolar CCI-779 in vitro (IC(50), 20 +/- 3.4 micromol/L) and conferred a partial protection in cells exposed to micromolar CCI-779. Treatment of cancer cells with micromolar but not nanomolar concentrations of CCI-779 caused a marked decline in global protein synthesis and disassembly of polyribosomes. The profound inhibition of protein synthesis was accompanied by rapid increase in the phosphorylation of translation elongation factor eEF2 and the translation initiation factor eIF2 alpha. These findings suggest that high-dose CCI-779 inhibits mTOR signaling through an FKBP12-independent mechanism that leads to profound translational repression. This distinctive high-dose drug effect could be directly related to the antitumor activities of CCI-779 and other rapalogues in human cancer patients.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Division; Cell Line; Cell Line, Tumor; Colonic Neoplasms; Female; Humans; Kidney; Lung Neoplasms; Male; Prostatic Neoplasms; Protein Biosynthesis; Protein Kinases; Sirolimus; Tacrolimus Binding Protein 1A; TOR Serine-Threonine Kinases

The receptor for epidermal growth factor (EGFR) is overexpressed in many cancers. One important signaling pathway regulated by EGFR is the phosphatidylinositol 3'-kinase (PI3K)-phosphoinositide-dependent kinase 1-Akt pathway. Activation of Akt leads to the stimulation of antiapoptotic pathways, promoting cell survival. Akt also regulates the mammalian target of rapamycin (mTOR)-S6K-S6 pathway to control cell growth in response to growth factors and nutrients. Recent reports have shown that the sensitivity of non-small-cell lung cancer cell lines to EGFR inhibitors such as erlotinib (Tarceva, OSI Pharmaceuticals) is dependent on inhibition of the phosphatidylinositol 3'-kinase-phosphoinositide-dependent kinase 1-Akt-mTOR pathway. There can be multiple inputs to this pathway as activity can be regulated by other receptors or upstream mutations. Therefore, inhibiting EGFR alone may not be sufficient for substantial inhibition of all tumor cells, highlighting the need for multipoint intervention. Herein, we sought to determine if rapamycin, an inhibitor of mTOR, could enhance erlotinib sensitivity for cell lines derived from a variety of tissue types (non-small-cell lung, pancreatic, colon, and breast). Erlotinib could inhibit extracellular signal-regulated kinase, Akt, and S6 only in cell lines that were the most sensitive. Rapamycin could fully inhibit S6 in all cell lines, but this was accompanied by activation of Akt phosphorylation. However, combination with erlotinib could down-modulate rapamycin-stimulated Akt activity. Therefore, in select cell lines, inhibition of both S6 and Akt was achieved only with the combination of erlotinib and rapamycin. This produced a synergistic effect on cell growth inhibition, observations that extended in vivo using xenograft models. These results suggest that combining rapamycin with erlotinib might be clinically useful to enhance response to erlotinib.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Dose-Response Relationship, Drug; Drug Synergism; ErbB Receptors; Erlotinib Hydrochloride; Female; HCT116 Cells; HT29 Cells; Humans; Lung Neoplasms; Mice; Mice, Transgenic; Pancreatic Neoplasms; Protein Kinase Inhibitors; Quinazolines; Sirolimus

Rapamycin has antiangiogenic activity against tumors. This has been discussed while addressing the problem of cancer in organ transplantation. Here we investigated effective dosing schedules against tumors and angiogenesis. Growth of established CT-26 colon adenocarcinoma tumors was measured in Balb/c mice treated with total equivalent rapamycin doses (1.5 mg/kg/day) given once a day, once every 3 days, or by continuous infusion. Tumors were most inhibited with continuous rapamycin infusion, and less by bolus dosing. Interestingly, however, continuous dosing produced the lowest rapamycin blood levels (15 ng/ml). As rapamycin-sensitive p70S6-kinase intracellular signaling is critical for angiogenesis, p70S6-kinase activation was measured in endothelial cells by Western blotting. Maximal p70S6-kinase inhibition occurred from 1-5 ng/ml rapamycin. These same rapamycin concentrations optimally blocked vessel-sprouting from cultured aortic rings. Therefore, low-level rapamycin dosing most effectively controls tumors in mice. Importantly, antiangiogenic rapamycin levels are compatible with immunosuppressive doses, supporting its potential use in transplant patients with cancer.

