Inhibitory Effect of mTOR Activator MHY1485 on Autophagy: Suppression of Lysosomal Fusion

Autophagy is a major degradative process responsible for the disposal of cytoplasmic proteins and dysfunctional organelles via the lysosomal pathway. During the autophagic process, cells form double-membraned vesicles called autophagosomes that sequester disposable materials in the cytoplasm and finally fuse with lysosomes. In the present study, we investigated the inhibition of autophagy by a synthesized compound, MHY1485, in a culture system by using Ac2F rat hepatocytes. Autophagic flux was measured to evaluate the autophagic activity. Autophagosomes were visualized in Ac2F cells transfected with AdGFP-LC3 by live-cell confocal microscopy. In addition, activity of mTOR, a major regulatory protein of autophagy, was assessed by western blot and docking simulation using AutoDock 4.2. In the result, treatment with MHY1485 suppressed the basal autophagic flux, and this inhibitory effect was clearly confirmed in cells under starvation, a strong physiological inducer of autophagy. The levels of p62 and beclin-1 did not show significant change after treatment with MHY1485. Decreased co-localization of autophagosomes and lysosomes in confocal microscopic images revealed the inhibitory effect of MHY1485 on lysosomal fusion during starvation-induced autophagy. These effects of MHY1485 led to the accumulation of LC3II and enlargement of the autophagosomes in a dose- and time- dependent manner. Furthermore, MHY1485 induced mTOR activation and correspondingly showed a higher docking score than PP242, a well-known ATP-competitive mTOR inhibitor, in docking simulation. In conclusion, MHY1485 has an inhibitory effect on the autophagic process by inhibition of fusion between autophagosomes and lysosomes leading to the accumulation of LC3II protein and enlarged autophagosomes. MHY1485 also induces mTOR activity, providing a possibility for another regulatory mechanism of autophagy by the MHY compound. The significance of this study is the finding of a novel inhibitor of autophagy with an mTOR activating effect.     

Quercetin induces protective autophagy in gastric cancer cells: involvement of Akt-mTOR- and hypoxia-induced factor 1α-mediated signaling

Quercetin, a dietary antioxidant present in fruits and vegetables, is a promising cancer chemopreventive agent that inhibits tumor promotion by inducing cell cycle arrest and promoting apoptotic cell death. In this study, we examined the biological activities of quercetin against gastric cancer. Our studies demonstrated that exposure of gastric cancer cells AGS and MKN28 to quercetin resulted in pronounced pro-apoptotic effect through activating the mitochondria pathway. Meanwhile, treatment with quercetin induced appearance of autophagic vacuoles, formation of acidic vesicular organelles (AVOs), conversion of LC3-I to LC3-II, recruitment of LC3-II to the autophagosomes as well as activation of autophagy genes, suggesting that quercetin initiates the autophagic progression in gastric cancer cells. Furthermore, either administration of autophagic inhibitor chloroquine or selective ablation of atg5 or beclin 1 using small interfering RNA (siRNA) could augment quercetin-induced apoptotic cell death, suggesting that autophagy plays a protective role against quercetin-induced apoptosis. Moreover, functional studies revealed that quercetin activated autophagy by modulation of Akt-mTOR signaling and hypoxia-induced factor 1α (HIF-1α) signaling. Finally, a xenograft model provided additional evidence for occurrence of quercetin-induced apoptosis and autophagy in vivo. Together, our studies provided new insights regarding the biological and anti-proliferative activities of quercetin against gastric cancer, and may contribute to rational utility and pharmacological study of quercetin in future anti-cancer research.     

