Antiproliferative Effects Of Rapamycin Research Articles

Cytotoxic and Cytostatic Effects of Targeting mTOR and Hedgehog Pathways in Acute Myeloid Leukemia

Objectives: Acute myeloid leukemia (AML) is a highly aggressive heterogeneous hematopoietic malignancy characterized by a rapid and abnormal proliferation of immature myeloid leukemia cells in the bone marrow and peripheral blood. Aberrant alterations in signal transduction pathways are strongly associated with the progression of AML. This study aimed to investigate cell viability and the cell cycle in AML cells by targeting the Hedgehog and mTOR signaling pathways with rapamycin and GANT61. Materials and Method: The antiproliferative effect of rapamycin and GANT61 was assessed by the MTT cell viability assay in two AML cell lines: CMK and MOLM-13. The effect of the inhibitors on cell-cycle distribution was determined using propidium iodide staining and measured with flow cytometry. Results: Rapamycin, an mTOR inhibitor, and GANT61, a Gli-1 inhibitor, decreased the cell proliferation of CMK and MOLM-13 cells. The IC20 values, which is the drug concentration that inhibits cell growth by 20%, were combined and administered to the cells. The results show the drugs to have a combinatorial inhibitory effect on CMK cells but not on MOLM-13 cells. In addition, the combination of drugs arrested the cells during the G0/G1 phase. Conclusion: This study suggests a novel combination therapy approach for AML via mTOR and Hedgehog signaling pathway inhibition using rapamycin and GANT61, respectively. It also suggest further studies be performed to reveal the mechanism of action.

Transcranial sonography depicts a larger substantia nigra echogenic area in renal transplant patients on calcineurin inhibitors than on rapamycin

BackgroundAfter kidney transplantation neurologic manifestations may develop, including Parkinson’s disease (PD). An enlarged substantia nigra (SN) by transcranial sonography has been recognized as a marker of PD.MethodsIn renal transplant recipients (RTRs = 95) and controls (n = 20), measurement of mesencephalon, SN, third ventricle, spleen and carotid intima-media thickness (cIMT) and middle cerebral artery (MCA), kidney and spleen arteries Doppler resistive index (RI) were performed.ResultsRTRs had larger SN, third ventricle and cIMT and higher renal RI than controls. The SN was larger in the CNIs group than in controls and rapamycin group, while the third ventricle was similar between patients but larger than in controls. In RTRs, SN showed a direct linear correlation with spleen and the third ventricle with age, cIMT and RI of the MCA, kidney and spleen. In CNIs group the SN correlated positively with age and cIMT, while the third ventricle reproduced RTRs correlations. Rapamycin group showed a direct linear relationship between the third ventricle and age and RI of the MCA, kidney and spleen; SN showed no correlations.ConclusionRTRs on CNIs present a larger SN area than on rapamycin, probably due to the antiproliferative effect of rapamycin. This finding might be relevant when interpreting TCS in RTRs.

PROX1, a Key Mediator of the Anti-Proliferative Effect of Rapamycin on Hepatocellular Carcinoma Cells.

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

Abstract #4396 Behavioral conditioning of anti-proliferative effects of rapamycin in experimental CNS malignancy

Abstract #4396 Behavioral conditioning of anti-proliferative effects of rapamycin in experimental CNS malignancy

Rapamycin Prolongs Graft Survival and Induces CD4+IFN-γ+IL-10+ Regulatory Type 1 Cells in Old Recipient Mice.

Although the elderly represents a rapidly growing population among transplant recipients, age-specific aspects have not been considered sufficiently in clinical trials. Moreover, age-specific effects of immunosuppressive therapies remain poorly understood. Here, we assessed the impact of rapamycin on alloimmune responses in old recipients using a fully major histocompatibility complex-mismatched murine transplantation model. Old untreated recipients displayed a prolonged skin graft survival compared to their young counterparts, an observation that confirmed data of our previous experiments. Rapamycin led to a significant prolongation of graft survival in both young and old recipients. However, graft survival was age-dependent and extended in old versus young recipients (19 days vs 12 days, P = 0.004). This age-specific effect was not linked to changes in frequencies or subset composition of either cluster of differentiation (CD)8 or CD4 T cells. Moreover, antiproliferative effects of rapamycin on CD8 and CD4 T cells as assessed by in vivo bromdesoxyuridine incorporation were comparable and age-independent. In contrast, the systemic production of IL-10 was markedly elevated in old recipients treated with rapamycin. In parallel to this shift in cytokine balance, IFN-γ/IL-10 double-positive regulatory type 1 cells emerged during T helper type 1 differentiation of old T helper cells in presence of rapamycin. Similarly, CD4IFN-γIL-10 cells expanded among Foxp3-negative cells after in vivo treatment of old recipients with rapamycin. Our results highlight novel aspects of age-dependent immunosuppressive effects of rapamycin, with relevance for age-specific immunosuppressive regimens.