Topics: Adenocarcinoma; Angiogenesis Inhibitors; Animals; Aorta, Thoracic; Cell Line, Tumor; Cells, Cultured; Colonic Neoplasms; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelium, Vascular; Humans; Male; Mice; Mice, Inbred BALB C; Neoplasms; Rats; Rats, Inbred Lew; Sirolimus

Topics: Adenoviridae; Animals; Clinical Trials as Topic; Colonic Neoplasms; Drug Screening Assays, Antitumor; Everolimus; Humans; Immunosuppressive Agents; Newcastle disease virus; Sirolimus; Viral Vaccines; Virus Replication

Selectively replicating adenoviruses have the potential to cure cancer but have shown little efficacy in clinical trials. We have tested the ability of the mTOR kinase inhibitor RAD001 (everolimus) to enhance the response of xenografts to an oncolytic adenovirus. The virus has Tcf sites inserted in the early viral promoters and replicates selectively in cells with activation of the Wnt signaling pathway. To enhance tumor cell infection, an integrin targeting peptide (CDCRGDCFC) was inserted into the fiber gene of the virus. RAD001 combines three useful properties: it inhibits tumor cell growth directly, blocks angiogenesis, and suppresses the immune response. RAD001 does not block viral protein expression, DNA replication, or cytopathic effect in tumor cells in vitro. After 6 weeks of daily RAD001 treatment, ongoing viral DNA replication could be detected in tumor xenografts, showing that RAD001 does not inhibit virus replication in vivo. I.v. injection of virus alone produced a small delay in xenograft growth, whereas combination therapy substantially prolonged the survival of the mice. We suggest that collapsing the tumor vasculature after the initial infection traps the virus and facilitates local spread within the tumor. Unlike conventional drugs, which require continued access to the tumor through the vascular system, oncolytic viruses are in principle less sensitive to late reductions in perfusion because they are produced locally within the tumor.

Topics: Adenoviridae; Animals; Cell Line, Tumor; Colonic Neoplasms; Combined Modality Therapy; Everolimus; Female; HCT116 Cells; HeLa Cells; HT29 Cells; Humans; Immunosuppressive Agents; Integrins; Intercellular Signaling Peptides and Proteins; Mice; Mice, Nude; Peptides; Protein Kinase Inhibitors; Signal Transduction; Sirolimus; Virus Replication; Wnt Proteins; Xenograft Model Antitumor Assays

We have constructed an oncolytic adenovirus expressing the Escherichia coli nitroreductase gene nfsB from an internal ribosome entry site (IRES) in the adenovirus L5 major late transcript. The virus (Tcf-NTR) has Tcf transcription factor-binding sites in the E1A, E1B, and E4 promoters, which restrict viral replication to cells that have activation of the Wnt signaling pathway. This virus was compared with an E1B-55K-deleted virus expressing nitroreductase (NTR) from a cytomegalovirus (CMV) promoter in the E1B-55K region [CRAd-NTR(PS1217H6)]. Both viruses express NTR in colorectal cancer cell lines and show increased cytopathic effect in the presence of the prodrug CB1954. Unlike the Tcf-NTR virus, the CMV-NTR virus expresses NTR in human lung fibroblasts and sensitizes these normal cells to CB1954. The in vivo activity of the viruses was tested in SW620 xenografts in nude mice by intravenous injection of 1,011 particles of virus followed 1 week later by intraperitoneal injections of CB1954. The CMV-NTR virus produced minimal effects in this model. The median time to form 1,000-mm(3) tumors in mice treated with the Tcf-NTR virus plus CB1954 was increased from 14 to 26 days (p=0.003), but this was due mainly to the direct oncolytic effect of the virus. Combination therapy with 3 x 10(11) particles of Tcf-NTR virus (given intravenously) and the mammalian target of rapamycin (mTOR) inhibitor RAD001 (everolimus) (given orally) significantly improved survival (median, >50 days), and addition of CB1954 to this regimen further delayed tumor growth. These results show that the Tcf-NTR virus is more tumor selective and active than the CMV-NTR virus. At the level of transduction that can be achieved currently with oncolytic viruses given intravenously, drugs such as RAD001, which do not require activation by the virus, produce greater increases in efficacy than prodrugs such as CB1954.