Effects of mTORC1 inhibition on proteasome activity and levels

The mechanistic target of rapamycin (mTOR) regulates numerous extracellular and intracellular signals involved in the maintenan-ce of cellular homeostasis and cell growth. mTOR also functions as an endogenous inhibitor of autophagy. Under nutrient-rich conditions, mTOR complex 1 (mTORC1) phosphorylates the ULK1 complex, preventing its activation and subsequent autophagosome formation, while inhibition of mTORC1 using either rapamycin or nutrient deprivation induces autophagy. Autophagy and proteasomal proteolysis provide amino acids necessary for protein translation. Although the connection between mTORC1 and autophagy is well characterized, the association of mTORC1 inhibition with proteasome biogenesis and activity has not been fully elucidated yet. Proteasomes are long-lived cellular organelles. Their spatiotemporal rather than homeostatic regulation could be another adaptive cellular mechanism to respond to starvation. Here, we reviewed several published reports and the latest research from our group to examine the connection between mTORC1 and proteasome. We have also investigated and described the effect of mTORC1 inhibition on proteasome activity using purified proteasomes. Since mTORC1 inhibitors are currently evaluated as treatments for several human diseases, a better understanding of the link between mTORC1 activity and proteasome function is of utmost importance.     

Quercetin induces protective autophagy in gastric cancer cells: Involvement of Akt-mTOR- and hypoxia-induced factor 1α-mediated signaling

Quercetin, a dietary antioxidant present in fruits and vegetables, is a promising cancer chemopreventive agent that inhibits tumor promotion by inducing cell cycle arrest and promoting apoptotic cell death. In this study, we examined the biological activities of quercetin against gastric cancer. Our studies demonstrated that exposure of gastric cancer cells AGS and MKN28 to quercetin resulted in pronounced pro-apoptotic effect through activating the mitochondria pathway. Meanwhile, treatment with quercetin induced appearance of autophagic vacuoles, formation of acidic vesicular organelles (AVOs), conversion of LC3-I to LC3-II, recruitment of LC3-II to the autophagosomes as well as activation of autophagy genes, suggesting that quercetin initiates the autophagic progression in gastric cancer cells. Furthermore, either administration of autophagic inhibitor chloroquine or selective ablation of atg5 or beclin 1 using small interfering RNA (siRNA) could augment quercetin-induced apoptotic cell death, suggesting that autophagy plays a protective role against quercetin-induced apoptosis. Moreover, functional studies revealed that quercetin activated autophagy by modulation of Akt-mTOR signaling and hypoxia-induced factor 1α (HIF-1α) signaling. Finally, a xenograft model provided additional evidence for occurrence of quercetin-induced apoptosis and autophagy in vivo. Together, our studies provided new insights regarding the biological and anti-proliferative activities of quercetin against gastric cancer, and may contribute to rational utility and pharmacological study of quercetin in future anti-cancer research.     

Blocking autophagy overcomes resistance to dual histone deacetylase and proteasome inhibition in gynecologic cancer

Histone deacetylase (HDAC) inhibitors and proteasome inhibitors have been approved by the FDA for the treatment of multiple myeloma and lymphoma, respectively, but have not achieved similar activity as single agents in solid tumors. Preclinical studies have demonstrated the activity of the combination of an HDAC inhibitor and a proteasome inhibitor in a variety of tumor models. However, the mechanisms underlying sensitivity and resistance to this combination are not well-understood. This study explores the role of autophagy in adaptive resistance to dual HDAC and proteasome inhibition. Studies focus on ovarian and endometrial gynecologic cancers, two diseases with high mortality and a need for novel treatment approaches. We found that nanomolar concentrations of the proteasome inhibitor ixazomib and HDAC inhibitor romidepsin synergistically induce cell death in the majority of gynecologic cancer cells and patient-derived organoid (PDO) models created using endometrial and ovarian patient tumor tissue. However, some models were not sensitive to this combination, and mechanistic studies implicated autophagy as the main mediator of cell survival in the context of dual HDAC and proteasome inhibition. Whereas the combination of ixazomib and romidepsin reduces autophagy in sensitive gynecologic cancer models, autophagy is induced following drug treatment of resistant cells. Pharmacologic or genetic inhibition of autophagy in resistant cells reverses drug resistance as evidenced by an enhanced anti-tumor response both in vitro and in vivo. Taken together, our findings demonstrate a role for autophagic-mediated cell survival in proteasome inhibitor and HDAC inhibitor-resistant gynecologic cancer cells. These data reveal a new approach to overcome drug resistance by inhibiting the autophagy pathway.Subject terms: Gynaecological cancer, Preclinical research     

