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.     

Metformin inhibits melanoma development through autophagy and apoptosis mechanisms

Metformin is the most widely used antidiabetic drug because of its proven efficacy and limited secondary effects. Interestingly, recent studies have reported that metformin can block the growth of different tumor types. Here, we show that metformin exerts antiproliferative effects on melanoma cells, whereas normal human melanocytes are resistant to these metformin-induced effects. To better understand the basis of this antiproliferative effect of metformin in melanoma, we characterized the sequence of events underlying metformin action. We showed that 24 h metformin treatment induced a cell cycle arrest in G0/G1 phases, while after 72 h, melanoma cells underwent autophagy as demonstrated by electron microscopy, immunochemistry, and by quantification of the autolysosome-associated LC3 and Beclin1 proteins. In addition, 96 h post metformin treatment we observed robust apoptosis of melanoma cells. Interestingly, inhibition of autophagy by knocking down LC3 or ATG5 decreased the extent of apoptosis, and suppressed the antiproliferative effect of metformin on melanoma cells, suggesting that apoptosis is a consequence of autophagy. The relevance of these observations were confirmed in vivo, as we showed that metformin treatment impaired the melanoma tumor growth in mice, and induced autophagy and apoptosis markers. Taken together, our data suggest that metformin has an important impact on melanoma growth, and may therefore be beneficial in patients with melanoma.     

Oleanolic Acid Suppresses Migration and Invasion of Malignant Glioma Cells by Inactivating MAPK/ERK Signaling Pathway

Mitogen-activated protein kinases/Extracellular signal-regulated kinase (MAPK/ERK) pathway is essential for migration and invasion of malignant glioma. It is efficient to inhibit migration and invasion of glioma cells by targeting this pathway. Oleanolic acid (OA) has been well demonstrated to suppress survival, growth and angiogenesis of glioma cells. However, it is still unknown if OA affects the migration and invasion of glioma cells. We utilized U-87 MG glioma cell lines and primary glioma cells from patients to study the effect of OA on migration and invasion of glioma cells with multidisciplinary approaches. In this study, we found that OA significantly decreased the ability of glioma cells to migrate and invade. Epithelial-mesenchymal transition (EMT) of glioma cells was also suppressed by OA treatment. Furthermore, MAPK/ERK pathway was greatly inhibited in glioma cells under OA treatment. MAPK/ERK reactivation induced by a recombinant lentiviral vector, Lv-MEK, was able to rescue the inhibitory effect of OA on migration and invasion of glioma cells. Taken together, we provided evidences that OA was a MAPK/ERK pathway-targeting anti-tumor agent. Although the concentrations we used exceeded its physiological level, OA may be used to prevent migration and invasion of glioma cells by developing its derivatives with enhanced bioactivity.     

PLCγ1 inhibition-driven autophagy of IL-1β-treated chondrocyte confers cartilage protection against osteoarthritis,involving AMPK,Erk and Akt

Previous studies identified the involvement of phosphoinositide-specific phospholipase C (PLC) γ1 in some events of chondrocytes. This study aims to investigate whether and how PLCγ1 modulates autophagy to execute its role in osteoarthritis (OA) progression. Rat normal or human OA chondrocytes were pretreated with IL-1β for mimicking or sustaining OA pathological condition. Using Western blotting, immunoprecipitation, qPCR, immunofluorescence and Dimethylmethylene blue assays, and ELISA and transmission electron microscope techniques, we found that PLCγ1 inhibitor U73122 enhanced Collagen II, Aggrecan and GAG levels, accompanied with increased LC3B-II/I ratio and decreased P62 expression level, whereas autophagy inhibitor Chloroquine partially diminished its effect. Meanwhile, U73122 dissociated Beclin1 from Beclin1-IP3R-Bcl-2 complex and blocked mTOR/ULK1 axis, in which the crosstalk between PLCγ1, AMPK, Erk and Akt were involved. Additionally, by haematoxylin and eosin, Safranin O/Fast green, and immunohistochemistry staining, we observed that intra-articular injection of Ad-shPLCγ1-1/2 significantly enhanced Collagen and Aggrecan levels, accompanied with increased LC3B and decreased P62 levels in a rat OA model induced by anterior cruciate ligament transection and medial meniscus resection. Consequently, PLCγ1 inhibition-driven autophagy conferred cartilage protection against OA through promoting ECM synthesis in OA chondrocytes in vivo and in vitro, involving the crosstalk between PLCγ1, AMPK, Erk and Akt.     

