Inhibition Of mTOR Signaling Pathway Research Articles

Risk of hematological and gastrointestinal toxicities in patients with advanced neuroendocrine tumors treated with everolimus: A meta-analysis of phase 3 randomized controlled trials.

e16184Background: Neuroendocrine tumors (NET) are uncommon and can develop from multiple primary sites. Inhibition of mTOR signaling pathway has shown antiproliferative effects on NET and hence, ev...

Promising horizon to alleviate Alzeheimer’s disease pathological hallmarks via inhibiting mTOR signaling pathway: A new application for a commonplace analgesic

Alzheimer’s disease (AD) is a very prevalent and burdensome disease of elderlies but albeit extensive studies, mechanisms underlying its pathology and consequently its definite treatment is ambiguous. Intra and extra-cellular aggregation of abnormal proteins and impaired autophagy machinery, two closely related events taking place in AD brains proposed to be directly controlled by mTOR signaling pathway.On the other hand, tramadol that is a very well tolerated opioid analgesic has been revealed to inhibit mTOR upstream controllers through interaction with specific types of muscarinic, serotonergic, nicotinic and NMDA receptors, although it seems to induce the opposite effect via µ-opioid receptor.Putting all the pieces of experimental evidence together, we hypothesize that tramadol might alleviate AD pathological hallmarks at least in cellular level through decreasing activity of PI3K-AKT and ERK and also mTOR signaling pathways respectively and results in autophagy activation as well as tau-dephosphorylation.

Anti-Leukemia Effect and Mechanism of Oridonin on Imatinib-Sensitive and Imatinib-Resistant K562 Cells

To explore the molecular mechanism of resistance to imatinib in K562 cells(K562-R) and the anti-proliferative effect of oridonin (OR), as well as its mechanism in imatinib-sensitive and imatinib-resistant K562 cells (K562-S and K562-R cells). The expression of p-Lyn in K562-S and K562-R cells were detected by Western blot. The anti-proliferative effect of OR in K562-S and K562-R cells was assayed by MTT, the morphological changes were examined with microscope, the cell apoptosis was detected by flow cytometry, the expressions of BCL-2 and Akt/mTOR signaling pathway were detected by Western blot. The over-expression of p-Lyn was detcected in K562-R cells, OR inhibited the proliferation of K562-S and K562-R cells and the value of IC50 was 4.23±1.30, 4.97±2.23 µmol/L, respectively. The apoptotic rate was obviously enhanced after OR treatment for 24 h, compared with control group. OR down-regulated the expression of p-Lyn, mTOR signaling pathway and BCL-2 protein. Over-expression of p-Lyn may be involved in the mechanism of resistance to imatinib. OR can inhibit the proliferation of K562-S and K562-R cells through down-regulating p-Lyn, inhibiting mTOR signaling pathway and down-regulating expression of BCL-2 protein.

Prognostic significance and function of mammalian target of rapamycin in tongue squamous cell carcinoma

Despite improvement in preoperative imaging, surgical technique, and adjuvant therapy, the prognosis of patients with tongue squamous cell carcinoma (SCC) is still unsatisfactory. The mammalian target of rapamycin (mTOR) play a key role in the regulation of tumor cell proliferation and survival. However, the significance of mTOR on the prognosis of tongue SCC remains largely undefined. In the present study, immunohistochemistry was performed to evaluate the expression of phosphorylated mTOR (p-mTOR) in 160 surgically resected tongue SCC, and correlated with survival. Univariate analysis revealed that p-mTOR overexpression (P = 0.006) was associated with inferior overall survival. In multivariate comparison, p-mTOR overexpression (P = 0.002, hazard ratio = 2.082) remained independently associated with worse overall survival. In vitro study, tongue cancer cells treated with everolimus, the specific mTOR inhibitor, or transfected with mTOR-mediated siRNAs dramatically attenuated the abilities of cell proliferation by MTT and BrdU assays. In 4-NQO-induced tongue cancer murine model, mTOR inhibitors significantly decreased the incidence of tongue SCC. In conclusion, p-mTOR overexpression was independently associated with poor prognosis of patients with tongue SCC. In vitro and vivo, mTOR inhibition showed the promising activity in tongue SCC. Our results suggest that inhibition of mTOR signaling pathway may be a novel therapeutic target for tongue SCC.

