Formation Of Acidic Vesicular Organelles Research Articles

Induction of Autophagy and Inhibition of Oncoprotein Aurora‐A Activity in Curcumin‐Enhanced Chemosensitivity of Grade III Human Bladder Cancer T24 Cells to FDA‐Approved Anticancer Drugs

Curcumin is an active anticancer compound in turmeric and curry. Autophagy (programmed cell death type II) is involved in the degradation and recycling of proteins and intracellular components in response to starvation and stress. Aurora-A is a mitosis-related serine-threonine kinase. Overexpression of Aurora-A increases drug resistance and promotes metastasis of cancer cells. Our previous report showed that curcumin significantly inhibited Aurora-A gene expression and subsequently kinase activity, which caused failure of various mitotic events and G2/M mitotic arrest in Aurora-A-overexpressing human bladder cancer T24 cells. Here we reveal that curcumin enhanced the chemosensitivity of T24 cells to cisplatin plus gemcitabine, the FDA-approved standard first chemotherapy for patients with locally advanced and metastatic bladder cancer. A decrease in cell viability and the occurrence of a synergy effect were observed. Addition of curcumin promoted apoptosis and G2/M arrest, and decreased Aurora-A activity. Curcumin-enhanced chemosensitivity was paralleled by significant increases of autophagy, which were characterized by the expression of autophagy marker LC3-II using immunoblotting and the formation of acidic vesicular organelles using flow cytometry. Curcumin-induced autophagy was observed via activation of Raf/MEK/ERK pathway, but not via increase in Atg12-Atg5 formation or inhibition of AKT/mTOR pathway. Our data suggest a combination of curcumin with the FDA-approved anticancer drugs for patients with bladder cancer.

Sensitivity of apoptosis-resistant colon cancer cells to tanshinones is mediated by autophagic cell death and p53-independent cytotoxicity

BackgroundMultidrug resistance (MDR) develops in nearly all patients with colon cancer. The reversal of MDR plays an important role in the success of colon cancer chemotherapy. One of the commonest mechanisms conferring MDR is the suppression of apoptosis in cancer cells. PurposeThis study investigated the sensitivity of cryptotanshinone (CTS) and dihydrotanshinone (DTS), two lipophilic tanshinones from a traditional Chinese medicine Salvia miltiorrhiza, in apoptosis-resistant colon cancer cells. MethodsCell viability was measured by MTT assay. Cell cycle distribution and apoptosis were determined by flow cytometry. Protein levels were analyzed by western blot analysis. The formation of acidic vesicular organelles was visualized by acridine orange staining. ResultsExperimental results showed that multidrug-resistant colon cancer cells SW620 Ad300 were sensitive to both CTS and DTS in terms of cell death, but with less induction of apoptosis when compared with the parental cells SW620, suggesting that other types of cell death such as autophagy could occur. Indeed, the two tanshinones induced more LC3B-II accumulation in SW620 Ad300 cells with increased autophagic flux. More importantly, cell viability was increased after autophagy inhibition, indicating that autophagy induced by the two tanshinones was pro-cell death. Besides, the cytotoxic actions of the two tanshinones were p53-independent, which could be useful in inhibiting the growth of apoptosis-resistant cancer cells with p53 defects. ConclusionThe current findings strongly indicate that both CTS and DTS could inhibit the growth of apoptosis-resistant colon cancer cells through induction of autophagic cell death and p53-independent cytotoxicity. They are promising candidates to be further developed as therapeutic agents in the adjuvant therapy for colon cancer, especially for the apoptosis-resistant cancer types.

