Anticancer Effect Of Rapamycin Research Articles

Abstract P6-07-04: Rapamycin inhibits the stemness of mammary epithelial cells in the premalignant tissues of MMTV-ErbB2 transgenic mice

Abstract Rapamycin, a well-studied mTOR inhibitor, has been demonstrated to inhibit mammary carcinogenesis at multiple stages, including initiation, invasion, and metastasis, in preclinical animal models. Nevertheless, the cancer preventative potential and underlying mechanisms remain unclear, especially in individual breast cancer subtypes like ErbB2/Her2-positive breast cancers. ErbB2 amplification/overexpression is a particular clinical concern because it occurs in approximately one-third of human breast cancers and is associated with poor prognosis. Therefore, we used MMTV-ErbB2 transgenic mice as our model system to test the efficacy of rapamycin in the prevention of ErbB2-mediated mammary tumor development. Our initial data provided proof of concept regarding the anti-cancer effects of rapamycin in vivo. Indeed, rapamycin (1.5 mg/kg/day for 12 days) significantly reduced the volume and weight of syngeneic 78617 cell-derived mammary tumors in MMTV-ErbB2 mice, despite observed decreases in CD4+ and CD8+ immune cells. Since advanced mammary gland development can serve as an indicator of breast cancer risk, we investigated the effects of rapamycin on mammary gland development in MMTV-ErbB2 mice that were treated with low-dose rapamycin (1 mg/kg/day) between weeks 10 and 20 of age. As such, rapamycin significantly attenuated mammary morphogenesis at 20 weeks of age, as indicated by decreased branching density, ductal elongation, and proliferative index of the premalignant mammary glands. Flow cytometric analysis of isolated primary mammary epithelial cells (MECs) was performed using CD24 and CD49f markers to identify MEC populations. We found that rapamycin has a significant impact on MEC stemness based on changes in luminal (CD24highCD49flow), mammary stem cell (MaSC)-enriched (CD24highCD49fhigh), and myoepithelial/basal (CD24low/highCD49fhigh) MEC populations. We also used CD61 and CD49f markers to identify a population enriched with luminal progenitor cells (CD61highCD49fhigh) that was selectively inhibited by rapamycin. Consistent with our flow cytometric analyses, rapamycin inhibited the luminal progenitor cell-enriched population, self-renewal, and anchorage-independent cell growth of primary MECs, as demonstrated by colony-forming cell, mammosphere, and 3D culture assays, respectively. These functional stem cell assays further corroborate that rapamycin suppresses the stemness of primary MECs. Molecular analysis of MECs demonstrated that rapamycin inhibited mTOR signaling, as expected. Importantly, rapamycin also significantly suppressed the receptor tyrosine kinase/ErbB2, estrogen receptor, Wnt/β-catenin, and TGFβ/Smad3 signaling pathways prior to malignant transformation. Collectively, our study provides evidence that rapamycin has potential cancer preventative effects in the mammary glands of MMTV-ErbB2 mice during the premalignant risk window. These rapamycin-induced anti-cancer effects ultimately highlight the promising clinical significance of rapamycin for the prevention and treatment of human ErbB2-overexpressing breast cancers. Citation Format: Yang X, Zhao Q, Parris AB, Howard EW, Zhao M, Guo Z, Xing Y, Ma Z. Rapamycin inhibits the stemness of mammary epithelial cells in the premalignant tissues of MMTV-ErbB2 transgenic mice [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-07-04.

Abstract 1308: Enhanced antitumor activity of rapamycin and genipin, a UCP-2 inhibitor, in lung cancer