Gambogic acid improves non-small cell lung cancer progression by inhibition of mTOR signaling pathway

Gambogic acid (GA) has been shown to inhibit cancer cell proliferation, induce apoptosis, and enhance reactive oxygen species accumulation. However, whether GA could improve multidrug resistance through modulating autophagy has never been explored. We demonstrated that the combination of GA and cisplatin (CDDP) resulted in a stronger growth inhibition effect on A549 and NCI-H460 cells using the MTT assay. Furthermore, treatment with GA significantly increased autophagy in these cells. More importantly, GA-induced cell death could be largely abolished by 3-methyladenine (3-MA) or chloroquine (CQ) treatment, suggesting that GA-induced cell death was dependent on autophagy. Western blot analysis showed that GA treatment suppressed the activation of Akt, mTOR, and S6. In addition, using a GA and rapamycin combination induced more cell death compared to either GA or rapamycin alone. In summary, GA may have utility as an adjunct therapy for non-small cell lung cancer (NSCLC) patients through autophagy-dependent cell death, even when cancer cells have developed resistance to apoptosis.

Inhibition of mTOR signaling pathway by aqueous extract of Siberian ginseng

Inhibition of mTOR signaling pathway by aqueous extract of Siberian ginseng

MiR-199a-3p enhances cisplatin sensitivity of cholangiocarcinoma cells by inhibiting mTOR signaling pathway and expression of MDR1.

Several studies have reported reduced miRNA-199a-3p (miR-199a-3p) in different human malignancies, however, little is known about miR-199a-3p in cholangiocarcinoma cells. In this study, we demonstrate the essential role and mechanism of miR-199a-3p in regulating cisplatin sensitivity in cholangiocarcinoma cell lines. Using a CCK-8 cell counting assay we found that expression of miR-199a-3p was positively correlated with cisplatin sensitivity in cholangiocarcinoma cell lines. MiR-199a-3p overexpression could decrease the proliferation rate and increase apoptosis of cholangiocarcinoma cells in the presence of cisplatin, while miR-199a-3p inhibition had the opposite effect. Further study demonstrated that mTOR was the target gene of miR-199a-3p, and that miR-199a-3p mimics could inhibit expression of mTOR, which consequently reduced the phosphorylation of its downstream proteins 4EBP1 and p70s6k. Rescue experiments proved that miR-199a-3p could increase the cisplatin sensitivity of cholangiocarcinoma cell lines by regulating mTOR expression. Moreover, we also found that miR-199a-3p overexpression could reduce cisplatin induced MDR1 expression by decreasing the synthesis and increasing the degradation of MDR1, thus enhancing the effectiveness of cisplatin in cholangiocarcinoma. In conclusion, miR-199a-3p could increase cisplatin sensitivity of cholangiocarcinoma cell lines by inhibiting the activity of the mTOR signaling pathway and decreasing the expression of MDR1.

Long Non-Coding RNA (lncRNA) Urothelial Carcinoma-Associated 1 (UCA1) Enhances Tamoxifen Resistance in Breast Cancer Cells via Inhibiting mTOR Signaling Pathway.

BackgroundLong non-coding RNA (lncRNA) UCA1 is an oncogene in breast cancer. The purpose of this study was to investigate the role of UCA1 in tamoxifen resistance of estrogen receptor positive breast cancer cells.Material/MethodsTamoxifen sensitive MCF-7 cells were transfected for UCA1 overexpression, while tamoxifen resistant LCC2 and LCC9 cells were transfected with UCA siRNA for UCA1 knockdown. qRT-PCR was performed to analyze UCA1 expression. CCK-8 assay, immunofluorescence staining of cleaved caspase-9, and flow cytometric analysis of Annexin V/PI staining were used to assess tamoxifen sensitivity. Western blot analysis was performed to detect p-AKT and p-mTOR expression.ResultsLncRNA UCA1 was significantly upregulated in tamoxifen resistant breast cancer cells compared to tamoxifen sensitive cells. LCC2 and LCC9 cells transfected with UCA1 siRNA had significantly higher ratio of apoptosis after tamoxifen treatment. UCA1 siRNA significantly decreased the protein levels of p-AKT and p-mTOR in LCC2 and LCC9 cells. Enforced UCA1 expression substantially reduced tamoxifen induced apoptosis in MCF-7 cells, while rapamycin treatment abrogated the protective effect of UCA1.ConclusionsUCA1 upregulation was associated with tamoxifen resistance in breast cancer. Mechanistically, UCA1 confers tamoxifen resistance to breast cancer cells partly via activating the mTOR signaling pathway.