Adenovirus expressing dual c-Met-specific shRNA exhibits potent antitumor effect through autophagic cell death accompanied by senescence-like phenotypes in glioblastoma cells.

c-Met, a cognate receptor tyrosine kinase of hepatocyte growth factor, is overexpressed and/or mutated in number of tumors. Therefore, abrogation of c-Met signaling may serve as potential therapeutic targets. In this study, we generated Ads expressing single shRNA specific to c-Met (shMet) (dl/shMet4 and dl/shMet5) or dual shRNAs specific to c-Met (dl/shMet4+5); and examined the therapeutic potential of these newly engineered Ads in targeting c-Met, and delineated their mechanism of action in vitro and in vivo. Ads expressing shMet induced knock-down in c-Met, and phenotypically resulted in autophagy-like features including appearance of membranousvacuoles, formation of acidic vesicular organelles, and cleavage and recruitment of microtubule-associated protein1 light chain 3 to autophagosomes. Ads expressing shMet also suppressed Akt phosphorylation and increased number of senescence-related gene products including SM22, TGase II, and PAI-1. These changes resulted in inhibition of cell proliferation and G2/M arrest of U343 cells. In vivo, intratumoral injection with dl/shMet4+5 resulted in a significant reduction of tumor growth with corresponding increasing overall survival. Histopathological analysis of these treated tumors revealed that Atg5 was highly up-regulated, indicating the therapeutic induction of autophagy. In sum, these results reveal that autophagic cell death induced by shMet-expressing Ads provide a novel strategy for targeting c-Met-expressing tumors through non-apoptotic mechanism of cell death.

In Vitro and in Vivo Atheroprotective Effects of Gossypetin against Endothelial Cell Injury by Induction of Autophagy.

Oxidized low-density lipoprotein (ox-LDL) contributes to the pathogenesis of atherosclerosis by promoting vascular endothelial cell injury.Gossypetin, a naturally occurring hexahydroxyflavone, has been shown to possess antimutagenic, antioxidant, antimicrobial, and antiatherosclerotic effects. In this study, the atheroprotective role of gossypetin was examined in endothelial cells. The protective effect of gossypetin against ox-LDL-induced injury in human umbilicalvein endothelial cells (HUVECs) was first noted at 0.1−0.5 μM. Gossypetin showed potential in reducing ox-LDL-dependent apoptosis, as demonstrated by morphological and biochemical features, including formation of apoptotic bodies,distribution of hypodiploid phase, and activation of caspase-3. Next, the ox-LDL induced formation of acidic vesicular organelles and the upregulation of autophagyrelated genes (LC3 and Beclin-1) were enhanced by gossypetin. Gossypetin triggered autophagic flux was further confirmed by an increase in the level of LC3-II under pretreatment conditions with an autophagy inhibitor, chloroquine (CQ). In addition, silencing Beclin-1 inhibited both the gossypetin-mediated protective affects and the autophagic process. Molecular data indicated that the autophagic effect of gossypetin might be mediated via the class III PI3K/Beclin-1 and PTEN/class I PI3K/Akt signaling cascades, as demonstrated by the use of a class III PI3K inhibitor, 3-methyladenine (3-MA), and a PTEN inhibitor, SF1670. Finally, gossypetin improved atherosclerotic lesions and endothelial injury in vivo. These data imply that gossypetin upregulates the autophagic pathway, which led to subsequent reduction of ox-LDL-induced atherogenic endothelial cell injury and apoptosis, and provide a new mechanism for the antiatherosclerotic activity of gossypetin.

Phellinus Linteus Extract Induces Autophagy and Synergizes With 5-Fluorouracil to Inhibit Breast Cancer Cell Growth