Simultaneous inhibition of the ubiquitin-proteasome system and autophagy enhances apoptosis induced by ER stress aggravators in human pancreatic cancer cells

In contrast to normal tissue, cancer cells display profound alterations in protein synthesis and degradation. Therefore, proteins that regulate endoplasmic reticulum (ER) homeostasis are being increasingly recognized as potential therapeutic targets. The ubiquitin-proteasome system and autophagy are crucially important for proteostasis in cells. However, interactions between autophagy, the proteasome, and ER stress pathways in cancer remain largely undefined. This study demonstrated that withaferin-A (WA), the biologically active withanolide extracted from Withania somnifera, significantly increased autophagosomes, but blocked the degradation of autophagic cargo by inhibiting SNARE-mediated fusion of autophagosomes and lysosomes in human pancreatic cancer (PC) cells. WA specifically induced proteasome inhibition and promoted the accumulation of ubiquitinated proteins, which resulted in ER stress-mediated apoptosis. Meanwhile, the impaired autophagy at early stage induced by WA was likely activated in response to ER stress. Importantly, combining WA with a series of ER stress aggravators enhanced apoptosis synergistically. WA was well tolerated in mice, and displayed synergism with ER stress aggravators to inhibit tumor growth in PC xenografts. Taken together, these findings indicate that simultaneous suppression of 2 key intracellular protein degradation systems rendered PC cells vulnerable to ER stress, which may represent an avenue for new therapeutic combinations for this disease.     

Microtubules facilitate autophagosome formation and fusion of autophagosomes with endosomes

Nutrient deprivation of eukaryotic cells provokes a variety of stress responses, including autophagy. Autophagy is carried out by autophagosomes which sequester cytosolic components and organelles for degradation after fusion with protease-containing endosomes. To determine the role of microtubules in autophagy, we used nocodazole and vinblastine to disrupt microtubules and independently measured formation and fusion of autophagsosomes in primary rat hepatocytes. By measuring the translocation of GFP-LC3, an autophagosomal marker, to autophagosomes and the lipidation of GFP-LC3, we quantified the rate and magnitude of autophagosome formation. Starvation increased both the rate of autophagosome formation over the basal level and the total number of autophagosomes per cell. Maximal autophagosome formation required an intact microtubule network. Fusion of autophagosomes with endosomes, assayed by acquisition of protease-inhibitor sensitivity as well as overlap with LysoTracker Red-positive endosomes, required intact microtubules. Live-cell imaging demonstrated that autophagosomes were motile structures, and their movement also required microtubules. Interestingly, vinblastine stimulated autophagosome formation more than twofold before any discernable change in the microtubule network was observed. Stimulation of autophagosome formation by vinblastine was independent of nutrients and mTOR activity but was inhibited by depletion of the Autophagy proteins Atg5 and Atg6, known to be required for autophagy.     

Quercetin Inhibits Angiogenesis Mediated Human Prostate Tumor Growth by Targeting VEGFR- 2 Regulated AKT/mTOR/P70S6K Signaling Pathways

