The anticancer effects of curcumin via targeting the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway.

The mammalian target of rapamycin (mTOR) is a protein kinase that has been considered as a key regulator of a large number of cellular processes, including cell growth, proliferation, differentiation, survival, and motility. Overactivation of mTOR (especially mTORC1) signaling is related to oncogenic cellular processes. Therefore targeting mTORC1 signaling is a new promising strategy in cancer therapy. In this regard, various studies have shown that curcumin, a polyphenol produced from the turmeric rhizome, has anti-inflammatory, antioxidant and anticancer properties. Curcumin may exert its anticancer function, at least in part, by suppressing mTOR-mediated signaling pathway in tumor cells. However, the exact underlying mechanisms by which curcumin blocks the mTORC1 signaling remain unclear. According to literature, curcumin inhibits insulin-like growth factor 1 (IGF-1)/phosphoinositide 3-kinase (PI3K)/Akt/mTORC1 pathway which leads to apoptosis and cell cycle arrest via suppression of erythroblastosis virus transcription factor 2 and murine double minute 2 oncoprotein. In addition, activation of unc-51-like kinase 1 by curcumin, as a downstream target of IGF-1/PI3K/Akt/mTORC1 axis, enhances autophagy. Curcumin induces AMP-activated protein kinase, a negative regulator of mTORC1, via inhibition of F0F1-ATPase. Interestingly, curcumin suppresses IκB kinase ß, the upstream kinase in mTORC1 pathway. Moreover, evidence revealed that curcumin downregulates the E3-ubiquitin ligases NEDD4, neural precursor cell-expressed developmentally downregulated 4. NEDD4 is frequently overexpressed in a wide range of cancers and degrades the phosphatase and tensin homolog, which is a negative regulator of mTORC1. Finally another suggested mechanism is suppression of MAOA/mTORC1/hypoxia-inducible factor 1α signaling pathway by curcumin.

Rapamycin restrains platelet procoagulant responses via FKBP-mediated protection of mitochondrial integrity.

BACKGROUND AND PURPOSE: Rapamycin is a potent immunosuppressant and anti-proliferative agent used clinically to prevent organ transplant rejection and for coating coronary stents to counteract restenosis. Rapamycin complexes with the immunophilin FKBP12, which subsequently binds and inhibits mTORC1. Despite several reports demonstrating that rapamycin affects platelet-mediated responses, the underlying mechanism of how it alters platelet function is poorly characterised. This study aimed to elucidate the effect of rapamycin on platelet procoagulant responses. EXPERIMENTAL APPROACH: The effect of rapamycin on platelet activation and signalling was investigated alongside the catalytic mTOR inhibitors KU0063794 and WYE-687, and the FKBP12-binding macrolide FK506. KEY RESULTS: Rapamycin affects platelet procoagulant responses by reducing externalisation of the procoagulant phospholipid phosphatidylserine, formation of balloon-like structures and local generation of thrombin. Catalytic mTOR kinase inhibitors did not alter platelet procoagulant processes, despite having a similar effect as rapamycin on Ca2+ signalling, demonstrating that the effect of rapamycin on procoagulant responses is independent of mTORC1 inhibition and not linked to a reduction in Ca2+ signalling. FK506, which also forms a complex with FKBP12 but does not target mTOR, reduced platelet procoagulant responses to a similar extent as rapamycin. Both rapamycin and FK506 prevented the loss of mitochondria integrity induced by platelet activation, one of the central regulatory events leading to PS externalisation. CONCLUSIONS AND IMPLICATIONS: Rapamycin suppresses platelet procoagulant responses by protecting mitochondrial integrity in a manner independent of mTORC1 inhibition. Rapamycin and other drugs targeting FKBP immunophilins could aid the development of novel complementary anti-platelet therapies.

Iridoids of Valeriana fauriei contribute to alleviating hepatic steatosis in obese mice by lipophagy.

Nonalcoholic fatty liver disease (NAFLD) is a common risk factor for metabolic syndrome that increases the risk of future cardiovascular disease, stroke, and diabetes. Recently, autophagy has been proposed as a means to prevent NAFLD. We investigated whether substances with autophagy-inducing activity alleviate NAFLD. The Valeriana fauriei (V. fauriei) was selected as a potential autophagy inducer among various natural materials using a Cyto-ID autophagy detection kit. V. fauriei 70 % ethanol extract (VFE) increased LC3II levels in the presence of the lysosomal inhibitor and reduced the GFP/mCherry puncta ratio, suggesting that VFE enhanced autophagy. VFE reduced oleic acid (OA)-induced lipid accumulation and increased the number of autophagosome in hepatocytes. Autophagy induction by VFE is due to inhibition of mTORC1 activity. VFE supplementation reduced fatty liver by downregulating lipogenesis-related genes and increased the autophagy, as revealed by TEM and IHC analysis in the fatty liver. We identified iridoids as main compounds of VFE; didrovaltrate (DI), valeriotriate B (VAL B), valeriotetrate C (VAL C), valtrate (VAL), and valechlorine (VC) were shown to enhance autophagy. These compounds also reduced OA-induced lipid accumulation in an Atg5-dependent manner. Taken together, VFE and its iridoids might be effective in alleviating fatty liver by acting as autophagy enhancers to break down LDs.