Age-Specific Prolongation of Graft Survival in Recipients Treated With Rapamycin is Linked to CD4 + IFN-g + IL-10+ Cells

Introduction: Although the elderly represent a rapidly growing population among transplant recipients, age-specific aspects have not been considered sufficiently in clinical trials. Moreover, age-specific effects of immunosuppressive therapies remain poorly understood. Methods: Here, we assessed the impact of Rapamycin on alloimmune responses in aging using a fully MHC-mismatched (DBA/2 on B57BL/6) murine transplantation model. Results: Old untreated recipients displayed a prolonged skin graft survival compared to their young counterparts (median survival time 9 vs. 7 days, p = 0.006). Rapamycin led to a marked and significant prolongation of graft survival specifically in old but not young recipients (19 days vs. 12 days, p = 0.004). This age-specific effect was not linked to changes in frequencies or subset composition of either CD8+ or CD4+ T cells. Moreover, anti-proliferative effects of Rapamycin on CD8+ and CD4+ T cells as assessed by in-vivo BrdU-incorporation were comparable and age-independent. In contrast, the systemic production of IL-10 was markedly elevated in old recipients treated with Rapamycin. This shift in cytokine balance was linked to an emergence of IFN-γ/IL-10 double-positive regulatory type 1 cells during Th1-differentiation of old T helper cells in presence of Rapamycin, a process that was independent of regulatory T cells. Conclusion: These results demonstrate a novel pathway of age-dependent alloimmunity for an immunosuppressive drug currently in widespread clinical use and with relevance for an age-specific immunosuppression.FigureFigure

Rapamycin regulates the proliferation of Huh7, a hepatocellular carcinoma cell line, by up-regulating p53 expression

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

Improvement of the antiproliferative effect of Rapamycin on tumor cell lines by poly (monomethylitaconate)-based pH-sensitive, plasma stable liposomes

pH-responsive polymers produce liposomes with pH-sensitive property which can release their encapsulated drug under mild acidic conditions found inside the cellular endosomes, inflammatory tissues and cancerous cells. The aim of this study was preparing pH-sensitive and plasma stable liposomes in order to enhance the selectivity and antiproliferative effect of Rapamycin. In the present study we used PEG-poly (monomethylitaconate)-CholC6 (PEG-PMMI-CholC6) copolymer and Oleic acid (OA) to induce pH-sensitive property in Rapamycin liposomes. pH-sensitive liposomal formulations bearing copolymer PEG-PMMI-CholC6 and OA were characterized in regard to physicochemical stability, pH-responsiveness and stability in human plasma. The ability of pH-sensitive liposomes in enhancing the cytotoxicity of Rapamycin was evaluated in vitro by using colon cancer cell line (HT-29) and compared with its cytotoxicity on human umbilical vein endothelial cell (HUVEC) line. Both formulations were found to release their contents under mild acidic conditions rapidly. However, unlike OA-based liposomes, the PEG-PMMI-CholC6 bearing liposomes preserved their pH-sensitivity in plasma. Both types of pH-sensitive Rapamycin-loaded liposomes exhibited high physicochemical stability and could deliver antiproliferative agent into HT-29 cells much more efficiently in comparison with conventional liposomes. Conversely, the antiproliferative effect of pH-sensitive liposomes on HUVEC cell line was less than conventional liposomes. This study showed that both OA and PEG-PMMI-CholC6-based vesicles could submit pH-sensitive property, however, only PEG-PMMI-CholC6-based liposomes could preserve pH-sensitive property after incubation in plasma. As a result pH-sensitive PEG-PMMI-CholC6-based liposomal formulation can improve the selectivity, stability and antiproliferative effect of Rapamycin.