Ketamine Exhibits Different Neuroanatomical Profile After Mammalian Target of Rapamycin Inhibition in the Prefrontal Cortex: the Role of Inflammation and Oxidative Stress.

Studies indicated that mammalian target of rapamycin (mTOR), oxidative stress, and inflammation are involved in the pathophysiology of major depressive disorder (MDD). Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, has been identified as a novel MDD therapy; however, the antidepressant mechanism is not fully understood. In addition, the effects of ketamine after mTOR inhibition have not been fully investigated. In the present study, we examined the behavioral and biochemical effects of ketamine in the prefrontal cortex (PFC), hippocampus, amygdala, and nucleus accumbens after inhibition of mTOR signaling in the PFC. Male adult Wistar rats received pharmacological mTOR inhibitor, rapamycin (0.2nmol) or vehicle into the PFC and then a single dose of ketamine (15mg/kg, i.p.). Immobility was assessed in forced swimming tests, and then oxidative stress parameters and inflammatory markers were evaluated in the brain and periphery. mTOR activation in the PFC was essential to ketamine's antidepressant-like effects. Ketamine increased lipid damage in the PFC, hippocampus, and amygdala. Protein carbonyl was elevated in the PFC, amygdala, and NAc after ketamine administration. Ketamine also increased nitrite/nitrate in the PFC, hippocampus, amygdala, and NAc. Myeloperoxidase activity increased in the hippocampus and NAc after ketamine administration. The activities of superoxide dismutase and catalase were reduced after ketamine administration in all brain areas studied. Inhibition of mTOR signaling pathways by rapamycin in the PFC was required to protect against oxidative stress by reducing damage and increasing antioxidant enzymes. Finally, the TNF-α level was increased in serum by ketamine; however, the rapamycin plus treatment group was not able to block this increase. Activation of mTOR in the PFC is involved in the antidepressant-like effects of ketamine; however, the inhibition of this pathway was able to protect certain brain areas against oxidative stress, without affecting inflammation parameters.

Aspirin may inhibit angiogenesis and induce autophagy by inhibiting mTOR signaling pathway in murine hepatocarcinoma and sarcoma models.

Aspirin is known to have inhibitory effects on growth development in various types of tumor. In previous studies, it was observed to inhibit angiogenesis by downregulating the expression of vascular endothelial growth factor-A (VEGF-A). In the present study, murine H22 hepatocarcinoma and S180 sarcoma models were used to ascertain whether aspirin could inhibit angiogenesis and promote autophagy in tumors. Tumor-bearing mice were randomly divided into four groups with 10 mice per group: i) no treatment; ii) low-dose aspirin (100 mg/kg); iii) high-dose aspirin (400 mg/kg); iv) everolimus group (4 mg/kg). The effects of high-dose aspirin were validated through preliminary experiments. The drug treatment was administered every day for 14 days. The tumor size was measured every other day and then the tumor growth curve was plotted, and the tumor inhibitory rates were calculated. The expression levels of phosphorylated mammalian target of rapamycin (p-mTOR), hypoxia-inducible factor-1α (HIF-1α), VEGF-A, UNC-51-like kinase-1 (ULK1) and microtubule-associated protein 1 light chain 3A (LC3A) were detected by immunohistochemistry and western blot analysis, respectively. We observed that tumor growth delay was achieved in both H22 hepatocarcinoma and S180 sarcoma models following treatment with aspirin. The tumor growth inhibition rates induced by low and high-dose aspirin and everolimus were 19.6, 33.6 and 53.7% (P<0.05) in H22 hepatocarcinoma, and 25.7, 40.6 and 48.7% (P<0.05) in S180 sarcoma. The immunohistochemistry and western blot analysis data from the models revealed that the expression of p-mTOR, HIF-1α and VEGF-A was decreased, while the expression of ULK1 and LC3A was increased following treatment with aspirin and everolimus. The changes were more apparent in the high-dose aspirin and everolimus groups (P<0.01). The inhibitory action of aspirin and everolimus on tumor angiogenesis may be through inhibiting the expression of p-mTOR, HIF-1α and VEGF-A. Alternatively, aspirin may induce autophagy by inhibiting the mTOR signaling target and then increasing ULK1 and LC3A.