Angiogenesis is a crucial step in the growth and metastasis of cancers, since it enables the growing tumor to receive oxygen and nutrients. Cancer prevention using natural products has become an integral part of cancer control. We studied the antiangiogenic activity of quercetin using ex vivo, in vivo and in vitro models. Rat aortic ring assay showed that quercetin at non-toxic concentrations significantly inhibited microvessel sprouting and exhibited a significant inhibition in the proliferation, migration, invasion and tube formation of endothelial cells, which are key events in the process of angiogenesis. Most importantly, quercetin treatment inhibited ex vivo angiogenesis as revealed by chicken egg chorioallantoic membrane assay (CAM) and matrigel plug assay. Western blot analysis showed that quercetin suppressed VEGF induced phosphorylation of VEGF receptor 2 and their downstream protein kinases AKT, mTOR, and ribosomal protein S6 kinase in HUVECs. Quercetin (20 mg/kg/d) significantly reduced the volume and the weight of solid tumors in prostate xenograft mouse model, indicating that quercetin inhibited tumorigenesis by targeting angiogenesis. Furthermore, quercetin reduced the cell viability and induced apoptosis in prostate cancer cells, which were correlated with the downregulation of AKT, mTOR and P70S6K expressions. Collectively the findings in the present study suggest that quercetin inhibits tumor growth and angiogenesis by targeting VEGF-R2 regulated AKT/mTOR/P70S6K signaling pathway, and could be used as a potential drug candidate for cancer therapy.     

Anti-cancer agent siramesine is a lysosomotropic detergent that induces cytoprotective autophagosome accumulation

A sigma-2 receptor ligand siramesine induces lysosomal leakage and cathepsin-dependent death of cancer cells in vitro and displays potent anti-cancer activity in vivo. The mechanism by which siramesine destabilizes lysosomes is, however, unknown. Here, we show that siramesine induces a rapid rise in the lysosomal pH that is followed by lysosomal leakage and dysfunction. The rapid accumulation of siramesine into cancer cell lysosomes, its ability to destabilize isolated lysosomes, and its chemical structure as an amphiphilic amine indicate that it is a lysosomotropic detergent. Notably, siramesine triggers also a substantial Atg6- and Atg7-dependent accumulation of autophagosomes that is associated with a rapid and sustained inhibition of mammalian target of rapamycin complex 1 (mTORC1; an inhibitor of autophagy). Siramesine fails, however, to increase the degradation rate of long-lived proteins. Thus, the massive accumulation of autophagosomes is likely to be due to a combined effect of activation of autophagy signaling and decreased autophagosome turnover. Importantly, pharmacological and RNA interference-based inhibition of autophagosome formation further sensitizes cancer cells to siramesine-induced cytotoxicity. These data identify siramesine as a lysosomotropic detergent that triggers cell death via a direct destabilization of lysosomes and cytoprotection by inducing the accumulation of autophagosomes. Threrefore, the combination of siramesine with inhibitors of autophagosome formation appears as a promising approach for future cancer therapy.     

Anti-cancer agent siramesine is a lysosomotropic detergent that induces cytoprotective autophagosome accumulation

A σ-2 receptor ligand siramesine induces lysosomal leakage and cathepsin-dependent death of cancer cells in vitro and displays potent anti-cancer activity in vivo. The mechanism by which siramesine destabilizes lysosomes is, however, unknown. Here, we show that siramesine induces a rapid rise in the lysosomal pH that is followed by lysosomal leakage and dysfunction. The rapid accumulation of siramesine into cancer cell lysosomes, its ability to destabilize isolated lysosomes, and its chemical structure as an amphiphilic amine indicate that it is a lysosomotropic detergent. Notably, siramesine triggers also a substantial Atg6- and Atg7-dependent accumulation of autophagosomes that is associated with a rapid and sustained inhibition of mammalian target of rapamycin complex 1 (mTORC1; an inhibitor of autophagy). Siramesine fails, however, to increase the degradation rate of long-lived proteins. Thus, the massive accumulation of autophagosomes is likely to be due to a combined effect of activation of autophagy signaling and decreased autophagosome turnover. Importantly, pharmacological and RNA interference-based inhibition of autophagosome formation further sensitizes cancer cells to siramesine-induced cytotoxicity. These data identify siramesine as a lysosomotropic detergent that triggers cell death via a direct destabilization of lysosomes and cytoprotection by inducing the accumulation of autophagosomes. Threrefore, the combination of siramesine with inhibitors of autophagosome formation appears as a promising approach for future cancer therapy.     