Topics: Adenoviridae; Animals; Antibiotics, Antineoplastic; Aziridines; Cell Line; Colonic Neoplasms; Combined Modality Therapy; Drug Resistance, Neoplasm; Escherichia coli Proteins; Fibroblasts; Genetic Therapy; Genetic Vectors; Humans; Male; Mice; Mice, Nude; Nitroreductases; Oncolytic Viruses; Prodrugs; Sirolimus; Transduction, Genetic

Colorectal neoplasms remain a leading cause of cancer-related deaths. A recognized weakness of conventional 5-fluorouracil (5-FU) therapy relates to expression of the intracellular enzyme, thymidine phosphorylase (TP). Although TP promotes 5-FU cytotoxicity, TP-derived 2-deoxy-D-ribose (dRib) counterproductively stimulates tumor angiogenesis. Here, the newly discovered antiangiogenic drug rapamycin was combined with 5-FU to counteract the potential escape mechanism of dRib-induced angiogenesis.. Orthotopic tumor growth was assessed in rapamycin and 5-FU-treated BALB/c mice with TP-expressing CT-26 colon adenocarcinoma cells. To examine liver metastasis, green-fluorescent protein-transfected CT-26 cells were visualized by fluorescence microscopy after intraportal injection. Cell counting and Ki67 staining were used to determine in vitro and in vivo cell expansion, respectively. In vitro angiogenic effects of dRib were assessed with endothelial cell migration and aortic ring assays. Western blotting detected dRib effects on p70/S6 kinase activation.. Rapamycin treatment of mice bearing orthotopic tumors inhibited tumor growth more than did 5-FU, and mice treated with both drugs typically developed no tumors. In the liver metastasis assay, combination therapy blocked metastatic expansion of solitary tumor cells. Interestingly, complex drug activities were suggested by tumor-cell proliferation being more sensitive to 5-FU than to rapamycin in vitro, but more sensitive to rapamycin in vivo. With regard to angiogenesis, dRib-induced endothelial cell migration and aortic ring formation were completely abrogated by rapamycin, correlating with blockage of dRib-induced p70/S6 kinase activation in endothelial cells.. Inhibition of dRib-induced angiogenesis with rapamycin counteracts a potential TP-based escape mechanism for colorectal cancer under 5-FU therapy, introducing a novel, clinically feasible, combination treatment option for this common neoplasm.

Topics: Adenocarcinoma; Animals; Antimetabolites, Antineoplastic; Aorta, Thoracic; Colonic Neoplasms; Deoxyribose; Disease Models, Animal; Fluorouracil; Immunosuppressive Agents; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, SCID; Muscle, Smooth, Vascular; Neovascularization, Pathologic; Rats; Rats, Inbred ACI; Sirolimus; Thymidine Phosphorylase

Cancer is an increasingly recognized problem associated with immunosuppression. Recent reports, however, suggest that the immunosuppressive agent rapamycin has anti-cancer properties that could address this problem. Thus far, rapamycin's effects on immunity and cancer have been studied separately. Here we tested the effects of rapamycin, versus cyclosporine A (CsA), on established tumors in mice simultaneously bearing a heart allograft. In one tumor-transplant model, BALB/c mice received subcutaneous syngenic CT26 colon adenocarcinoma cells 7 days before C3H ear-heart transplantation. Rapamycin or CsA treatment was initiated with transplantation. In a second model system, a B16 melanoma was established in C57BL/6 mice that received a primary vascularized C3H heart allograft. In vitro angiogenic effects of rapamycin and CsA were tested in an aortic ring assay. Results show that CT26 tumors grew for 2 weeks before tumor complications occurred. However, rapamycin protected allografts, inhibited tumor growth, and permitted animal survival. In contrast, CsA-treated mice succumbed to advancing tumors, albeit with a functioning allograft. Rapamycin's antitumor effect also functioned in severe combined immunodeficient BALB/c mice. Similar effects of the drugs occurred with B16 melanomas and primary vascularized C3H allografts in C57BL/6 mice. Furthermore, in this model, rapamycin inhibited the tumor growth-enhancing effects of CsA. Moreover, in vitro experiments showed that CsA promotes angiogenesis by a transforming growth factor-beta-related mechanism, and that this effect is abrogated by rapamycin. This study demonstrates that rapamycin simultaneously protects allografts from rejection and attacks tumors in a complex transplant-tumor situation. Notably, CsA protects allografts from rejection, but cancer progression is promoted in transplant recipients.