Abstract mTOR is constitutively activated in lung cancer. Deletion of TOR1 increases expression of OXPHOS proteins, and mitochondrial uncoupling proteins 2 (UCP-2) expression promotes chemoresistance. Therefore, we evaluated whether adding genipin, a UCP-2 inhibitor, to rapamycin has better antitumor effect in lung cancer. Combined with genipin and rapamycin have a synergistic effect with more cellular apoptosis in A549, H460 and CL1-0 cells. The similar phenomenon was found in the combination treatment with genipin and everolimus or BGT226 (a dual PI3K/mTOR inhibitor) in this three lung cancer cells. It is said that NrF2 may affect UCP-2 expression, and we found NrF2 was activated in lung cancer A549, H460 and CL1-0 cells treated with rapamycin. Everolimus and dual PI3K/mTOR inhibitor, BGT226, also induced NrF2 nuclear translocation in these lung cancer cells. It is said that under oxidant stress, p62 activates NrF2 through P62 Ser351-phosphorylation. We found that rapamycin induced ROS generation, p62 serine phosphorylation and NrF2 downstream mRNA and protein expression, including UCP-2. ROS inhibitor, NAC, and down-regulation of p62 by siRNA suppressed rapamycin-induced UCP-2 expression. Overexpression of p62 by cDNA transfection enhanced NrF2 activation and then UCP-2 expression in lung cancer cells. Moreover, rapamycin-induced increased UCP-2 expression, leading decreased mitochondrial membrane potential and mild increased oxygen consumption rate. Finally in A549 subcutaneous tumor mode in SCID mice, combined genipin and everolimus also demonstrated the greatest antitumor activity. Therefore, inhibition UCP-2 by genipin enhances anticancer effect of rapamycin in lung cancer. Citation Format: Wen-Pin Su, Jang-Yang Chang, Ching-Chuan Kuo, Wu-Chou Su. Enhanced antitumor activity of rapamycin and genipin, a UCP-2 inhibitor, in lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1308.

Anticancer effect of rapamycin on MCF-7 via downregulation of VEGF expression.

The importance of mTOR signaling in tumor biology is widely accepted and a number of agents that selectively target mTOR are being developed in cancer therapy. On the other hand, it has been demonstrated that mTOR can act as an angiogenic agent. Thus, we hypothesized that the mTOR inhibitor-induced anticancer effect is affected by expression of a key angiogenic factor, vascular endothelial growth factor (VEGF) and investigated the anticancer effect underlying mTOR using an in vitro assay. The mTOR inhibitor rapamycin dose-dependently reduced the cell viability of the breast cancer cell line, MCF-7, but did not reduce the cell viability of the colon cancer cell line, HT-29. Rapamycin reduced the VEGF expression in the culture medium of MCF-7, while rapamycin did not contribute VEGF expression in the culture medium of HT-29. VEGF stimulated cell viability and VEGF inhibition reduced cell viability of MCF-7, and rapamycin dose-dependently restored the cell viability of MCF-7 reduced by rapamycin. These findings suggest that mTOR acts as a direct anticancer agent and that the mTOR-inhibitor-induced anticancer effect involved the reduced expression of VEGF in MCF-7. Our results imply that mTOR regulates the expression of VEGF and is involved in breast cancer progression.

Anticancer Benefits of Early Versus Late Use of Rapamycin in a Rat Model of Urothelial Carcinoma

BackgroundWe previously reported both in vivo and in vitro effects of rapamycin on urothelial carcinoma. Clinically, the use of rapamycin could not completely prevent the recurrence of urothelial carcinoma. Therefore, we designed this study to compare the difference of efficacy between early and late use of rapamycin in a rat model of urothelial carcinoma. MethodsThe rat model of urothelial carcinoma was induced by adding 0.05% N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) to the drinking water for up to 20 weeks in male Fisher-344 rats. Rapamycin was fed orally from the 1st day, 5th week, 9th week, 13th week, and 17th week. The antitumor effects of different periods of rapamycin treatment were assessed grossly and microscopically. ResultsPapillary tumors of urinary bladder were successfully induced in the BBN group. Simultaneous use of rapamycin and BBN from the 1st day of treatment significantly reduced the tumor growth in urinary bladder: 80% of the rats had no tumor and 20% had low-grade tumors. Adding rapamycin from the 5th week was associated with more tumor growth: 20% of the rats had no tumors, 20% had low-grade tumors, and 60% had high-grade tumors. Moreover, in the groups with rapamycin treatment from the 9th week, 13th week, and 17th week, all rats developed high-grade papillary tumors in urinary bladder, as did the control group that received no rapamycin. ConclusionsThe study results suggest that the anticancer effect of rapamycin on urothelial carcinoma is stage dependent. Early use of rapamycin provides better anticancer effect, whereas late use of rapamycin fails to inhibit the cancer growth.