Inositol hexakisphosphate kinases promote autophagy

We and other authors have previously reported that increasing cellular diphosphoinositol pentakisphosphate (InsP(7)) levels increases cell sensitivity to cell death. In the present study, we elucidated the relationship between inositol hexakisphosphate kinases (InsP(6)Ks), which form InsP(7), and autophagy using InsP(6)Ks overexpression and disruption systems. A large number of autophagosomes were induced in cells transfected with InsP(6)Ks, as revealed by the conversion of LC3-I to LC3-II, which was examined using immunoblotting, immunocytochemistry, and immuno-electron microscopy for LC3; consequently, the rate of cell death was higher among these cells than among cells transfected with a control vector, as shown using propidium iodide staining. However, the reduction of InsP(6)Ks levels using RNAi suppressed the formation of autophagosomes. Moreover, the number of autophagosomes and the rate of cell death were significantly higher among cells transfected with InsP(6)Ks subjected to staurosporine-induced stress than among cells transfected with InsP(6)Ks subjected to normal conditions. The cell death induced by InsP(6)Ks was not completely suppressed by z-VAD-fmk, a pan-caspase inhibitor. The phosphorylation of mammalian target of rapamycin (mTOR) was also depressed in cells overexpressing InsP(6)Ks, suggesting that the mTOR pathway regulates autophagosomes generated by InsP(6)Ks. These findings imply that InsP(6)Ks promote autophagy and induce caspase-independent cell death. This phenomenon opens a new pathway of autophagy via InsP(6)Ks.     

Concomitant reduction of c-Myc expression and PI3K/AKT/mTOR signaling by quercetin induces a strong cytotoxic effect against Burkitt’s lymphoma

Burkitt’s lymphoma is an aggressive B cell lymphoma whose pathogenesis involves mainly c-Myc translocation and hyperexpression, in addition to antigen-independent BCR signaling and, in some cases, EBV infection. As result of BCR signaling activation, the PI3K/AKT/mTOR pathway results constitutively activated also in the absence of EBV, promoting cell survival and counterbalancing the pro-apoptotic function that c-Myc may also exert. In this study we found that quercetin, a bioflavonoid widely distributed in plant kingdom, reduced c-Myc expression and inhibited the PI3K/AKT/mTOR activity in BL, leading to an apoptotic cell death. We observed a higher cytotoxic effect against the EBV-negative BL cells in comparison with the positive ones, suggesting that this oncogenic gammaherpesvirus confers an additional resistance to the quercetin treatment. Besides cell survival, PI3K/AKT/mTOR pathway also regulates autophagy: we found that quercetin induced a complete autophagic flux in BL cells, that contributes to c-Myc reduction in some of these cells. Indeed, autophagy inhibition by chloroquine partially restored c-Myc expression in EBV-positive (Akata) and EBV-negative (2A8) cells that harbor c-Myc mutation. Interestingly, chloroquine did not affect the quercetin-mediated reduction of c-Myc expression in Ramos cells, that have no c-Myc mutation in the coding region, although autophagy was induced.These results suggest that mutant c-Myc could be partially degraded through autophagy in BL cells, as previously reported for other mutant oncogenic proteins.     

Identification of ML-9 as a lysosomotropic agent targeting autophagy and cell death

The growing number of studies suggested that inhibition of autophagy enhances the efficacy of Akt kinase inhibitors in cancer therapy. Here, we provide evidence that ML-9, a widely used inhibitor of Akt kinase, myosin light-chain kinase (MLCK) and stromal interaction molecule 1 (STIM1), represents the ‘two-in-one'' compound that stimulates autophagosome formation (by downregulating Akt/mammalian target of rapamycin (mTOR) pathway) and inhibits their degradation (by acting like a lysosomotropic agent and increasing lysosomal pH). We show that ML-9 as a monotherapy effectively induces prostate cancer cell death associated with the accumulation of autophagic vacuoles. Further, ML-9 enhances the anticancer activity of docetaxel, suggesting its potential application as an adjuvant to existing anticancer chemotherapy. Altogether, our results revealed the complex effect of ML-9 on autophagy and indentified ML-9 as an attractive tool for targeting autophagy in cancer therapy through dual inhibition of both the Akt pathway and the autophagy.     