Phellinus linteus (PL) is a medicinal mushroom due to its several biological properties, including anticancer activity. However, the mechanisms of its anticancer effect remain to be elucidated. We evaluated the inhibitory effects of the ethanolic extract from the PL combined with 5-FU on MDA-MB-231 breast cancer cell line and to determine the mechanism of cell death. Individually, PL extract and 5-FU significantly inhibited the proliferation of MDA-MB-231 cells in a dose-dependent manner. PL extract (30 mg/mL) in combination with 5-FU (10 μg/mL) synergistically inhibited MDA-MB-231 cells by 1.8-fold. PL did not induce apoptosis, as demonstrated by the DNA fragmentation assay, the sub-G1 population, and staining with annexin V-FITC and propidium iodide. The exposure of MDA-MB-231 cells to PL extracts resulted in several confirmed characteristics of autophagy, including the appearance of autophagic vacuoles revealed by monodansylcadaverine staining, the formation of acidic vesicular organelles, autophagosome membrane association of microtubule-associated protein light chain 3 (LC3) characterized by cleavage of LC3 and its punctuate redistribution, and ultrastructural observation of autophagic vacuoles by transmission electron microscopy. We concluded that PL extracts synergized with low doses of 5-FU to inhibit triple-negative breast cancer cell growth and demonstrated that PL extract can induce autophagy-related cell death.

Sonodynamic therapy induces the interplay between apoptosis and autophagy in K562 cells through ROS

Sonodynamic therapy (SDT) is a relatively new approach in the treatment of various cancers including leukemia cells. The aim of this study is to investigate the occurrence of apoptosis and autophagy after treated by protoporphyrin IX (PpIX)-mediated SDT (PpIX-SDT) on human leukemia K562 cells as well as the relationship between them. Firstly, mitochondrial-dependent apoptosis was observed through morphological observation and biochemical analysis. Meanwhile, SDT was shown to induce autophagy in K562 cells, which caused an increase in EGFP-LC3 puncta cells, a conversion of LC3 II/I, formation of acidic vesicular organelles (AVOs) and co-localization between LC3 and LAMP2 (a lysosome marker). Besides, pretreatment with autophagy inhibitor 3-MA or bafilomycin A1 was shown to provide protection against autophagy and to enhance SDT-induced apoptosis and necrosis, while the apoptosis suppressor z-VAD-fmk failed to affect formation of autophagic vacuoles or partially prevented SDT-induced cytotoxicity, which suggested that SDT-induced autophagy functioned as a survival mechanism. Additionally, this study reported apparent apoptosis and autophagy with dependence on intracellular reactive oxygen species (ROS) production. Preliminary data showed that ROS scavenger N-acetylcysteine (NAC) effectively blocked the SDT induced accumulation of ROS, reversed sono-damage, cell apoptosis and autophagy. Taken together, these data indicate that autophagy may be cytoprotective in our experimental system, and the ROS caused by PpIX-SDT treatment may play an important role in initiating apoptosis and autophagy.

Autophagy in resin monomer-initiated toxicity of dental mesenchymal cells: a novel therapeutic target of N-acetyl cysteine.

Dental restorative biomaterials are commonly used to restore the teeth impaired by caries, erosion, or fracture. Resin monomers released from polymerized restorative composite materials could disturb cell viability and cause toxicity of oral eukaryotic cells, which remains a medical challenge. However, the intracellular processes or underlying mechanisms of resin monomer-mediated toxicity and the potential preventive/therapeutic strategy are still far from clear. The present study aimed to determine the role of autophagy in resin monomer triethylene glycol dimethacrylate (TEGDMA)-induced cytotoxicity and explore autophagy as a potential therapeutic target of anti-oxidant N-acetyl cysteine (NAC) in vitro and ex vivo. The results showed that TEGDMA exposure resulted in several specific features of autophagy in human dental mesenchymal cells (DMCs), including the formation of acidic vesicular organelles, appearance of autophagic vacuoles, and LC3-II accumulation. By pharmacological and genetic approaches, the inhibition of autophagy significantly prevented TEGDMA-induced apoptosis in DMCs. Moreover, the autophagy activated by TEGDMA occurred via the AMPK/mTOR pathway, which could be abrogated by NAC pretreatment. More importantly, the tooth slice organ culture model provided further evidence of autophagy involvement in TEGDMA-triggered dental mesenchymal tissue toxicity and as a therapeutic target of NAC ex vivo. Our findings provide novel insights into the mechanisms of resin monomer-mediated toxicity and highlight autophagy as a promising therapeutic target of NAC for improving dental restorative biomaterials that enable dental tissue protection.