Silymarin Inhibits Endothelial Progenitor Cellsʼ Senescence and Protects Against the Antiproliferative Activity of Rapamycin: Preliminary Study

Rapamycin, an antiproliferative agent used on drug-eluting stents, induces endothelial progenitor cells (EPCs) senescence through telomerase inactivation and may impair the reendothelization of an injured arterial wall, leading to thrombosis. We examined whether silymarin, a complex of flavonolignans with hepatoprotective and antioxidative properties, can protect EPCs against rapamycin-induced senescence. Mononuclear cells were isolated from peripheral blood of healthy volunteers. EPCs were cultured in endothelial cell growth medium-2 in the presence or absence of rapamycin (0.1 ng/mL) and/or silymarin (12.5–50 μg/mL). EPCs senescence–associated b-galactosidase activity, telomerase activity, and prolifertive activity were measured. The influence on tubular-like structure formation in vitro was investigated, and colony-forming assay on methylcellulose plates was performed. Silymarin increased telomerase activity 3-fold, reduced the number of senescent cells, and increased EPC proliferative activity (up to 64%) in comparison with cells cultured with rapamycin alone. Moreover, silymarin partially prevented impairment of tubular-like structure formation in Matrigel by rapamycin. These findings suggest that silymarin counteracts the inhibitory effects of rapamycin in EPCs. Silymarin may protect EPCs against the antiproliferative effects of rapamycin and restore their reconstructive ability.

Low-dose rapamycin reduces kidney volume angiomyolipomas and prevents the loss of renal function in a patient with tuberous sclerosis complex

Tuberous sclerosis complex (TSC) is caused by constitutively activated mammalian target of rapamycin (mTOR) resulting in non-malignant tumours of several organs including renal angiomyolipomas (AMLs). AMLs may originate renal failure, hypertension and spontaneous life-threatening bleeding. Recent reports suggest a possible beneficial role of the mTOR inhibitor rapamycin for TSC. However, safety and efficiency of rapamycin in TSC patients as an anti-proliferative agent are still undefined. A 40-year-old man with sporadic TSC and a history of spontaneous bleeding from his left kidney AMLs received low-dose rapamycin for 12months, and this was associated with a reduction in bilateral kidney AML volume, stabilization and even improvement of renal function. There was also a reduction of facial angiofibromas, improvement of blood pressure control and absence of AML bleeding over this time period. Brain lesion images remained stable, and no significant rapamycin-associated side effects were noted. To the best of our knowledge, this is the first report of a case of reduction in renal AML volume together with preservation of renal function in a patient with TSC receiving low-dose rapamycin. These data suggest that it could be the result of the anti-angiogenic, anti-fibrotic and anti-proliferative effects of rapamycin.

Abstract C55: Involvement of oxidative stress in the antitumor effect of mTOR inhibitors in mantle cell lymphoma