Enhancing perifosine's anticancer efficacy by preventing autophagy

Our long-term research goal is to develop efficacious regimens for cancer therapy through our understanding of cancer biology and drug mechanisms. Perifosine is an alkylphospholipid exhibiting antitumor activity and is currently being tested in clinical trials. Its activity is partly associated with its ability to inhibit Akt activity. In an effort to understand the mechanism by which perifosine exerts its anticancer activity, our recent work shows that perifosine, in addition to inhibition of Akt, inhibits mTOR signaling through a different mechanism than classical mTOR inhibitors such as rapamycin via facilitating the degradation of major components in the mTOR axis including mTOR, raptor and rictor. Accordingly, perifosine substantially induces autophagy in addition to apoptosis. The combination of perifosine with a lysosomal inhibitor enhances apoptosis and inhibition of the growth of xenografts in nude mice, suggesting that perifosine-induced autophagy protects cells from undergoing apoptosis. Thus, our findings highlight a novel mechanism accounting for perifosine's anticancer activity involving degradation-mediated mTOR inhibition and also suggest a potential strategy to enhance perifosine's anticancer efficacy by preventing autophagy.     

Autophagy Suppresses RIP Kinase-Dependent Necrosis Enabling Survival to mTOR Inhibition

mTOR inhibitors are used clinically to treat renal cancer but are not curative. Here we show that autophagy is a resistance mechanism of human renal cell carcinoma (RCC) cell lines to mTOR inhibitors. RCC cell lines have high basal autophagy that is required for survival to mTOR inhibition. In RCC4 cells, inhibition of mTOR with CCI-779 stimulates autophagy and eliminates RIP kinases (RIPKs) and this is blocked by autophagy inhibition, which induces RIPK- and ROS-dependent necroptosis in vitro and suppresses xenograft growth. Autophagy of mitochondria is required for cell survival since mTOR inhibition turns off Nrf2 antioxidant defense. Thus, coordinate mTOR and autophagy inhibition leads to an imbalance between ROS production and defense, causing necroptosis that may enhance cancer treatment efficacy.     

Trehalose Inhibits Protein Aggregation Caused by Transient Ischemic Insults Through Preservation of Proteasome Activity,Not via Induction of Autophagy

Protein aggregation has been proved to be a pathological basis accounting for neuronal death caused by either transient global ischemia or oxygen glucose deprivation (OGD), and inhibition of protein aggregation is emerging as a potential strategy of preventing brain damage. Trehalose was found to inhibit protein aggregation caused by neurodegenerative diseases via induction of autophagy, whereas its effect is still elusive on ischemia-induced protein aggregation. In this study, we investigated this issue by using rat model of transient global ischemia and SH-SY5Y model of OGD. We found that pretreatment with trehalose inhibited transient global ischemia-induced neuronal death in the hippocampus CA1 neurons and OGD-induced death in SH-SY5Y cells, which was associated with inhibition of the formation of ubiquitin-labeled protein aggregates and preservation of proteasome activity. In vitro study showed that the protection of trehalose against OGD-induced cell death and protein aggregation in SH-SY5Y cells was reversed when proteasome activity was inhibited by MG-132. Further studies revealed that trehalose prevented OGD-induced reduction of proteasome activity via suppression of both oxidative stress and endoplasmic reticulum stress. Particularly, our results showed that trehalose inhibited OGD-induced autophagy. Therefore, we demonstrated that proteasome dysfunction contributed to protein aggregation caused by ischemic insults and trehalose prevented protein aggregation via preservation of proteasome activity, not via induction of autophagy.     