Therapeutic effects of antibiotic drug mefloquine against cervical cancer through impairing mitochondrial function and inhibiting mTOR pathway.

Targeting mitochondria is an attractive strategy for cancer therapy due to the essential roles of mitochondria in cancer cell energy metabolism. In this study, we show that mefloquine, an antibiotic drug, effectively targets cervical cancer cells through impairing mitochondrial function. Mefloquine dose-dependently induces apoptosis and inhibits proliferation and anchorage-independent colony formation of multiple cervical cancer cell lines. Mefloquine alone inhibits cervical tumor growth in vivo and its combination with paclitaxel is synergistic in inhibiting tumor growth. Mechanistically, mefloquine inhibits mitochondrial function via inhibiting mitochondrial respiration, decreasing membrane potential, increasing ROS generation, and decreasing ATP level. We further show that mefloquine suppresses activation of mTOR signaling pathway in HeLa cells. However, the inhibitory effects of mefloquine on survival, colony formation, and ATP are abolished in mitochondrial respiration-deficient HeLa ρ0 cells, demonstrating that mefloquine acts on cervical cancer cells via targeting mitochondrial respiration. Inhibition of mTOR signaling pathway by mefloquine was also reversed in HeLa ρ0 cells, suggesting deactivation of mTOR pathway as a consequence of mitochondria function disruption. Our work suggests that mefloquine is a potential candidate for cervical cancer treatment. Our work also highlights the therapeutic value of anti-mitochondria and establishes the association of mitochondrial function and the activation of mTOR signaling pathway in cervical cancer cells.

MEK Inhibition Sensitizes Precursor B-Cell Acute Lymphoblastic Leukemia (B-ALL) Cells to Dexamethasone through Modulation of mTOR Activity and Stimulation of Autophagy.

Resistance to glucocorticosteroids (GCs) is a major adverse prognostic factor in B-ALL, but the molecular mechanisms leading to GC resistance are not completely understood. Herein, we sought to elucidate the molecular background of GC resistance in B-ALL and characterize the therapeutic potential of targeted intervention in these mechanisms. Using exploratory bioinformatic approaches, we found that resistant cells exhibited significantly higher expression of MEK/ERK (MAPK) pathway components. We found that GC-resistant ALL cell lines had markedly higher baseline activity of MEK and small-molecule MEK1/2 inhibitor selumetinib increased GCs-induced cell death. MEK inhibitor similarly increased in vitro dexamethasone activity in primary ALL blasts from 19 of 22 tested patients. To further confirm these observations, we overexpressed a constitutively active MEK mutant in GC-sensitive cells and found that forced MEK activity induced resistance to dexamethasone. Since recent studies highlight the role GC-induced autophagy upstream of apoptotic cell death, we assessed LC3 processing, MDC staining and GFP-LC3 relocalization in cells incubated with either DEX, SEL or combination of drugs. Unlike either drug alone, only their combination markedly increased these markers of autophagy. These changes were associated with decreased mTOR activity and blocked 4E-BP1 phosphorylation. In cells with silenced beclin-1 (BCN1), required for autophagosome formation, the synergy of DEX and SEL was markedly reduced. Taken together, we show that MEK inhibitor selumetinib enhances dexamethasone toxicity in GC-resistant B-ALL cells. The underlying mechanism of this interaction involves inhibition of mTOR signaling pathway and modulation of autophagy markers, likely reflecting induction of this process and required for cell death. Thus, our data demonstrate that modulation of MEK/ERK pathway is an attractive therapeutic strategy overcoming GC resistance in B-ALL patients.

Probucol inhibits neural cell apoptosis via inhibition of mTOR signaling pathway after spinal cord injury

Autophagy plays an essential role in neurodevelopment, axonal guidance, neuropathic pain remission, and neuronal survival. Inhibiting the mammalian target of rapamycin (mTOR) signaling pathway can induce the occurrence of autophagy. In this study, we initially detected the effect of probucol on autophagy after spinal cord injury (SCI) by intraperitoneally injecting spinal cord-injured rats with probucol for 7days. The levels of Beclin1 and LC3B were evidently enhanced at 7days post-operation. However, the increase in the phosphorylated AMP-activated protein kinase (AMPK) protein and the decrease in ribosomal protein S6 kinase p70 subtype (p70S6K) phosphorylation level simultaneously occurred after SCI. Moreover, the expression levels of apoptosis-related proteins of Caspase-3, Caspase-9, and Bax were significantly reduced. Immunofluorescence results indicated that the expression of Caspase-3 protein was evidently decreased and that of Beclin-1 protein was increased by probucol. Nissl staining and Basso, Beattie, and Bresnahan scores showed that the quantity and function of motor neurons were visibly preserved by probucol after SCI. This study showed that probucol inhibited the mTOR signaling pathway to induce autophagy, reduce neural cell apoptosis and promote recovery of neurological function after SCI.