Intermittent supplementation with rapamycin as a dietary restriction mimetic

Aging is a complex process associated with accumulation of damage, loss of function and increased vulnerability to disease, leading ultimately to death. Despite the complicated etiology of aging, an important discovery of recent years has been that simple genetic alterations can cause a substantial increase in healthy lifespan in laboratory model organisms [1]. Many of these longevity-extending mutations down-regulate the activity of the mTOR/S6K pathway [2-5] suggesting that reduced Tor/S6K signaling promotes entry into alternative phases normally entered during periods of starvation. In fact, dietary restriction (DR), a reduction in food intake without malnutrition, lowers Tor/S6K signaling and extends the average and maximum life span of a variety of organisms including yeast, flies, worms, fish, and rodents [1]. In both rodents and monkeys, it delays loss of function and reduces the incidence of major diseases [1] and in humans it causes a reduction in several metabolic factors and markers of diseases associated with including diabetes, and cardiovascular disease and cancer [6]. In yeast, down-regulation of the Tor/S6 pathway was shown to be important for the effects of DR on longevity and stress resistance [7]. Recently, it has been demonstrated that supplementation with rapamycin (an inhibitor of mTOR) started both at 9 and 20 months of life determines a small but significant extension of average and maximal life span in genetically heterogeneous male and female mice [8, 9]. However, in these studies rapamycin supplementation did not change the distribution of causes of death, and in particular did not reduced cancer. Interestingly, in recent issue of Cell Cycle Anisimov and et al. reported that lifelong intermittent administration (three times a week for 2 weeks, followed by a 2 week break) of rapamycin started at 2 mos of age significantly increased maximal lifespan and delayed spontaneous cancer in normal inbread female 129/Sv mice [10]. This study is in agreement with the effect of inhibition of Tor/S6K signaling in protecting yeast against age-dependent DNA mutations [11] and of mutations in GH or GHR in reducing cancer incidence in mice and humans [1, 12]. Similarly, DR without malnutrition extends lifespan and powerfully protects against cancer in mice [13]. Rapamycin is an immunosuppressant and antiproliferative agent that is clinically used to prevent rejection in organ transplantation, primarily in renal transplant patients. Rapamycin by inhibiting the activation of mTORC1, inhibits effector T-cell proliferation and dendritic cell maturation, but does not supress TReg cells induction [14]. Data from experimental studies indicate that rapamacyin prolongs allograft survival and reverses acute rejection of kidney allografts in in rodents and humans [15]. Besides the well-known immunosuppressive and anti-rejection properties of rapamycin, there is accumulating scientific evidence supporting a potential anti-atherogenic, anti-fibrotic, antiangiogenic, and anticancer effect of rapamycin [16]. Nevertheless, chronic subministration of rapamycin is associated with a number of side effects in some renal transplant patients, including impaired wound-healing, lymphoceles, delayed graft function, anemia, pneumonitis, hypercholesterolemia and proteinuria [15, 16]. However, these side effects are dose-dependent, and it is not known if lower doses and intermittent subministration may limit some of these effects and potentiate the beneficial effects. Interestingly in this paper rapamycin was able to extend lifespan and reduce cancer risk in mice also when used with an intermittent schedule. More studies are needed to understand benefits and side-effects of rapamycin supplementation in different strains of mice and in monkeys as a candidate cancer-preventive and life-extension pharmacological agent. However, the efficacy of intermittent rapamycin treatment in cancer prevention and life span extension shown by Anisimov et al. is very promising since it is likely to reduce the side effects associated with chronic treatment.