Potent anti-cancer effect of 3'-hydroxypterostilbene in human colon xenograft tumors.

Here we report that 3′-hydroxypterostilbene (HPSB), a natural pterostilbene analogue, was more potent than pterostilbene against the growth of human cancer cells (COLO 205, HCT-116, and HT-29) with measured IC50 values of 9.0, 40.2, and 70.9 µM, respectively. We found that HPSB effectively inhibited the growth of human colon cancer cells by inducing apoptosis and autophagy. Autophagy occurred at an early stage and was observed through the formation of acidic vesicular organelles and microtubule-associated protein 1 light chain 3-II production. At the molecular levels, the results from western blot analysis showed that HPSB significantly down-regulated phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinases (MAPKs) signalings including decreased the phosphorylation of mammalian target of rapamycin (mTOR). Significant therapeutic effects were demonstrated in vivo by treating nude mice bearing COLO 205 tumor xenografts with HPSB (10 mg/kg i.p.). These inhibitory effects were accompanied by mechanistic down-regulation of the protein levels of cyclooxygenase-2 (COX-2), matrix metallopeptidase-9 (MMP-9), vascular endothelial growth factor (VEGF), and cyclin D1, as well as by the induction of apoptosis in colon tumors. Our findings suggest that HPSB could serve as a novel promising agent for colon cancer treatment.

HIV-1 and Morphine Regulation of Autophagy in Microglia: Limited Interactions in the Context of HIV-1 Infection and Opioid Abuse

Microglia are the predominant resident central nervous system (CNS) cell type productively infected by HIV-1, and play a key role in the progression of HIV-associated dementia (HAD). Moreover, neural dysfunction and progression to HAD are accelerated in opiate drug abusers. In the present study, we examined the role of the autophagy pathway in the neuropathogenesis of HIV-1 using primary human microglial cells and determined whether opiates converge at this point. Infection of microglia with the HIV-1SF162 macrophage-tropic strain resulted in increased Beclin1 expression, accompanied by an increase of LC3 protein levels and accumulation of LC3 reporter RFP+ GFP+ (yellow) puncta, suggesting that HIV-1 infection triggers autophagosome formation without promoting protein degradation by the lysosome. Conversely, coexposure with HIV-1 and morphine significantly decreased virus-induced Beclin1 expression and autophagosome formation. Exploration of the possible mechanism(s) used by morphine to disrupt the autophagic process unveiled a significant increase in intracellular pH, which coincided with a reduction in the formation of acidic vesicular organelles and in autophagolysosome formation. Small interfering RNA targeting BECN1, a gene critical for autophagosome formation, significantly reduced viral replication and the virus-induced inflammatory responses. Conversely, morphine-enhanced viral replication and inflammatory responses were not affected by gene silencing with siBeclin1, suggesting that the interactive effect of morphine in HIV-1 pathogenesis is mediated through a Beclin1-independent mechanism. These novel findings may have important implications on the connections between autophagy and HIV-1 pathogenesis mediated by microglial cells in opioid-abusing individuals. About 50% of individuals infected with HIV-1 will develop some sort of neurocognitive impairment that cannot be prevented nor eradicated by antiretroviral therapy. The neuropathogenesis is mostly due to inflammatory responses by infected microglia, the resident immune cells of the brain. Cognitive disorders may also be associated with drugs of abuse. In fact, opioid drug users have an increased risk of developing neurocognitive disorders with increased progression to dementia. Although the mechanism(s) by which opioids exacerbate the neuropathogenesis of HIV-1 are not entirely known, it is well accepted that glia are critical to opiate responses. This study gives us new insight into possible autophagic mechanism(s) in microglia that control HIV-1 replication and virus-induced inflammation in the context of opioid abuse and should greatly improve our knowledge in the pathogenesis of HIV-1 resulting from substance abuse to provide a better understanding for the design of candidate antiviral therapies targeting drug-abusing individuals.