Discovery of Small-Molecule Selective mTORC1 Inhibitors via Direct Inhibition of Glucose Transporters.

The mechanistic target of rapamycin (mTOR) is a central regulator of cellular metabolic processes. Dysregulation of this kinase complex can result in a variety of human diseases. Rapamycin and its analogs target mTORC1 directly; however, chronic treatment in certain cell types and in vivo results in the inhibition of both mTORC1 and mTORC2. We have developed a high-throughput cell-based screen for the detection of phosphorylated forms of the mTORC1 (4E-BP1, S6K1) and mTORC2 (Akt) substrates and have identified and characterized a chemical scaffold that demonstrates a profile consistent with the selective inhibition of mTORC1. Stable isotope labeling of amino acids in cell culture-based proteomic target identification revealed that class I glucose transporters were the primary target for these compounds yielding potent inhibition of glucose uptake and, as a result, selective inhibition of mTORC1. The link between the glucose uptake and selective mTORC1 inhibition are discussed in the context of a yet-to-be discovered glucose sensor.

Comparison of anti-peritoneal fibrotic effects between an mTORC1-specific blocker and a PI3K/mTOR dual-blocker.

OBJECTIVE: To compare the anti-peritoneal fibrotic effects between a mammalian target of rapamycin complex 1-specific blocker and a phosphatidyl-inositol 3-kinase/mammalian target of rapamycin dual-blocker. METHODS: A total of 40 male Sprague-Dawley rats were randomly divided into five groups with eight animals per group. The normal group (N group) did not receive any intervention. The normal saline group (NS group) received an intraperitoneal injection of normal saline at 1 ml/100 g daily. The model group (3 W group), rapamycin (RAPA) group and BEZ235 (PI3K/mTOR dual-blocker) group all received an intraperitoneal injection of 0.1% chlorhexidine gluconate at 1 ml/100g daily. And the RAPA and BEZ235 groups also received a 0.5 mg/d RAPA or 2.5 mg/d BEZ235 gavage every day, respectively. Rats in each group were sacrificed after 3 weeks. RESULTS: Immunohistochemistry, real-time PCR and western blotting analysis of fibrosis-related indicators (FN, Col 1, and α-SMA) confirmed that RAPA and BEZ235 significantly inhibited peritoneal fibrosis and that these two drugs had similar effects. The p-Akt, p-mTOR, p-p70S6K expression levels were significantly up-regulated in the 3 W group compared to the NS group, confirming that the mTOR pathway was significantly activated during peritoneal fibrosis. RAPA significantly inhibited the phosphorylation of mTOR and p70S6K but did not have significant effects on p-Akt upstream of mTOR. BEZ235 had significant inhibitory effects on all signaling molecules (p-Akt, p-mTOR, and p-p70S6K) in the mTOR pathway. CONCLUSION: RAPA did not up-regulate p-Akt in a negative feedback fashion. Both drugs effectively inhibited peritoneal fibrosis.

PPARα-Mediated Positive-Feedback Loop Contributes to Cold Exposure Memory.

Fluctuations in food availability and shifts in temperature are typical environmental changes experienced by animals. These environmental shifts sometimes portend more severe changes; e.g., chilly north winds precede the onset of winter. Such telltale signs may be indicators for animals to prepare for such a shift. Here we show that HEK293A cells, cultured under starvation conditions, can "memorize" a short exposure to cold temperature (15 °C), which was evidenced by their higher survival rate compared to cells continuously grown at 37 °C. We refer to this phenomenon as "cold adaptation". The cold-exposed cells retained high ATP levels, and addition of etomoxir, a fatty acid oxidation inhibitor, abrogated the enhanced cell survival. In our standard protocol, cold adaptation required linoleic acid (LA) supplementation along with the activity of Δ-6-desaturase (D6D), a key enzyme in LA metabolism. Moreover, supplementation with the LA metabolite arachidonic acid (AA), which is a high-affinity agonist of peroxisome proliferator-activated receptor-alpha (PPARα), was able to underpin the cold adaptation, even in the presence of a D6D inhibitor. Cold exposure with added LA or AA prompted a surge in PPARα levels, followed by the induction of D6D expression; addition of a PPARα antagonist or a D6D inhibitor abrogated both their expression, and reduced cell survival to control levels. We also found that the brief cold exposure transiently prevents PPARα degradation by inhibiting the ubiquitin proteasome system, and starvation contributes to the enhancement of PPARα activity by inhibiting mTORC1. Our results reveal an innate adaptive positive-feedback mechanism with a PPARα-D6D-AA axis that is triggered by a brief cold exposure in cells. "Cold adaptation" could have evolved to increase strength and resilience against imminent extreme cold temperatures.

A Novel mTORC1/2 Inhibitor (MTI-31) Inhibits Tumor Growth, Epithelial-Mesenchymal Transition, Metastases, and Improves Antitumor Immunity in Preclinical Models of Lung Cancer.