Abstract Existing treatments for mantle cell lymphoma (MCL) are non-curative, demonstrating a need for a refined treatment approach. MCLs are aggressive lymphomas, usually characterized by over-expression of cyclin D1. Recent clinical trials have shown promising results with the use of mammalian target of rapamycin (mTOR) inhibitors. While cyclin D1 can be regulated through the mTOR pathway, treatment with mTOR inhibitors often inhibit MCL growth without altering cyclin D1. Thus, the anti-tumor activity of mTOR inhibitors is likely mediated through other key targets. One potential target is C/EBPβ-LIP, a transcription factor regulated through the mTOR pathway. One target of C/EBPβ gene regulation is manganese superoxide dismutase (MnSOD), an antioxidant defense enzyme with tumor suppressor activity in some cell types. MnSOD catalyzes the dismutation of superoxide to hydrogen peroxide. Thus, it removes one form of reactive oxygen species (ROS) while generating another. Previous studies in our laboratory have shown that C/EBPβ-LIP levels decrease with mTOR inhibitor treatment in proportion to increasing MnSOD levels. We hypothesize that the anti-tumor effect of mTOR inhibitors in MCL is mediated by the increase in MnSOD protein expression and accumulation of ROS, specifically hydrogen peroxide. Here we investigated the effect of rapamycin on MnSOD and the levels of ROS in two human MCL-derived cell lines. High MnSOD levels correlated with increased sensitivity of cells treated with rapamycin. To investigate the biological effects of increased MnSOD expression, we measured overall ROS levels (DCFH) and the rate of hydrogen peroxide efflux with the fluorescent probe Amplex Red, which is indicative of changes to hydrogen peroxide levels in the cells. These results showed that both overall ROS and hydrogen peroxide levels increased in parallel with MnSOD expression. These increases in hydrogen peroxide were not due to decreased levels of catalase or glutathione peroxidase. Treatment with the general ROS scavenger, N-acetyl cysteine, reduced overall ROS, cellular levels of hydrogen peroxide, and the growth inhibitory effect of rapamycin. These findings indicate that rapamycin-induced cytostatic effect is characterized by generation of ROS, specifically hydrogen peroxide. This is likely due to the increased levels of MnSOD. By understanding the key signaling molecules in the anti-proliferative effects of rapamycin, we may be able to identify additional predictive markers to improve the therapeutic value of mTOR inhibitors, or study drug combinations that will enhance the effect of ROS-induced cytotoxicity. Citation Information: Cancer Res 2009;69(23 Suppl):C55.

Abstract PL01-04: The role of regulatory T cells in K-Ras driven lung cancer

Abstract PL01-04 Background K-Ras mutations occur in up to 30% of human lung adenocarcinomas (Salgia and Skarin 1998) and occur almost exclusively in patients with a history of smoking. These mutations confer resistance to commonly used cytotoxic chemotherapies as well as targeted agents (Eberhard, Johnson et al. 2005). In preclinical mouse models of lung cancer, K-Ras mutations are necessary for tobacco carcinogen-driven lung tumorigenesis and are sufficient to cause lung adenocarcinomas in transgenic mice (Johnson, Mercer et al. 2001). Inflammation is characteristic of smoking-related lung cancer and preclinical models of K-Ras-driven lung tumorigenesis. Specific components of the inflammatory response such as regulatory T cells (Treg) regulate inflammation and are thought to contribute to tumorigenesis. Because inhibitors of mTOR such as the immunosuppressant rapamycin can prevent K-Ras mediated murine lung tumorigenesis (Wislez, Spencer et al. 2005; Granville, Warfel et al. 2007), we hypothesized that the anti-tumor effects of rapamycin might be related to alterations in lung-associated inflammation. Methods and Findings Lung tumorigenesis was studied in three different murine models that depend on mutant K-Ras; a tobacco carcinogen-driven model, a syngeneic inoculation model and a transgenic model. Splenic and lung-associated T cells were studied using flow cytometry and immunohistochemistry. Exposure of A/J mice to the tobacco-carcinogen NNK that causes K-Ras mutations (Belinsky, Devereux et al. 1989) tripled the fraction of lung-associated Treg prior to tumor development. Induction of lung tumors and induction of lung associated Foxp3+ cells by NNK was dose dependent. When administered to achieve trough levels comparable to that in humans, rapamycin prevented the induction of lung associated Foxp3+ cells by NNK, coincident with a 90% decrease in lung tumors. In A/J mice inoculated with syngeneic lung adenocarcinoma cells that were resistant to the anti-proliferative effects of rapamycin, rapamycin neither inhibited tumor growth nor decreased Treg. In contrast, depletion of Treg in inoculated mice using an anti-CD25 antibody prevented lung tumorigenesis by 80%. Transgenic mice that express mutant K-Ras (K-RasLA2) also develop lung tumors with infiltrating Foxp3+ cells, and when K-RasLA2 mice were crossed to genetically engineered mice that bear a loss of function mutation in Foxp3, lung tumorigenesis was inhibited by 75%. Conclusions These studies show that Treg are required for K-Ras-mediated lung tumorigenesis, and provide a strong rationale to clinically evaluate rapamycin or other agents that decrease Treg, to determine if decreasing Treg will inhibit K-Ras driven lung cancer. Citation Information: Cancer Prev Res 2008;1(7 Suppl):PL01-04.