153P - Autophagy Induced By Tyrosine Kinase Inhibitors in Non-Small Cell Lung Carcinoma and Tumor Microenvironment

ABSTRACT Aim: This study aimed to investigate whether autophagy could confer acquired resistance to TKI in NSCLC, and the role of TME in autophagy induction and TKI sensitivity. Methods: Two NSCLC cell lines (HCC827 and HCC4006) with EGFR mutations (exon 19 deletions) were selected. MTT and annexin-V binding assays were performed to determine cell proliferation and apoptotic cell death upon TKI treatment respectively. Autophagy was determined by conversion of LC3I to LC3II, formation of ATG5/12 conjugation and p62 degradation using Western blot. Acidic vesicular organelle (AVO) formation was shown by acridine orange staining. Autophagy inhibitor (chloroquine) was used to study the functional significance of TKI-induced autophagy. TME was established by co-culturing human lung fibroblast MRC-5 with HCC827 cells directly. The cells were separated by cell sorter after staining surface marker for HCC827 cells. Then, ELISA and real time PCR were performed to detect the production of cytokines, mRNA and autophagic molecules respectively. Results: MTT assay after 72 hours of treatment confirmed that both cell lines were sensitive to erlotinib (IC50 Conclusions: EGFR-TKI induced both apoptosis and autophagy in EGFR-mutated NSCLC cell lines. Inhibition of autophagy with chloroquine enhanced TKI-induced cell death. In the presence of TME, autophagy was induced in both cancer and stromal components, partially mediated via inflammatory factors. Autophagy inhibition under TME could also enhance apoptotic effect of TKI. Disclosure: All authors have declared no conflicts of interest.

The effects of alpha lipoic acid in preventing oxidative stress-induced retinal pigment epithelial cell injury.

This study evaluated the protective effect of alpha lipoic acid (ALA) in human adult retinal pigment epithelium cells (ARPE-19). An RPE oxidative stress model was established in ARPE-19 cells. Cell apoptosis was detected by Annexin V/PI staining and Hoechst33342 staining. Autophagy activation was evaluated by formation of acidic vesicular organelles (AVO) as well as protein expression of Atg5 and LC3. Akt phosphorylation and Bax protein expression were measured using western blot. Exogenous H2O2 significantly increased intracellular ROS concentration and decreased the viability of ARPE-19 cells in a dose-dependent way. H2O2 (12.5 μmol/L) induced early stage apoptosis, significantly decreased Akt phosphorylation, and increased Bax protein level 4 h after stimulation. H2O2 (12.5 μmol/L) significantly increased AVO formation, mRNA and protein levels of Atg-5, and protein expression of LC3 I and II. Treatment of ARPE-19 cells with 37.5 μmol/L ALA significantly blocked increased intracellular ROS level, apoptosis, AVO formation, as well as elevation of Atg5, LC3 I, and II protein levels induced by 12.5 μmol/L H2O2. Exogenous H2O2 induces apoptosis and activation of autophagy in human adult retinal pigment epithelium cells through Akt-Bax signaling. ALA is effective in protecting RPE cells from H2O2-induced cell death.

Apogossypolone inhibits the proliferation of LNCaP cells in vitro and in vivo.