Normal tissue depresses while tumor tissue enhances human T cell responses in vivo to a novel self/tumor melanoma antigen,OA1

Antitumor T cells often recognize targets that are nonmutated "self" tissue differentiation Ags, but the relative impact of Ag expression by normal and transformed tissue for a human self/tumor Ag has not been studied. To examine the influence of self-tolerance mechanisms on the function of self/tumor-specific T cell responses in humans, we sought to identify an Ag that was expressed, processed, and presented in an MHC-restricted fashion by tumor cells, but for which there was the human equivalent of a "knockout." In this study, we report the first immunological characterization of a melanoma/melanocyte differentiation Ag, called OA1, which meets these criteria. This Ag, an X chromosome-encoded melanoma/melanocyte differentiation Ag, was completely deleted in a male patient. Using a newly identified HLA-A*2402-restricted epitope (LYSACFWWL) to study T cell tolerance, we found that OA1-specific T cell reactivity was more than five SD higher in the knockout patient that in normal controls. These data provide compelling evidence for T cell tolerance to OA1 in humans. Most surprisingly, we found elevated levels of OA1-specific T cells in patients with metastatic malignant melanoma, indicating that the tumor-bearing state partially reversed tolerance observed in normal (non-"knockout") individuals. Taken together, these findings indicated that tolerance can exist for self/tumor Ags in humans, and that this tolerance could be partially abrogated by the growth of the tumor, increasing the reactivity of tumor Ag-specific T cells. Thus, the tumor-bearing state reverses, in part, the tolerance of T cells that results from the normal expression of tissue differentiation Ags.     

AMP-activated protein kinase (AMPK)/Ulk1-dependent autophagic pathway contributes to C6 ceramide-induced cytotoxic effects in cultured colorectal cancer HT-29 cells

Colorectal cancer is the second leading cause of cancer-related deaths. Drug resistance and/or off-target toxicity against normal cells limit the effectiveness of current chemotherapies for the treatment of colorectal cancer. In the current study, we studied the potential cytotoxic effects of short-chain and cell-permeable C6 ceramide in cultured colorectal cancer HT-29 cells and focused on the underlying mechanisms. We observed that C6 ceramide-induced HT-29 cell death and growth inhibition in a dose- and time-dependent manner. However, no significant apoptosis was observed in C6 ceramide-treated HT-29 cells. Our data support that autophagy contributed to C6 ceramide-induced cytotoxic effects, as autophagy inhibitors, 3-methyladenine (3-MA) and hydroxychloroquine, inhibited C6 ceramide’s effect; however, autophagy activators, everolimus (RAD001) and temsirolimus, mimicked C6 ceramide effects and induced HT-29 cell death. Further, we indentified that AMP-activated protein kinase (AMPK)/Ulk1 signaling was required for autophagy induction by C6 ceramide, and AMPK silencing by a specific short hairpin RNA suppressed C6 ceramide-induced autophagy and cytotoxic effects. Reversely, forced activation of AMPK by its activator AICAR or by genetic manipulation caused autophagic death in HT-29 cells, which was inhibited by 3-MA. Our results suggest that autophagy, but not apoptosis, is a major contributor for C6 ceramide-induced cytotoxic effects in HT-29 cells, and activation of AMPK/Ulk1 is required for the process.     