Topics: Adenocarcinoma; Animals; Antibiotics, Antineoplastic; Colonic Neoplasms; Cyclosporine; Drug Therapy, Combination; Graft Rejection; Heart Transplantation; Immunosuppressive Agents; Male; Melanoma; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, SCID; Sirolimus; Skin Neoplasms; Transplantation, Homologous

Recently, we showed that autocrine transforming growth factor alpha (TGFalpha) controls the epidermal growth factor receptor (EGFR)-mediated basal expression of integrin alpha2, cell adhesion and motility in highly progressed HCT116 colon cancer cells. We also reported that the expression of basal integrin alpha2 and its biological effects are critically controlled by the constitutive activation of the ERK/MAPK pathway (Sawhney, R. S., Sharma, B., Humphrey, L. E., and Brattain, M. G. (2003) J. Biol. Chem. 278, 19861-19869). In the present report, we further examine the downstream signaling mechanisms underlying EGFR/ERK signaling and integrin alpha2 function in HCT116 cells. Selective MEK inhibitors attenuated TGFalpha-mediated basal activation of p70S6K (S6K) specifically at Thr-389, indicating that this S6K site is downstream of ERK/MAPK signaling. Cells were treated with the selective protein kinase C (PKC) inhibitor bisindolylmaleimide to determine the role of PKC in S6K activation. The Thr-421 and Ser-424 phosphorylation sites of S6K were specifically inhibited by bisindolylmaleimide, which also blocked integrin alpha2 expression, cell adhesion, and motility. These data establish a novel cell motility function of S6K via PKC activation in a cancer cell. In addition, we examined whether mammalian target of rapamycin signaling controls S6K activation. Rapamycin inhibited constitutive S6K phosphorylation specifically at Thr-389, Thr-421, and Ser-424 sites. The assignment of these phosphorylation sites on S6K to biological functions was unequivocally confirmed by transfection of cells with specific single phosphorylation site dominant negative mutants. These experiments show for the first time that autocrine TGFalpha regulates cell adhesion function by multiple signaling pathways via specific phosphorylation sites of S6K in cancer cells.

Topics: Autocrine Communication; Binding Sites; Biotinylation; Blotting, Western; Cell Adhesion; Cell Culture Techniques; Cell Line, Tumor; Cell Movement; Colonic Neoplasms; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation; Genes, Dominant; Humans; Immunoblotting; Indoles; Integrin alpha2; Maleimides; Mitogen-Activated Protein Kinase Kinases; Mutation; Phosphorylation; Plasmids; Protein Kinase C; Ribosomal Protein S6 Kinases, 70-kDa; Serine; Signal Transduction; Sirolimus; Threonine; Time Factors; Transfection; Transforming Growth Factor alpha

Topics: Adenocarcinoma; Angiogenesis Inhibitors; Animals; Antibiotics, Antineoplastic; Colonic Neoplasms; Disease Models, Animal; Immunosuppressive Agents; Male; Mice; Mice, Inbred BALB C; Sirolimus

The aim of this study was to determine whether constitutive ErbB2 activation controls growth and apoptosis in colon cancer cells. Growth arrested GEO cells showed constitutive activation of ErbB2 in the absence of exogenous growth factors or serum supplementation. Higher levels of heregulin and ErbB2 activation were observed in the growth-arrested state and cell cycle re-entry was independent of exogenous growth factors. Blockade of ErbB2 activation by heregulin neutralizing antibodies and by AG879 resulted in prevention of cell cycle re-entry. This indicated that autocrine heregulin activity was responsible for growth factor independence and for cell cycle re-entry. Activation of ErbB2 was the result of heregulin mediated interaction with ErbB3 and generated downstream activation of the ERK and the PI3K/AKT pathways. Heregulin neutralizing antibody treatment of growth arrested GEO cells also generated apoptosis as reflected by PARP cleavage and DNA fragmentation indicating a cell survival signal was also induced by the constitutively activated ErbB2. The activation of AKT but not the MAPK pathway was responsible for cell survival in these cells.