The aim of the present study was to investigate the anti-tumor effect of apogossypolone (ApoG2) on human LNCaP cells in vitro and in vivo. Cell viability was evaluated using an MTT assay. Cell autophagy and apoptosis were detected by flow cytometry and using a terminal deoxynucleotidyl transferase dUTP nick end labeling assay, respectively. Morphological autophagy alterations were observed by transmission electron microscopy. The formation of acidic vesicular organelles was assessed by acridine orange staining and fluorescence microscopy. Quantitative polymerase chain reaction (qPCR) was conducted to detect the expression levels of apoptosis-associated protein B-cell lymphoma 2 (Bcl-2) and Bak. The models of transplantation tumors in nude mice were established via subcutaneous injection of LNCaP cells. Growth of LNCaP cells was inhibited by ApoG2 treatment. Flow cytometry demonstrated that ApoG2 induced apoptosis in LNCaP cells. The Bcl-2 expression was decreased while Bak expression was increased. In addition, activation of cysteine aspartate protease (caspase)-3 and -8 was observed and 3-methyladenine (3-MA) enhanced apoptosis of LNCaP cells. Furthermore, nude mice treated with ApoG2 demonstrated a significant decrease in tumor volume and a significant increase in cell viability. Immunohistochemical analysis of tumor tissues demonstrated that ApoG2 enhanced caspase-3, -8, LC-3B and beclin-1 expression and reduced the expression of Bcl-2. ApoG2 was able to effectively suppress the growth of LNCaP cells through the induction of autophagy and apoptosis.

Wogonoside induces autophagy-related apoptosis in human glioblastoma cells.

Wogonoside, a bioactive flavonoid extracted from the root of Scutellaria baicalensis Georgi, has shown preclinical anticancer efficacy in various cancer models. However, the effects of wogonoside on glioblastoma cells remain unclear. In the present study, we found that wogonoside exhibited a cytotoxic effect on human glioblastoma cells. The suppression of cell viability was due to the induction of mitochondrial apoptosis. Furthermore, the presence of autophagic hallmarks, including an increase in punctate microtubule associated protein1 light chain3 (LC3) dots, changes in cellular morphology and increased levels of autophagy-related proteins were observed in the wogonoside-treated cells. Wogonoside treatment also enhanced autophagic flux as reflected by the increased acidic vesicular organelle (AVO) formation, p62 degradation and LC3 turnover. Notably, blockade of autophagy by a chemical inhibitor or RNA interference decreased the anticancer effect of wogonoside. In addition, the p38 mitogen-activated protein kinase (MAPK) signaling pathway, the phosphatidylinositide3-kinase/protein kinase B/mammalian target of rapamycin/p70S6 kinase (PI3K/AKT/mTOR/p70S6K) signaling pathway and reactive oxygen species (ROS) participated in wogonoside-induced autophagy and apoptosis. These findings support the initiation of further studies of wogonoside as a candidate for the treatment of human malignant glioma.

The novel pterostilbene derivative ANK-199 induces autophagic cell death through regulating PI3 kinase class III/beclin 1/Atg‑related proteins in cisplatin‑resistant CAR human oral cancer cells.

Pterostilbene is an effective chemopreventive agent against multiple types of cancer cells. A novel pterostilbene derivative, ANK-199, was designed and synthesized by our group. Its antitumor activity and mechanism in cisplatin-resistant CAR human oral cancer cells were investigated in this study. Our results show that ANK-199 has an extremely low toxicity in normal oral cell lines. The formation of autophagic vacuoles and acidic vesicular organelles (AVOs) was observed in the ANK-199-treated CAR cells by monodansylcadaverine (MDC) and acridine orange (AO) staining, suggesting that ANK-199 is able to induce autophagic cell death in CAR cells. Neither DNA fragmentation nor DNA condensation was observed, which means that ANK-199-induced cell death is not triggered by apoptosis. In accordance with morphological observation, 3-MA, a specific inhibitor of PI3K kinase class III, can inhibit the autophagic vesicle formation induced by ANK-199. In addition, ANK-199 is also able to enhance the protein levels of autophagic proteins, Atg complex, beclin 1, PI3K class III and LC3-II, and mRNA expression of autophagic genes Atg7, Atg12, beclin 1 and LC3-II in the ANK-199-treated CAR cells. A molecular signaling pathway induced by ANK-199 was therefore summarized. Results presented in this study show that ANK-199 may become a novel therapeutic reagent for the treatment of oral cancer in the near future (patent pending).