Fibroblast growth factor 21 (FGF21) alleviates senescence,apoptosis, and extracellular matrix degradation in osteoarthritis via the SIRT1-mTOR signaling pathway

Osteoarthritis (OA) is a complex condition that involves both apoptosis and senescence and currently cannot be cured. Fibroblast growth factor 21 (FGF21), known for its role as a potent regulator of glucose and energy metabolism, protects from various diseases, possibly by mediating autophagy. In the present study, the role of FGF21 in the progression of OA was investigated in both in vitro and in vivo experiments. In vitro, the results revealed that FGF21 administration alleviated apoptosis, senescence, and extracellular matrix (ECM) catabolism of the chondrocytes induced by tert-butyl hydroperoxide (TBHP) by mediating autophagy flux. Furthermore, CQ, an autophagy flux inhibitor, could reverse the protective effect of FGF21. It was observed that the FGF21-induced autophagy flux enhancement was mediated by the nuclear translocation of TFEB, which occurs due to the activation of the SIRT1-mTOR signaling pathway. The in vivo experiments demonstrated that FGF21 treatment could reduce OA in the DMM model. Taken together, these findings suggest that FGF21 protects chondrocytes from apoptosis, senescence, and ECM catabolism via autophagy flux upregulation and also reduces OA development in vivo, demonstrating its potential as a therapeutic agent in OA.Subject terms: Macroautophagy, Senescence     

Suppression of Autophagy Enhanced Growth Inhibition and Apoptosis of Interferon-β in Human Glioma Cells

Interferon-beta (IFN-β) is a cytokine with anti-viral, anti-proliferative, and immunomodulatory effects. In this study, we investigated the effects of IFN-β on the induction of autophagy and the relationships among autophagy, growth inhibition, and apoptosis induced by IFN-β in human glioma cells. We found that IFN-β induced autophagosome formation and conversion of microtubule associated protein 1 light chain 3 (LC3) protein, whereas it inhibited cell growth through caspase-dependent cell apoptosis. The Akt/mTOR signaling pathway was involved in autophagy induced by IFN-β. A dose- and time-dependent increase of p-ERK 1/2 expression was also observed in human glioma cells treated with IFN-β. Autophagy induced by IFN-β was suppressed when p-ERK1/2 was impaired by treatment with U0126. We also demonstrated that suppression of autophagy significantly enhanced growth inhibition and cell apoptosis induced by IFN-β, whereas inhibition of caspase-dependent cell apoptosis impaired autophagy induced by IFN-β. Collectively, these findings indicated that autophagy induced by IFN-β was associated with the Akt/mTOR and ERK 1/2 signaling pathways, and inhibition of autophagy could enhance the growth inhibitory effects of IFN-β and increase apoptosis in human glioma cells. Together, these findings support the possibility that autophagy inhibitors may improve IFN-β therapy for gliomas.     

The MEK/ERK pathway is essential for maintenance of cytoprotective autophagy in <Emphasis Type="Italic">E1A</Emphasis>+<Emphasis Type="Italic">cHA</Emphasis>-<Emphasis Type="Italic">RAS</Emphasis> transformants after exposure to radiation

Autophagy is a conserved process of protein and organelle degradation that serves to maintain cell viability. Autophagy is frequently induced in response to stress or to exposure to DNA-damaging agents or retinoids, as well as to starvation and deficiency of growth factors. In this work, autophagy induced in E1A+cHA-RAS transformed cells in response to X-ray radiation was studied, with a focus on the role of the MEK/ERK signaling pathway in the regulation of radiation-induced autophagy. It was found that inhibition of the MEK/ERK pathway diminished cell viability and altered the sequence of events in radiation-induced autophagy. In particular, it caused aberrations in its final stages, leading to cytoplasmic accumulation of the p62/SQSTM1 adaptor protein in autophagic cavities of unclear origin. Thus, the MEK/ERK pathway activity is essential for the induction and maintenance of autophagy, increasing the viability of exposed cells in response to radiation.     