PURPOSE: We aimed to investigate efficacy and mechanism of MTI-31 (LXI-15029), a novel mTORC1/mTORC2 inhibitor currently in human trial (NCT03125746), in non-small cell lung cancer (NSCLC) models of multiple driver mutations and tyrosine kinase inhibitor (TKI)-resistance. EXPERIMENTAL DESIGN: Gene depletion, inhibitor treatment, immunological, flow cytometry, cellular, and animal studies were performed to determine in vitro and in vivo efficacy in NSCLC models of driver mutations and elucidate roles by mTOR complexes in regulating migration, epithelial-mesenchymal transition (EMT), metastasis, intracranial tumor growth, and immune-escape. RESULTS: MTI-31 potently inhibited cell proliferation (IC50 <1 µmol/L) and in vivo tumor growth in multiple NSCLC models of EGFR/T790M, EML4-ALK, c-Met, or KRAS (MED <10 mg/kg). In EGFR-mutant and/or EML4-ALK-driven NSCLC, MTI-31 or disruption of mTORC2 reduced cell migration, hematogenous metastasis to the lung, and abrogated morphological and functional traits of EMT. Disruption of mTORC2 inhibited EGFR/T790M-positive tumor growth in mouse brain and prolonged animal survival correlating a diminished tumor angiogenesis and recruitment of IBA1+ microglia/macrophages in tumor microenvironment. MTI-31 also suppressed programmed death ligand 1 (PD-L1) in EGFR- and ALK-driven NSCLC, mediated in part by mTORC2/AKT/GSK3ß-dependent proteasomal degradation. Depletion of mTOR protein or disruption of mTOR complexes profoundly downregulated PD-L1 and alleviated apoptosis in Jurkat T and primary human T cells in a tumor-T cell coculture system. CONCLUSIONS: Our results highlight mTOR as a multifaceted regulator of tumor growth, metastasis, and immune-escape in EGFR/ALK-mutant and TKI-resistant NSCLC cells. The newly characterized mechanisms mediated by the rapamycin-resistant mTORC2 warrant clinical investigation of mTORC1/mTORC2 inhibitors in patients with lung cancer.

HSP70 Acetylation Prevents Combined mTORC1/2 Inhibitor and Curcumin Treatment-Induced Apoptosis.

We previously reported that PP242 (dual inhibitor of mTORC1/2) plus curcumin induced apoptotic cell death through lysosomal membrane permeabilization (LMP)-mediated autophagy. However, the relationship between ER stress and apoptotic cell death by combined PP242 and curcumin treatment remains unknown. In the present study, we found that combined PP242 and curcumin treatment induced cytosolic Ca2+ release and ER stress. Interestingly, pretreatment with the chemical chaperones (TUDCA and 4-PBA) and knockdown of CHOP and ATF4 by siRNA did not abolish combined treatment-induced apoptosis in renal carcinoma cells. These results suggest that combined treatment with mTORC1/2 inhibitor and curcumin induces ER stress which is not essential for apoptotic cell death. Furthermore, overexpression of HSP70 significantly inhibited PP242 plus curcumin-induced LMP and apoptosis, but the protective effect was abolished by K77R mutation of acetylation site of HSP70. Taken together, our results reveal that regulation of HSP70 through K77 acetylation plays role in combined PP242 and curcumin treatment-induced apoptosis.

Blackcurrant anthocyanins stimulated cholesterol transport via post-transcriptional induction of LDL receptor in Caco-2 cells.

PURPOSES: We previously showed that polyphenol-rich blackcurrant extract (BCE) showed a hypocholesterolemic effect in mice fed a high fat diet. As direct cholesterol removal from the body via the intestine has been recently appreciated, we investigated the effect of BCE on the modulation of genes involved in intestinal cholesterol transport using Caco-2 cells as an in vitro model. METHODS: Caco-2 cells were treated with BCE to determine its effects on mRNA and protein expression of genes important for intestinal cholesterol transport, low-density lipoprotein (LDL) uptake, cellular cholesterol content, and cholesterol transport from basolateral to apical membrane of Caco-2 cell monolayers. Cells were also treated with anthocyanin-rich or -poor fraction of BCE to determine the role of anthocyanin on BCE effects. RESULTS: BCE significantly increased protein levels of LDL receptor (LDLR) without altering its mRNA, which consequently increased LDL uptake into Caco-2 cells. This post-transcriptional induction of LDLR by BCE was markedly attenuated in the presence of rapamycin, an inhibitor of mechanistic target of rapamycin complex 1 (mTORC1). In addition, BCE altered genes involved in cholesterol transport in the enterocytes, including apical and basolateral cholesterol transporters, in such a way that could enhance cholesterol flux from the basolateral to apical side of the enterocytes. Indeed, BCE significantly increased the flux of LDL-derived cholesterol from the basolateral to the apical chamber of Caco-2 monolayer. LDLR protein levels were markedly increased by anthocyanin-rich fraction, but not by anthocyanin-free fraction. CONCLUSION: mTORC1-dependent post-transcriptional induction of LDLR by BCE anthocyanins drove the transport of LDL-derived cholesterol to the apical side of the enterocytes. This may represent a potential mechanism for the hypocholesterolemic effect of BCE.