Rapamycin-induced remission of Kaposi's sarcoma is not associated with expansion of cytotoxic T-lymphocyte subsets.

We present a case of post-transplantation Kaposi's sarcoma (KS) successfully treated by conversion to rapamycin. Clinical and histological resolution was observed within 6 months of commencing rapamycin. Also, vascular endothelial growth factor (VEGF) staining in the biopsy samples resolved following rapamycin therapy. Interestingly there was no expansion in cytotoxic T-lymphocyte (CTL) subsets observed during this period, as might be expected if this remission was due to immune reconstitution following reduction in immunosuppression. These data suggest that the resolution of tumour with rapamycin could be the result of the antiangiogenic, antiproliferative effects of rapamycin.

Antiproliferative effect of rapamycin on human T-cell leukemia cell line Jurkat by cell cycle arrest and telomerase inhibition

To examine the ability of rapamycin to suppress growth and regulate telomerase activity in the human T-cell leukemia cell line Jurkat. Cell proliferation was assessed after exposure to rapamycin by 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide assay. Cell cycle progression and apoptosis were determined by flow cytometry. The proteins important for cell cycle progression and Akt/mammalian target of rapamycin signaling cascade were assessed by Western blotting. Telomerase activity was quantified by telomeric repeat amplication protocol assay. The human telomerase reverse transcriptase (hTERT) mRNA levels were determined by semi-quantitative RT-PCR. Rapamycin inhibited the proliferation of Jurkat, induced G1 phase arrest, unregulated the protein level of p21 as well as p27, and downregulated cyclinD3, phospho-p70s6k, and phospho-s6, but had no effect on apoptosis. Treatment with rapamycin reduced telomerase activity, and reduced hTERT mRNA and protein expression. Rapamycin displayed a potent antileukemic effect in the human Tcell leukemia cell line by inhibition of cell proliferation through G1 cell cycle arrest and also through the suppression of telomerase activity, suggesting that rapamycin may have potential clinical implications in the treatment of some leukemias.

Anti-Proliferative Effect of Rapamycin on Human T Cell Leukemia Cell Line Jurkat by Cell Cycle Arrest and Telomerase Inhibition.

Rapamycin exerts its biological activity by inhibiting the kinase mammalian target of rapamycin (mTOR), which is a key regulator of cell growth and survival in many cell types. Its constitutive activation has been involved in pathogenesis of various cancers; the critical functions of mTOR have led to the development of mTOR inhibitors (MTIs) as novel anticancer agents. Recently, the anticancer effects of rapamycin are presently evaluated in various solid tumors; however, the use of rapamycin in acute lymphoblastic leukemia (ALL) is poorly documented. We try to examine the ability of rapamycin to suppress growth and its mechanism in Human T Cell Leukemia cell line Jurkat, and meanwhile to explore its ability to regulate telomerase. Cell proliferation was assessed after exposure to rapamycin by MTT assay. Apoptotic cells were determined by flow cytometric detection of annexin V binding assay. And cell cycle was monitored by flow cytometric detection of DNA content assay. Proteins important for cell cycle progression and Akt/mTOR signaling cascade were assessed by Western blot. Telomerase activity was quantified by TRAP assay. hTERT mRNA levels were determined by semi-quantitative RT-PCR. Rapamycin significantly inhibited proliferation of Jurkat in a dose and time-dependent manner with IC50 values for 24hr, 48hr, and 72hr were 344nM, 92nM and 16nM. To further determine the mechanism of growth inhibition by rapamycin, we found rapamycin did not increase the amount of cells in annexin V+ fraction 72 hr after treatment, confirming that rapamycin did not promote apoptosis in Jurkat cells. However, G1 phase arrest was induced by rapamycin (10nM) since 16hr after treatment, and ratio of G1 continued to grow as treatment was prolonged to 32hr, which indicated rapamycin inhibited cell cycle progress. By Western blot analysis, we found rapamycin could up-regulate the level of cyclin-dependent kinase inhibitors (CDKIs) of p27Kip1 as well as p21waf1, down-regulate CyclinD3, but had no significantly effect on expression of Cdk4, Cdk6 or CyclinD2, which demonstrated that G1 cell cycle arrest induced by rapamycin in Jurkat cells, was mediated by affecting cyclin D3, P27Kip1and p21waf1. Moreover, we characterized the signaling pathways affected by rapamycin, and found phosphorylation of mTOR downstream cascade targets, sucha as p70S6K and S6 were significantly reduced by rapamycin, but phospho- Akt level was not affected, which in agreement with established models that rapamycin functions downstream of Akt in Akt/mTOR signaling. Interestingly, Activation of telomerase is seen in Jurkat cells and thought to be a critical element in leukemia pathogenesis. Treatment with rapamycin decreased telomerase activity dose-dependently, which was accompanied with down-regulation of the catalytic subunit, telomerase reverse transcriptase (TERT). However, the exact transcription factors, which modulate hTERT gene transcription and are targeted by the mTOR pathway, need to be further identified. Conclusion:Rapamycin displayed potent antileukemic effect in Human T Cell Leukemia cell line by inhibition of cell proliferation through G1 cell cycle arrest and also through suppression of telomerase activity, suggesting rapamycin may have potential clinical implications in treatment of some leukemia.