Growth suppression of human transformed cells by treatment with bark extracts from a medicinal plant, Terminalia arjuna

Summary We have investigated the effects of acetone and methanol extracts of a medicinal plant, Terminalia arjuna, on the growth of human normal fibroblasts (WI-38), osteosarcoma (U2OS), and glioblastoma (U251) cells in vitro. We found that both extracts at 30 μg and 60 μg/ml concentrations inhibit the growth of transformed cells; the growth of normal cells was least affected. Although the transformed cells appeared to have fragmented nucleus by Hoechst staining, no deoxyribonucleic acid laddering effect was observed. In response to the extract treatment, the tumor suppressor protein, p53, was induced in U2OS but not in U251 and WI-38 cells. A cyclin-dependent kinase inhibitor, p21^WAF1, was induced in transformed cells only. The study suggests that the bark extract of medicinal plant, T. arjuna, has components that can induce growth arrest of transformed cells by p53-dependent and-independent pathways.     

Stimulation of α7-nAChRs coordinates autophagy and apoptosis signaling in experimental knee osteoarthritis

Osteoarthritis (OA) is the most common chronic joint disease in the elderly population. Growing evidence indicates that a balance between autophagy and apoptosis in chondrocytes plays a key role in OA’s cartilage degradation. Thus, drugs targeting the balance between apoptosis and autophagy are potential therapeutic approaches for OA treatment. In previous studies, we found that the activation of α7 nicotinic acetylcholine receptors (α7-nAChRs) alleviated monosodium iodoacetate (MIA)-induced joint degradation and osteoarthritis pain. To explore the potential functions of α7-nAChRs in autophagy and apoptosis signaling in knee OA, we compared the expression of α7-nAChRs in human knee articular cartilage tissues from normal humans and OA patients. We found that knee joint cartilage tissues of OA patients showed decreased α7-nAChRs and an imbalance between autophagy and apoptosis. Next, we observed that α7-nAChRs deficiency did not affect cartilage degradation in OA development but reversed the beneficial effects of nicotine on mechanical allodynia, cartilage degradation, and an MIA-induced switch from autophagy to apoptosis. Unlike in vivo studies, we found that primary chondrocytes from α7-nAChRs knockout (KO) mice showed decreased LC3 levels under normal conditions and were more sensitive toward MIA-induced apoptosis. Finally, we found that α7-nAChRs deficiency increased the phosphorylation of mTOR after MIA treatment, which can also be observed in OA patients’ tissues. Thus, our findings not only confirmed that nicotine alleviated MIA-induced pain behavior and cartilage degradation via stimulating the α7-nAChRs/mTOR signal pathway but found the potential role of α7-nAChRs in mediating the balance between apoptosis and autophagy.Subject terms: Autophagy, Apoptosis     

Loss of protein targeting to glycogen sensitizes human hepatocellular carcinoma cells towards glucose deprivation mediated oxidative stress and cell death

Protein targeting to glycogen (PTG) is a ubiquitously expressed scaffolding protein that critically regulates glycogen levels in many tissues, including the liver, muscle and brain. However, its importance in transformed cells has yet to be explored in detail. Since recent studies have demonstrated an important role for glycogen metabolism in cancer cells, we decided to assess the effect of PTG levels on the ability of human hepatocellular carcinoma (HepG2) cells to respond to metabolic stress. Although PTG expression did not significantly affect the proliferation of HepG2 cells under normal culture conditions, we determined that PTG plays an important role during glucose deprivation. Overexpression of PTG protected cells from cell death in the absence of glucose, whereas knocking down PTG further promoted cytotoxicity, as measured by the release of lactate dehydrogenase (LDH) into the media. Additionally, we demonstrated that PTG attenuates glucose deprivation induced haeme oxygenase-1 (HO-1) expression, suggesting that PTG protects against glucose deprivation-induced oxidative stress. Indeed, treating cells with the antioxidant N-acetyl cysteine (NAC) rescued cells from cytotoxicity caused by glucose deprivation. Finally, we showed that loss of PTG resulted in enhanced autophagy. In control cells, glucose deprivation suppressed autophagy as determined by the increase in the levels of p62, an autophagy substrate. However, in knockdown cells, this suppression was relieved. Blockade of autophagy also attenuated cytotoxicity from glucose deprivation in PTG knockdown cells. Taken together, our findings identify a novel role for PTG in protecting hepatocellular carcinoma cells from metabolic stress, in part by regulating oxidative stress and autophagy.     