Saikosaponin a functions as anti-epileptic effect in pentylenetetrazol induced rats through inhibiting mTOR signaling pathway.

Saikosaponin a (SSa), which is one major bioactive compound isolated from radix bupleuri, has been demonstrated to exhibit the properties of anticonvulsant and antiepileptic in few reports. This study aims to clarify the molecular mechanism by which SSa protects against pentylenetetrazol (PTZ) induced epileptic seizure. PTZ induced rat and hippocampal neuron were established. Treated rats or hippocampal neuron with SSa, and mTOR, P70S6K, IL-1β and TNF-α were then determined. In PTZ induced rat, SSa significantly reduced seizure severity and duration while markedly elevated seizure latency, and it also down-regulated hippocampal p-mTOR, p-70S6K, L-1β and TNF-α expression. In hippocampal neurons exposed to PTZ, p-mTOR and p-70S6K expression levels were also decreased by SSa. Pre-incubated hippocampal neurons with leucine, an mTOR agonist, reversed the effects of SSa on decreasing cytokines expression and inhibiting cell apoptosis. The treatment of mTOR inhibitor rapamycin prevented against the increase of cytokines expression and hippocampal neuron apoptosis induced by PTZ. Leucine also canceled the alleviation of seizures and induction of hippocampal caspase-3 activity in PTZ induced rat with the treatment of SSa. SSa protects against PTZ induced epileptic seizure and hippocampal neuron apoptosis through inhibiting mTOR signaling pathway.

N-Hydroxyphthalimide exhibits antitumor activity by suppressing mTOR signaling pathway in BT-20 and LoVo cells.

BackgroundN-Hydroxyphthalimide (NHPI), an important chemical raw material, was found to have potent and selective anti-proliferative effect on human breast carcinoma BT-20 cells, human colon adenocarcinoma LoVo and HT-29 cells during our screening for anticancer compounds. The purpose of this study is to assess the antitumor efficacy of NHPI in vitro and in vivo and to explore the underlying antitumor mechanism.MethodsCell cytotoxicity of NHPI was evaluated using MTS assay and cell morphological analysis. After NHPI treatment, cell cycle, apoptosis and mitochondrial membrane potential were analyzed using flow cytometer. The subcellular localization of eukaryotic initiation factor 4E (eIF4E) was analyzed by immunofluorescence assay. The antitumor efficacy of NHPI in vivo was tested in BT-20 xenografts. The underlying antitumor mechanisms of NHPI in vitro and in vivo were investigated with western blot analysis in NHPI-treated cancer cells and tumor tissues. Statistical significance was determined using Student’s t-test.ResultsIn vitro, NHPI selectively inhibited the proliferation and induced G2/M phase arrest in BT-20 and LoVo cells, which was attributed to the inhibition of cyclin B1 and cdc2 expressions. Furthermore, NHPI induced apoptosis via mitochondrial pathway. Of note, NHPI effectively inhibited mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and mTOR complex 2 (mTORC2) signaling, and overcame the feedback activation of Akt and extracellular signal-regulated kinase (ERK) caused by mTORC1 inhibition in BT-20 and LoVo cells. In vivo, NHPI inhibited tumor growth and suppressed mTORC1 and mTORC2 signaling in BT-20 xenografts with no obvious toxicity.ConclusionsWe found for the first time that NHPI displayed antitumor activity which is associated with the inhibition of mTOR signaling pathway. Our findings suggest that NHPI may be developed as a promising candidate for cancer therapeutics by targeting mTOR signaling pathway and as such warrants further exploration.

Crosstalk between Ca2+ signaling and mitochondrial H2O2 is required for rotenone inhibition of mTOR signaling pathway leading to neuronal apoptosis.