Antiproliferative effects of rapamycin as a single agent and in combination with carboplatin and paclitaxel in head and neck cancer cell lines

Recent data suggested that combining targeted therapies with chemotherapy may counteract drug resistance. Activation of the PI3K/AKT/mTOR pathway downstream to kinase receptors, such as EGFR, was found in 57-81% of head and neck squamous cell carcinoma (HNSCC), and was eventually associated with a loss of PTEN function. mTOR was shown to modulate cell proliferation, apoptosis, invasion, and angiogenesis. This study aimed to evaluate molecular and cellular effects of rapamycin in a panel of cell lines either as single agent or in combination with cytotoxics commonly used in HNSCC. Antiproliferative effects of rapamycin, carboplatin, and paclitaxel were evaluated in a panel of three HNSCC cell lines (SCC61, SQ20B and HEP2). Cells were exposed to rapamycin for 48 h, to carboplatin for 48 h, or to paclitaxel for 24 h. Antiproliferative effects of simultaneous and sequential rapamycin-based combinations were studied using MTT assay and median effect plot analysis. Cell cycle effects were analysed using flow cytometry. Rapamycin induced concentration dependent antiproliferative effects in HNSCC cell lines with IC(50) of 5 +/- 1, 12 +/- 2 and 20 +/- 2 microM in SCC61, SQ20B, and HEP2 cells, respectively. Higher antiproliferative effects were observed in SCC61 cells overexpressing NOXA and cyclin D1 than in HEP2 that overexpressed MDR1 and BCL2. In our panel, antiproliferative effects of rapamycin were associated with G0/G1 cell cycle accumulation and apoptosis induction, at concentrations ranging 3-30 microM. Combinations of rapamycin with paclitaxel and carboplatin displayed synergistic and additive effects. Synergistic effects were observed with paclitaxel in SQ20B and HEP2 cells and with carboplatin in SQ20B cells, when cells were exposed to cytotoxics prior to rapamycin. Our results show that rapamycin displays antiproliferative effects and induces apoptosis in HNSCC cell lines, cellular effects being more potent in cells that do not express BCL2 and MDR1. Additive and synergistic effects were observed when rapamycin was combined with carboplatin and paclitaxel.

Rapamycin inhibits the growth of cholangiocarcinoma cells in vitro

15153 Background: In the present study, anti-neoplastic effect of rapamycin against cholangiocarcinoma was studied in vitro. Methods: Expression of mTOR in 4 cholangiocarcinoma cell lines, TFK1, HuCCT1, NOZW, and OZ was evaluated by real-time PCR. Then, the four cholangiocarcinoma cell lines were cultured with rapamycin (0, 25, 50, 100, 200 nM), gemcitabine (0, 0.5, 1, 2 μM), or both, and anti-proliferative effect was evaluated by MTT assay. Results: All the four cholangiocarcinoma cell lines expressed endogenous mTOR- mRNA. Level of expression was the highest in HuCCT1 (65.8), and the lowest in TFK1 (17.6). Then, rapamycin significantly inhibited the growth of all the four cholangiocarcinoma cell lines, in dose-dependent manner. Gemcitabine inhibited the growth of NOZW (48.4%) and HuCCT1 (48.9%), but less efficiently in TFK1 (5.9%) and OZ (27.4%). Furthermore, synergistic anti-proliferative effect of rapamycin and gemcitabine was observed in TFK1 (39.1%), NOZW (38.9%), and OZ (47.1%), not in HuCCT1 (18.9%). Conclusion: Rapamycin effectively inhibited the growth of the cholangiocarcinoma cell lines, and synergistic effect with gemcitabine was observed in three of the four cell lines. No significant financial relationships to disclose.