Augmentation of autophagy by mTOR-inhibition in myocardial infarction: When size matters

The extent of adverse myocardial remodeling contributes essentially to the prognosis after myocardial infarction (MI). Currently, therapeutic strategies that inhibit remodeling are limited to inhibition of neurohumoral activation. mTOR-dependent signaling mechanisms are centrally involved in the myocardial remodeling process. There exists a controversy as to whether autophagy is beneficial in the setting of myocardial infarction. We now provide evidence that induction of autophagy by inhibition of mTOR with everolimus (RAD) prevents adverse left ventricular remodeling and limits infarct size following myocardial infarction. mTOR inhibition increases autophagy and concomitantly decreases proteasome activity especially in the border zone of the infarcted myocardium. The induction of autophagy via mTOR inhibition is a novel potential therapeutic approach to limit infarct size and to attenuate adverse left ventricular remodeling following MI.     

Tumor cells can evade dependence on autophagy through adaptation

The autophagy-lysosome and the proteasome constitute the two major intracellular degradation systems. Suppression of the proteasome promotes autophagy for compensation and simultaneous inhibition of autophagy can selectively increase apoptosis in transformed cells, but not in untransformed or normal cells. Transformed cells are thus more dependent on autophagy for survival. However, it is unclear whether long-term autophagy inhibition/insufficiency would affect such dependency. To address this question, we transformed wild-type and autophagy-deficient cells lacking a key autophagy-related gene Atg5 with activated Ras. We found that such transformation did not make the autophagy-deficient tumor cells more susceptible to proteasome inhibitors than the wild type tumor cells, although the transformed cells were in general more sensitive to proteasome inhibition. We then compared the effect of acute versus constitutive knock-down of a key autophagy initiating molecule, Beclin 1, in an already transformed cancer cell line. In a wild-type U251 glioblastoma cell line (autophagy intact), increased sensitivity to proteasome inhibition was induced immediately after the knock-down of Beclin 1 expression with a specific siRNA (acute autophagy deficiency). On the other hand, when the tumor cell line was selected over a long period to achieve constitutive knock-down of Beclin 1, its sensitivity to proteasome inhibitors was no higher than that of the wild-type tumor cells. These results suggest that long-term autophagy deficiency either before or after oncogenic transformation can render the tumor cell survival independent of the autophagic activity, and the response to chemotherapy is no longer affected by the manipulation of the autophagy status.     

Berberine induces autophagic cell death and mitochondrial apoptosis in liver cancer cells: The cellular mechanism

Extensive studies have revealed that berberine, a small molecule derived from Coptidis rhizoma (Huanglian in Chinese) and many other plants, has strong anti‐tumor properties. To better understand berberine‐induced cell death and its underlying mechanisms in cancer, we examined autophagy and apoptosis in the human hepatic carcinoma cell lines HepG2 and MHCC97‐L. The results of this study indicate that berberine can induce both autophagy and apoptosis in hepatocellular carcinoma cells. Berberine‐induced cell death in human hepatic carcinoma cells was diminished in the presence of the cell death inhibitor 3‐methyladenine, or following interference with the essential autophagy gene Atg5. Mechanistic studies showed that berberine may activate mitochondrial apoptosis in HepG2 and MHCC97‐L cells by increasing Bax expression, the formation of permeable transition pores, cytochrome C release to cytosol, and subsequent activation of the caspases 3 and 9 execution pathway. Berberine may also induce autophagic cell death in HepG2 and MHCC97‐L cells through activation of Beclin‐1 and inhibition of the mTOR‐signaling pathway by suppressing the activity of Akt and up‐regulating P38 MAPK signaling. This is the first study to describe the role of Beclin‐1 activation and mTOR inhibition in berberine‐induced autophagic cell death. These results further demonstrate the potential of berberine as a therapeutic agent in the emerging list of cancer therapies with novel mechanisms. J. Cell. Biochem. 111: 1426–1436, 2010. © 2010 Wiley‐Liss, Inc.