Rotenone, a neurotoxic pesticide, induces loss of dopaminergic neurons related to Parkinson's disease. Previous studies have shown that rotenone induces neuronal apoptosis partly by triggering hydrogen peroxide (H2O2)-dependent suppression of mTOR pathway. However, the underlying mechanism is not fully understood. Here, we show that rotenone elevates intracellular free calcium ion ([Ca2+]i) level, and activates CaMKII, resulting in inhibition of mTOR signaling and induction of neuronal apoptosis. Chelating [Ca2+]i with BAPTA/AM, preventing extracellular Ca2+ influx using EGTA, inhibiting CaMKII with KN93, or silencing CaMKII significantly attenuated rotenone-induced H2O2 production, mTOR inhibition, and cell death. Interestingly, using TTFA, antimycin A, catalase or Mito-TEMPO, we found that rotenone-induced mitochondrial H2O2 also in turn elevated [Ca2+]i level, thereby stimulating CaMKII, leading to inhibition of mTOR pathway and induction of neuronal apoptosis. Expression of wild type mTOR or constitutively active S6K1, or silencing 4E-BP1 strengthened the inhibitory effects of catalase, Mito-TEMPO, BAPTA/AM or EGTA on rotenone-induced [Ca2+]i elevation, CaMKII phosphorylation and neuronal apoptosis. Together, the results indicate that the crosstalk between Ca2+ signaling and mitochondrial H2O2 is required for rotenone inhibition of mTOR-mediated S6K1 and 4E-BP1 pathways. Our findings suggest that how to control over-elevation of intracellular Ca2+ and overproduction of mitochondrial H2O2 may be a new approach to deal with the neurotoxicity of rotenone.

Effective targeting of colorectal cancer cells using TORC1/2 kinase inhibitors in vitro and in vivo.

We investigated the effects of TORC1/2 kinase inhibitors on colorectal cancer (CRC) cell lines. Using selective TORC1/2 inhibitors, rapamycin and PP242, we assessed their effect on the growth of CRC cells in vitro and tumor growth in vivo. Rapamycin and PP242 inhibit proliferation and induce apoptosis of CRC cells. They also enhance proapoptotic effect of conventional chemo drug doxorubicin in CRC cells in vitro. When combined with doxorubicin, rapamycin and PP242 almost completely inhibit tumor growth in vivo. Rapamycin and PP242 inhibit phosphorylation of Akt, ribosomal S6 kinase, 4EBP1 and mTOR. Our study suggests rapamycin and PP242 may be a useful therapeutic agent and inhibiting mTOR signaling pathway represents a new targeted therapy for CRC.

Abstract 1196: Enhancement of anti-melanoma effect of BRAF and MEK inhibition by metformin

Abstract Introduction: Metformin has been shown to inhibit human cancer cell growth via activation of MAP kinase with resultant inhibition of mTOR signaling pathway. Recent clinical evidence demonstrated the anti-melanoma effect of combined mutant BRAF inhibition and MEK inhibition. We determined the effect of metformin in combination with mutant BRAF and MEK inhibition on melanoma proliferation and the possible mechanisms. Methods: Human melanoma cells (SK-23, Mel888, Mel1861) were treated with Metformin (1-20 mM), PLX4032 (a mutant BRAF inhibitor, 1-20 uM), AZD6244 (a MEK inhibitor, 1-20 uM), or combination. Cell proliferation assay was performed using Cell Titer Blue assay. Western blotting was performed to determine the expression of PARP, caspases-9, LC3A/B,Beclin1, pERK, pMEK, AMPKa, p70S6, and p4E-BP1. GAPDH was used as internal control. Autophagy was further analyzed with immunofluorescence with LC-3 and NECN1 anti-antibody for visualization of lysosomal morphology and evaluated under the Nikon Eclipse Ti microscope (20×). DAPI was used to counter stain the nucleus. Apoptosis was further analyzed with flow cytometry using FITC Annexin V Apoptosis Detection Kit (BD Biosciences). Data were presented as means ± SD for the three separate experiments. For comparison between groups, the student's t test was used and p&amp;lt; 0.05 was considered to be statically significant. Results: There was dose-dependent inhibition of melanoma cell proliferation with either metformin(M), PLX4032(P), and AZD6244(A). Addition of M to PA combination had synergistic effect on the anti-melanoma proliferative effect of PA. Significantly increased expression (p&amp;lt;0.05) of apoptosis markers (PARP and caspases-9) and autophagy markers (LC3B and Beclin1) were observed in cells treated with PAM compared with PA. Enhanced induction of autophagy with the triple drugs was confirmed by visualization of autophagosome and autolysosome formation on immunofluorescence. Synergistic effect on apoptosis was confirmed on Annexin V assay. The PAM combination treatment also lead to significant enhancement of AMPK activation and increased phosphorylation of AMPKα at Thr-172 compared with metformin alone (p&amp;lt;0.01). The addition of M to PA combination resulted in inhibition of mTOR signaling with decreased phosphorylation of p70S6K and 4E-BP1 in treated cancer cells, when compared with PA or M alone. Conclusion: We have observed a synergistic effect on suppressing human melanoma proliferation with the addition of metformin to combined inhibition of mutant BRAF and MEK. The synergism is due to enhanced inhibition of mTOR signaling pathways. Citation Format: Yanping Zhang, Guangyong Peng, Eddy C. Hsueh. Enhancement of anti-melanoma effect of BRAF and MEK inhibition by metformin. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1196. doi:10.1158/1538-7445.AM2015-1196