Topics: Adenocarcinoma; Apoptosis; Autocrine Communication; Cell Cycle; Chromones; Colonic Neoplasms; Culture Media; Culture Media, Serum-Free; Dimerization; DNA Fragmentation; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Gene Expression Regulation, Neoplastic; Genes, erbB-2; Growth Substances; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Morpholines; Neoplasm Proteins; Neuregulin-1; Neutralization Tests; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Poly(ADP-ribose) Polymerases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptor, ErbB-3; Signal Transduction; Sirolimus; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

Xenobiotics frequently induce proteins involved in their detoxification. Because many drugs that are metabolized by human cytochromes P450 (CYP) 3A4 and 3A5 are also transported by the drug efflux pump P-glycoprotein, we determined whether expression of these proteins was altered by a variety of drugs in a cell line derived from a human colon adenocarcinoma, LS180/WT, and its adriamycin-resistant subline, LS180/AD50. P-glycoprotein and CYP3A4 were constitutively expressed in both LS180/AD50 and LS180/WT cells, and both proteins were up-regulated after treatment with many drugs, including rifampicin, phenobarbital, clotrimazole, reserpine, and isosafrole. However, there were some exceptions because P-glycoprotein was up-regulated by midazolam and nifedipine, whereas CYP3A4 was not. CYP3A5, which is also constitutively expressed in these cells, remained unchanged with most drug treatments but was up-regulated by reserpine and clotrimazole. The apparent coordinated coexpression of the CYP3A gene family and P-glycoprotein in the LS180 cells suggests that for common orally administered drugs, P-glycoprotein may play an important role in net drug absorption and drug/drug interactions of shared CYP3A4/P-glycoprotein substrates.

Topics: Adenocarcinoma; ATP Binding Cassette Transporter, Subfamily B, Member 1; Base Sequence; Blotting, Northern; Cell Line; Clotrimazole; Colonic Neoplasms; Cytochrome P-450 CYP2E1; Cytochrome P-450 Enzyme System; Dexamethasone; DNA Primers; Doxorubicin; Gene Expression Regulation, Neoplastic; Humans; Midazolam; Mixed Function Oxygenases; Molecular Sequence Data; Multigene Family; Phenobarbital; Phenytoin; Polymerase Chain Reaction; Rifampin; Tumor Cells, Cultured; Verapamil

We show that cell lines derived from childhood alveolar rhabdomyosarcoma (RMS) are very sensitive to the growth-inhibitory effects of the immunosuppressive agent rapamycin (RAP), compared to other human cell lines (50% inhibitory concentration range of 0.1-8 ng/ml, compared to 1280 to > 10,000 ng/ml). Our data suggest that the sensitivity of RMS lines is due to RAP inhibition of insulin-like growth factor 1 receptor-mediated signaling, which is essential for continued proliferation of RMS cells. The embryonal RMS line Rh1, which was resistant to RAP in serum-containing medium (50% inhibitory concentration, 4180 ng/ml), was highly sensitive under autocrine conditions of growth, indicating that resistance was due to paracrine signaling pathways insensitive to RAP action. FK506 reversed RAP action in all cell lines, indicating a dependence on complexing with the cytosolic FK506-binding protein for activity.

Topics: Carrier Proteins; Cell Division; Child; Colonic Neoplasms; Heat-Shock Proteins; Humans; Immunosuppressive Agents; Polyenes; Receptor, IGF Type 1; Rhabdomyosarcoma; Sirolimus; Tacrolimus; Tacrolimus Binding Proteins; Tumor Cells, Cultured

Rapamycin exhibits activity against several ascites and solid transplantable tumors; it is slightly active to inactive against leukemias. On a weight basis, rapamycin was less active than 5-fluorouracil, cyclophosphamide and adriamycin, but rapamycin's maximal activity against Colon 38 tumor was similar to that of 5-fluorouracil and cyclophosphamide. Its activity was such that it significantly inhibited tumor growth at any stage of development. In the active dose range, rapamycin appeared less toxic than the other drugs. In the Colon 38 tumor model, rapamycin at a given dose exhibited the same activity when administered ip, iv, im and sc; upon oral administration, its activity was reduced but not abolished. Rapamycin was compatible with 5-fluorouracil and cyclophosphamide. The sequential treatment 5-fluorouracil-rapamycin-cyclophosphamide was superior to the sequence 5-fluorouracil-adriamycin-cyclophosphamide in protecting Colon 38 tumor-bearing mice. 29-Demethoxyrapamycin exerted only marginal activity against P388 lymphocytic leukemia; it was inactive against B16 melanocarcinoma and Colon 38 solid tumor.

Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Colonic Neoplasms; Male; Mice; Mice, Inbred Strains; Neoplasms, Experimental; Polyenes; Sirolimus