Rubella virus perturbs autophagy.

Autophagy is a cellular catabolic process implicated in numerous physiological processes and pathological conditions, including infections. Viruses have evolved different strategies to modulate the autophagic process. Since the effects of rubella virus (RV) on autophagy have not yet been reported, we evaluated the autophagic activity in the Statens Seruminstitut Rabbit Cornea cell line infected with the To336 strain of RV. Our results showed that RV lowered the levels of microtubule-associated protein 1 light chain 3 B-II (LC3B-II) and the autophagy-related gene 12-autophagy-related gene 5 conjugate, inhibited the autophagic flux, suppressed the intracellular redistribution of LC3B, decreased both the average number and the size of autophagosomes per cell and impeded the formation of acidic vesicular organelles. Induction of autophagy by using rapamycin decreased both the viral yields and the apoptotic rates of infected cultures. Besides its cytoprotective effects, autophagy furnishes an important antiviral mechanism, inhibition of which may reorchestrate intracellular environment so as to better serve the unique requirements of RV replication. Together, our observations suggest that RV utilizes a totally different strategy to cope with autophagy than that evolved by other positive-stranded RNA viruses, and there is considerable heterogeneity among the members of the Togaviridae family in terms of their effects on the cellular autophagic cascade.

Inhibition of autophagy enhances DNA damage-induced apoptosis by disrupting CHK1-dependent S phase arrest

DNA damage has been shown to induce autophagy, but the role of autophagy in the DNA damage response and cell fate is not fully understood. BO-1012, a bifunctional alkylating derivative of 3a-aza-cyclopenta[a]indene, is a potent DNA interstrand cross-linking agent with anticancer activity. In this study, BO-1012 was found to reduce DNA synthesis, inhibit S phase progression, and induce phosphorylation of histone H2AX on serine 139 (γH2AX) exclusively in S phase cells. Both CHK1 and CHK2 were phosphorylated in response to BO-1012 treatment, but only depletion of CHK1, but not CHK2, impaired BO-1012-induced S phase arrest and facilitated the entry of γH2AX-positive cells into G2 phase. CHK1 depletion also significantly enhanced BO-1012-induced cell death and apoptosis. These results indicate that BO-1012-induced S phase arrest is a CHK1-dependent pro-survival response. BO-1012 also resulted in marked induction of acidic vesicular organelle (AVO) formation and microtubule-associated protein 1 light chain 3 (LC3) processing and redistribution, features characteristic of autophagy. Depletion of ATG7 or co-treatment of cells with BO-1012 and either 3-methyladenine or bafilomycin A1, two inhibitors of autophagy, not only reduced CHK1 phosphorylation and disrupted S phase arrest, but also increased cleavage of caspase-9 and PARP, and cell death. These results suggest that cells initiate S phase arrest and autophagy as pro-survival responses to BO-1012-induced DNA damage, and that suppression of autophagy enhances BO-1012-induced apoptosis via disruption of CHK1-dependent S phase arrest.

Ursolic acid induces autophagy in U87MG cells via ROS-dependent endoplasmic reticulum stress

Malignant gliomas are the most common primary brain tumors, and novel ways of treating gliomas are urgently needed. Ursolic acid (UA), a pentacyclic triterpenoid, has been reported to exhibit promising antitumor activity. Here, we evaluated the effects of UA on U87MG cells and explored the underlying molecular mechanisms. The results demonstrated that both G1-phase arrest and autophagy were induced by UA in U87MG cells. Evidence of UA-induced autophagy included the formation of acidic vesicular organelles, increase of autophagolysosomes and LC3-II accumulation. UA was also found to induce ER stress and an increase in intracellular calcium accompanied by ROS production. The increase in free cytosolic calcium induced by UA activated the CaMKK-AMPK-mTOR kinase signaling cascade, which ultimately triggered autophagy. Western blot analysis showed that UA promoted the phosphorylation of PERK and eIF2α; this was followed by the upregulation of the downstream protein CHOP, implying the involvement of the ER stress-mediated PERK/eIF2α/CHOP pathway in glioma cells. Meanwhile, UA activated IRE1α and subsequently increased the levels of phosphorylated JNK and Bcl-2, resulting in the dissociation of Beclin1 from Bcl-2. Furthermore, TUDCA and the silencing of either PERK or IRE1α partially blocked the UA-induced accumulation of LC3-II, suggesting that ER stress precedes the process of autophagy. Additionally, NAC attenuated the UA-induced elevation in cytosolic calcium, ER stress markers and autophagy-related proteins, indicating that UA triggered ER stress and autophagy via a ROS-dependent pathway. Collectively, our findings revealed a novel cellular mechanism triggered by UA and provide a molecular basis for developing UA into a drug candidate.