JUNB‐FBXO21‐ERK axis promotes cartilage degeneration in osteoarthritis by inhibiting autophagy

Osteoarthritis (OA) is a heterogeneous disease that is extremely hard to cure owing to its complex regulation network of pathogenesis, especially cartilage degeneration. FBXO21 is a subunit of ubiquitin E3 ligases that degrades P‐glycoprotein and EID1 by ubiquitination and activates the JNK and p38 pathways; however, its role in OA remains unknown. Here, the main objective of this study was to evaluate the potential effects and mechanism of FBXO21 in OA degeneration, we revealed that FBXO21 is upregulated in the cartilage of patients with OA, aging, and monosodium iodoacetate‐induced OA rats, and chondrocytes treated with interleukin‐1β, tumor necrosis factor‐α, and lipopolysaccharide. Moreover, the in vivo and in vitro knockdown of FBXO21 suppressed OA‐related cartilage degeneration, as evidenced by activated autophagy, upregulated anabolism, alleviated apoptosis, and downregulated catabolism. In contrast, its overexpression promoted OA‐related cartilage degeneration. In addition, using mass spectrometry and co‐immunoprecipitation assay, we demonstrated that the downstream mechanism of FBXO21 inhibits autophagy by interacting with and phosphorylating ERK. Furthermore, FBXO21 alleviated anabolism and enhanced apoptosis and catabolism by inhibiting autophagy in rat chondrocytes. Interestingly, for its upstream mechanism, JUNB promoted FBXO21 expression by directly targeting the FBXO21 promoter, thus further accelerating cartilage degeneration in SW1353 cells and rat chondrocytes. Overall, our findings reveal that the JUNB‐FBXO21‐ERK axis regulates OA apoptosis and cartilage matrix metabolism by inhibiting autophagy. Therefore, FBXO21 is an attractive target for regulating OA pathogenesis, and its knockdown may provide a novel targeted therapy for OA.     

DDIT3/CHOP promotes autophagy in chondrocytes via SIRT1-AKT pathway

Endoplasmic reticulum (ER) stress can initiate autophagy via unfolded protein response (UPR). As a key downstream gene of UPR, DDIT3/CHOP is expressed in chondrocytes. However, the regulation mechanism of DDIT3/CHOP on autophagy in chondrocytes remains unclear. In this study, the expression levels of autophagic markers Beclin1 and LC3B were found to decrease while p62 increase in the tibial growth plate and costal primary chondrocytes from DDIT3/CHOP KO mice. In vitro, overexpressing DDIT3/CHOP induced autophagy in ATDC5 chondrocytes, displaying an elevated immunofluorescence signal of LC3B and elevated numbers of autophagosomes and autolysosomes. Analysis of the gain- and loss-of-function indicated that the protein level of Beclin1 and the ratio of LC3BII/I increased in DDIT3/CHOP overexpression cells, whereas decreased in DDIT3/CHOP knockdown cells. The decreased level of p62 and additional accumulation of LC3BII caused by chloroquine (CQ) further indicated that DDIT3/CHOP enhanced autophagic flux. Mechanistically, we found that DDIT3/CHOP binds directly to the promoter of SIRT1 to promote its expression by CHIP, qRT-PCR, and Western blot analysis. In addition, SIRT1 enhanced autophagic activity in ATDC5 cells, and inhibition or activation of SIRT1 partially reversed the effect of overexpressing or downregulating DDIT3/CHOP on autophagy. Furthermore, AKT signaling was found to be responsible for DDIT3/CHOP-regulated autophagy in ATDC5 cells. SIRT1 knockdown reversed the effect of DDIT3/CHOP overexpression on AKT signaling. In conclusion, our data clarifies that DDIT3/CHOP promotes autophagy in ATDC5 chondrocytes through the SIRT1-AKT pathway. These results were also confirmed in the primary chondrocytes.     