Rapamycin inhibits Erk1/2-mediated neuronal apoptosis caused by cadmium.

Cadmium (Cd), an environmental contaminant, causes neurodegenerative disorders. Recently we have shown that rapamycin prevents Cd-induced neuronal cell death by inhibiting mTOR signaling pathway. Here we found that rapamycin exerted its prevention against Cd-induced neuronal cell death also partially via blocking Erk1/2 pathway. Inhibiting Erk1/2 with PD98059 or silencing Erk1/2 potentiated rapamycin's inhibition of Cd-induced phosphorylation of Erk1/2 and apoptosis in neuronal cells. Both PP2A and PTEN/Akt were involved in the regulation of Erk1/2 activation and cell death triggered by Cd. Inhibition of PP2A with okadaic acid or ectopic expression of dominant negative PP2A attenuated rapamycin's inhibition of Cd-induced phospho-Erk1/2 and apoptosis, whereas over-expression of wild-type PP2A enhanced rapamycin's effects; Over-expression of wild-type PTEN or dominant negative Akt, or inhibition of Akt with Akt inhibitor X strengthened rapamycin's inhibition of Cd-induced phospho-Erk1/2 and cell death. Furthermore, expression of a rapamycin-resistant and kinase-active mTOR (mTOR-T) blocked rapamycin's inhibitory effects on Cd-induced inhibition of PP2A, down-regulation of PTEN, and activation of Akt, leading to Erk1/2 activation and cell death, whereas silencing mTOR mimicked rapamycin's effects. The results uncover that rapamycin inhibits Cd activation of Erk1/2-mediated neuronal apoptosis through intervening mTOR-PP2A/PTEN signaling network.

Phosphorylated p70S6K expression is an independent prognosticator for patients with esophageal squamous cell carcinoma

Although marked improvements have been made in surgical technique and chemoradiotherapy, the prognosis for patients with esophageal squamous cell carcinoma (ESCC) is still unsatisfactory. The mammalian target of rapamycin (mTOR) and its downstream signaling, p70 ribosomal S6 protein kinase (p70S6K) and eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), seem to play central roles in the regulation of cancer cell proliferation and survival. The significance of mTOR and its downstream targets, p70S6K and 4E-BP1, on the prognosis of ESCC remains uncertain, but this pathway is of particular concern because effective inhibitors are already available. Immunohistochemistry performed to evaluate the expression of phosphorylated mTOR (p-mTOR), phosphorylated p70S6K (p-p70S6K), phosphorylated 4E-binding protein 1 (p-4E-BP1), and Ki-67 using 105 surgically resected ESCC correlated with treatment outcome. The effect of the mTOR signaling pathway inhibitor everolimus on ESCC cell lines were investigated invitro by the 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and invivo by a nude mouse xenograft model. Univariate analysis showed that p-mTOR overexpression (P=.022), p-p70S6K overexpression (P=.002), and Ki-67 labeling index >50% (P=.045) were associated with inferior overall survival (OS). In a multivariate comparison, p-p70S6K overexpression (P=.001; hazard ratio, 2.247) remained independently associated with worse OS. In cell lines and the xenograft model, everolimus significantly inhibited ESCC growth. Overexpression of p-p70S6K is associated independently with a poor prognosis among patients with ESCC. The mTOR signaling pathway inhibitor everolimus can inhibit ESCC growth invitro and invivo. Our findings suggest that inhibition of mTOR signaling pathway may be a promising novel target for ESCC.