Combining an mTOR antagonist and receptor tyrosine kinase inhibitors for the treatment of prostate cancer

Inhibition of the mammalian target of rapamycin (mTOR) signaling pathway is a potentially useful therapeutic strategy in the treatment of advanced prostate cancer. However mTOR antagonists used as single agents are not likely to result in dramatic clinical responses, so that it is useful to identify prospective agents that might be useful in combination. We treated CWR22Rv1 and LNCaP prostate cancer cells with an mTOR inhibitor, rapamycin, alone, or in combination with either of two receptor protein kinase (RTK) inhibitors. We assessed the effects of these treatments on cell survival and activation of down-stream mTOR target proteins. Treatment with either PD16839, an EGFr antagonist, or imatinib mesylate (Gleevec), a PDGFr, c-kit and bcr/abl antagonist, enhanced the anti-proliferative effects of rapamycin. We therefore assessed the effects of treatment with the RTK antagonist alone and in combination with rapamycin on mTOR targeted proteins. RTK antagonists alone had no effect or paradoxically increased phosphorylation of the mTOR targeted proteins, p70 S6 kinase and ribosomal S6. In contrast, when these cells were treated with either RTK antagonist in the presence of rapamycin, there was a dramatic decrease in phosphorylation of these two mTOR-targeted proteins. These effects were not mediated through phospho-AKT. Since two separate RTK antagonists had additive antiproliferative effects in combination with an mTOR antagonist and were associated with a dramatic decrease in mTOR targeted proteins in cells with or without PTEN expression, the strategy deserves further evaluation for the treatment of prostate cancer.

Rapamycin Inhibits Proliferation of Estrogen-Receptor-Positive Breast Cancer Cells

Estrogen-receptor (ER)-positive breast cancers comprise the majority of sporadic breast cancers. Although 50% respond to antihormonal treatment, both primary and acquired resistance limit the utility of this therapy, and other agents are needed. Rapamycin, an inhibitor of the mammalian Target of Rapamycin (mTOR), possesses antitumor activity against many tumors including breast tumors, and particularly against ER-positive breast cancer cell lines. The sensitivity of these cells to rapamycin has been attributed to activation of the PI3K/Akt/mTOR pathway by nongenomic ER signaling. The purpose of this study was to evaluate the efficacy of rapamycin against ER-positive breast cancer, particularly under 17beta-estradiol (E2)-dependent conditions, and to investigate mechanisms of rapamycin-sensitivity in ER-positive cells. Breast cancer cell lines were tested for sensitivity to rapamycin. Antiproliferative effects of rapamycin, alone and in combination with tamoxifen, were assessed under E2-dependent conditions. Western blot analysis was used to detect activation of mTOR by nongenomic ER signaling. Rapamycin effectively inhibits proliferation of the ER-positive MCF-7 cell line. In our system, this sensitivity is probably not due to nongenomic ER activation of the PI3K/Akt/mTOR pathway; rapid stimulation of mTOR occurred nonspecifically after medium replacement, and addition of E2 stimulated mTOR only after 1 h. Combining rapamycin and tamoxifen under E2-dependent conditions yielded additive/synergistic effects at effective concentrations. These results suggest that rapamycin may be an effective treatment for ER-positive breast cancer, either alone or in combination with tamoxifen, and also may be a potential therapy for tamoxifen-resistant cancers.

3P-0885 The antiproliferative effect of rapamycin is associated with inhibition of cholesterol ester synthesis

3P-0885 The antiproliferative effect of rapamycin is associated with inhibition of cholesterol ester synthesis