Combined inhibition of Dnmt and mTOR signaling inhibits formation and growth of colorectal cancer

Although the anticancer effects of rapamycin (RPM) and 5-aza-deoxycytidine (AZA) have been studied extensively, the combined effect of these two drugs on colorectal cancer (CRC) is still unknown. This study addresses the effect of AZA and RPM combination therapy on CRC and its influence on the mammalian target of rapamycin (mTOR) and its signal transduction pathway. Human CRC cell line HCT116 was treated with AZA alone, RPM alone, or concurrently with a combination of both drugs. Cell viability, apoptosis, and cell cycle distribution were analyzed. CRC was initiated in S-ICR mice, which were then treated with the drugs mentioned above, and tumor incidence and volume were measured. The activity of the mTOR signal transduction pathway was detected by Western blot analysis or immunohistochemistry. Combination treatment with AZA and RPM inhibited the growth of HCT116 cells, induced apoptosis, arrested the cell cycle, and reduced the incidence and tumor volume of CRC in mice, as well as inhibited the phosphorylation of components of the mTOR signal transduction pathway. These effects were more significant than those of single-drug treatments. Combination treatment with AZA and RPM inhibits the formation and growth of CRC. These findings may provide a novel strategy for CRC treatment.

Anti-Cancer Effects of Rapamycin on U937 Cell Line.

Purpose: Cytotoxicity of rapamycin alone or in combination with arsenic trioxide in lymphoma cell line U937 was assessed, for the study of Anti-cancer effects of rapamycin.Methods: cell vability and proliferation was analyzed by MTT and cell counting, clone formulating ability was analyzed by semisolid medium, cell cycle was analyzed by Propidium Iodide/RN-ase stain, the phosphorylation level of mammalian target of rapamycin (mTOR) was detected after marked by Phospho-mTOR antibody (Ser2448) and FITC. Expression of P27 was assessed by western- blot.Result: 1.MTT measurement showed with increasing concentrations of RAPA (10 nM 100 nM, 1000 nM) cell proliferation inhibition rate was follwed 16.2%, 25.5%, 47.8%. Clonogenic assay showed the U937 proliferation inhibition rate was 80.5% in a 7-day leukemia colony-forming assay with concentration of 10 nM RAPA.2 RAPA 10nM, As2O3 0.6uM, As2O3 0.9uM alone, RAPA 10nM in combination with As2O3 0.6uM and 0.9uM, cell proliferation inhibition rate was followed 13%, 26%, 35%, 43%, 54%. 3. Propidium Iodide/RN-ase measurement showed RAPA resulted in U937 cell arrested in G1-phase, and was blocked in S-phase. 4. FCM showed the phosphorylation level of mTOR down-regulated significantly after treated by rapamycin (10 nM),5.The expression of P27 enhanced after treatd by rapamycin (10 nM)Conclutions: From the experiment we can see rapamycin alone can inhibit the proliferation and clone formulating ability of U937 cell line. mTOR kinase is involved in the regulation of cell growth and proliferation. Rapamycin can down-regulate the phosphorylation level of mTOR, which can induce increasing expression of P27, As a result U937 arrest in G1-phase and block in S-phase. Interestingly rapamycin exert additive effect in proliferation experiment when combind with As2O3, higher than rapamycin or As2O3 alone.

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.

Rapamycin induces Smad activity in prostate cancer cell lines.

Rapamycin inhibits the FK506-binding protein 12 (FKBP12)/mammalian target of rapamycin (mTOR) complex and causes cell cycle arrest in G1. The precise mechanism of growth inhibition by rapamycin is only partly understood. Rapamycin led to growth inhibition in the human prostate cancer cell lines LNCaP and PC3 cells after 72 h, ID50: 93 and 50 nM, respectively. Filter cDNA array analysis showed down-regulation by more than 0.75x by rapamycin in PC3 cells and LNCaP cells of the following genes: follistatin, eukaryotic initiation factor-4E (eIF4E), glucose-6-phosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH)-A, ATP synthase, heat shock protein (HSP)-1. Upregulation by more than 1.5x was found for: bone morphogenetic protein (BMP)-4, FKBP12, carcinoma embryonic antigen (CEA) precursor, eukaryotic initiation factor (eIF)-3 p36 subunit, latent transforming growth factor (TGF) beta binding protein (LTBP)1. Rapamycin induced BMP4 and reduced follistatin expression in PC3 cells. This resulted in a dose-dependent nuclear expression of Smad4 and activated the SBE4 Smad-reporter, indicating activation of TGFbeta/BMP signaling. Combining rapamycin with PI3K inhibition (LY294002) increased growth inhibition. These findings illustrate that Smad signaling plays a role in the anticancer effects of rapamycin and show that combination with PI3K inhibition improves growth inhibition.