The role of autophagy in cytotoxicity induced by new oncogenic B-Raf inhibitor UI-152 in v-Ha-ras transformed fibroblasts

In human cancers, B-Raf is the most frequently mutated protein kinase in the MAPK signaling cascade, making it an important therapeutic target. We recently discovered a potent and selective B-Raf inhibitor, UI-152, by using a structure-based drug design strategy. In this study, we examined whether B-Raf inhibition by UI-152 may be an effective therapeutic strategy for eliminating cancer cells transformed with v-Ha-ras (Ras-NIH 3T3). UI-152 displayed selective cytotoxicity toward Ras-NIH 3T3 cells while having little to no effect on non-transformed NIH 3T3 cells. We found that treatment with UI-152 markedly increased autophagy and, to a lesser extent, apoptosis. However, inhibition of autophagy by addition of 3-MA failed to reverse the cytotoxic effects of UI-152 on Ras-NIH 3T3 cells, demonstrating that apoptosis and autophagy can act as cooperative partners to induce growth inhibition in Ras-NIH 3T3 cells treated with UI-152. Most interestingly, cell responses to UI-152 appear to be paradoxical. Here, we showed that although UI-152 inhibited ERK, it induced B-Raf binding to Raf-1 as well as Raf-1 activation. This paradoxical activation of Raf-1 by UI-152 is likely to be coupled with the inhibition of the mTOR pathway, an intracellular signaling pathway involved in autophagy. We also showed for the first time that, in multi-drug resistant cells, the combination of UI-152 with verapamil significantly decreased cell proliferation and increased autophagy. Thus, our findings suggest that the inhibition of autophagy, in combination with UI-152, offers a more effective therapeutic strategy for v-Ha-ras-transformed cells harboring wild-type B-Raf.     

Connexin 31.1 degradation requires the Clathrin‐mediated autophagy in NSCLC cell H1299

Connexins have relative short half‐lives. Connexin 31.1 (Cx31.1) was newly reported to be down‐regulated in non‐small cell lung cancer cell lines, and displayed tumour‐suppressive properties. However, no reports describing how a cell regulates Cx31.1 level were found. In this study, Cx31.1 was suggested to be degraded through both ubiquitin–proteasome system (UPS) and autophagy. Blockage of UPS with MG‐132 increased Cx31.1 level, but could not inhibit the degradation of Cx31.1 completely. In H1299 cells stably expressing Cx31.1, Cx31.1 reduced when autophagy was induced through starvation or Brefeldin A treatment. Knockdown of autophagy‐related protein ATG5 could increase the cellular level of Cx31.1 both under normal growth condition and starvation‐induced autophagy. Colocalization of Cx31.1 and autophagy marker light chain 3 (LC3) was revealed by immunofluorescence analysis. Coimmunoprecipitation and immunofluorescence showed that Cx31.1 might interact with clathrin heavy chain which was newly reported to regulate autophagic lysosome reformation (ALR) and controls lysosome homoeostasis. When clathrin expression was knockdown by siRNA treatment, the level of Cx31.1 increased prominently both under normal growth condition and starvation‐induced autophagy. Under starvation‐induced autophagy, LC3‐II levels were slightly accumulated with siCla. treatment compared to that of siNC, which could be ascribed to that clathrin knockdown impaired the late stage of autophagy, ALR. Taken together, we found autophagy contributed to Cx31.1 degradation, and clathrin might be involved in the autophagy of Cx31.1.     

Wogonin induces cross-regulation between autophagy and apoptosis via a variety of Akt pathway in human nasopharyngeal carcinoma cells

Autophagy as well as apoptosis is an emerging target for cancer therapy. Wogonin, a flavonoid compound derived from the traditional Chinese medicine of Huang‐Qin, has anticancer activity in many cancer cells including human nasopharyngeal carcinoma (NPC). However, the involvement of autophagy in the wogonin‐induced apoptosis of NPC cells was still uninvestigated. In this study, we found wogonin‐induced autophagy had interference on the process of apoptosis. Wogonin‐induced autophagy formation evidenced by LC3 I/II cleavage, acridine orange (AO)‐stained vacuoles and the autophagosome/autolysosome images of TEM analysis. Activation of autophagy with rapamycin resulted in increased wogonin‐mediated autophagy via inhibition of mTOR/P70S6K pathway. The functional relevance of autophagy in the antitumor activity was investigated by annexin V‐positive stained cells and PARP cleavage. Induction of autophagy by rapamycin ameliorated the wogonin‐mediated apoptosis, whereas inhibition of autophagy by 3‐methyladenine (3‐MA) or bafilomycin A1 increased the apoptotic effect. Interestingly, this study also found, in addition the mTOR/P70S6K pathway, wogonin also inhibited Raf/ERK pathway, a variety of Akt pathways. Inactivation of PI₃K/Akt by their inhibitors significantly induced apoptosis and markedly sensitized the NPC cells to wogonin‐induced apoptosis. This anticancer effect of Akt was further confirmed by SH6, a specific inhibitor of Akt. Importantly, inactivation of its downstream molecule ERK by PD98059, a MEK inhibitor, also induced apoptosis. This study indicated wogonin‐induced both autophagy and apoptosis through a variety of Akt pathways and suggested modulation of autophagy might provide profoundly the potential therapeutic effect. J. Cell. Biochem. 113: 3476–3485, 2012. © 2012 Wiley Periodicals, Inc.     

Upregulation of ATG3 contributes to autophagy induced by the detachment of intestinal epithelial cells from the extracellular matrix,but promotes autophagy-independent apoptosis of the attached cells

Detachment of nonmalignant intestinal epithelial cells from the extracellular matrix (ECM) triggers their growth arrest and, ultimately, apoptosis. In contrast, colorectal cancer cells can grow without attachment to the ECM. This ability is critical for their malignant potential. We found previously that detachment-induced growth arrest of nonmalignant intestinal epithelial cells is driven by their detachment-triggered autophagy, and that RAS, a major oncogene, promotes growth of detached cells by blocking such autophagy. In an effort to identify the mechanisms of detachment-induced autophagy and growth arrest of nonmalignant cells we found here that detachment of these cells causes upregulation of ATG3 and that ATG3 upregulation contributes to autophagy and growth arrest of detached cells. We also observed that when ATG3 expression is artificially increased in the attached cells, ATG3 promotes neither autophagy nor growth arrest but triggers their apoptosis. ATG3 upregulation likely promotes autophagy of the detached but not that of the attached cells because detachment-dependent autophagy requires other detachment-induced events, such as the upregulation of ATG7. We further observed that those few adherent cells that do not die by apoptosis induced by ATG3 become resistant to apoptosis caused by cell detachment, a property that is critical for the ability of normal epithelial cells to become malignant. We conclude that cell-ECM adhesion can switch ATG3 functions: when upregulated in detached cells in the context of other autophagy-promoting events, ATG3 contributes to autophagy. However, when overexpressed in the adherent cells, in the circumstances not favoring autophagy, ATG3 triggers apoptosis.