DHA2, a synthesized derivative of bisbibenzyl, exerts antitumor activity against ovarian cancer through inhibition of XIAP and Akt/mTOR pathway

The analysis of dihydroptychantol (DHA) and its chemically synthesized macrocyclic bisbibenzyl derivatives (DHA1, 2 and 3) led to the selection of DHA2 as a potential drug candidate for ovarian cancer. The exposure of ovarian cancer SKOV3 cells to DHA2 resulted in the downregulation of the anti-apoptotic X-linked inhibitor of apoptosis protein (XIAP) and Bcl-2 and led to caspase-independent cell death. The overexpression of XIAP reversed DHA2-induced cell death, and the depletion of XIAP had the opposite effect. DHA2 could induce autophagy, as evidenced by increases in the formation of acidic vesicular organelles and the processing of LC3B-I to LC3B-II. Pretreatment with autophagy inhibitors potentiated DHA2-mediated cell death. We showed that typical PI3K/Akt signaling was involved in DHA2-mediated cell death. The overexpression of Akt almost completely reversed DHA2-induced cell death, and the inactivation of Akt significantly facilitated DHA2-induced cell death. The administration of DHA2 to xenograft mice reduced tumor growth by causing Akt inactivation, the conversion of LC3B and proliferation inhibition. Our study demonstrates that the inhibition of XIAP and the inactivation of Akt/mTOR facilitate the efficacy of DHA2 in ovarian cancer cells.

Temozolomide induces autophagy via ATM‑AMPK‑ULK1 pathways in glioma.

Autophagy is a cytoprotective process, which occurs following temozolomide (TMZ) treatment, and contributes to glioma chemoresistance and TMZ treatment failure. However, the molecular mechanisms by which TMZ induces autophagy are largely unknown. In the current study, the ataxia‑telangiectasia mutated (ATM) inhibitor KU‑55933, adenosine monophosphate‑activated protein kinase (AMPK) inhibitor compoundC, and U87MG and U251 cell lines were employed to investigate the molecular mechanisms of TMZ‑induced autophagy in glioma, and to evaluate the effects of autophagy inhibition on TMZ cytotoxicity. KU‑55933 and compound C were observed to inhibit the activation of autophagy‑initiating kinase ULK1 and result in a significant decrease of autophagy as indicated by depressed LC3B cleavage and acidic vesicular organelle formation. The activation of AMPK‑ULK1 was ATM dependent. Autophagy inhibition via the AMPK inhibitor compoundC augmented TMZ cytotoxicity as observed by depressed cell viability, increased γH2AX‑marked double‑strand breaks (DSBs) and elevated numbers of apoptotic glioma cells. In conclusion, TMZ induced autophagy via ATM‑AMPK‑ULK1 pathways. TMZ chemoresistance may therefore be overwhelmed by targeting AMPK, particularly for the treatment of O6‑methylguanine DNA methyltransferase‑negative gliomas.

Justicidin A‐induced autophagy inhibits apoptosis of human hepatocellular carcinoma Hep 3B cells (644.7)

Justicidin A (JA) is a new and pure arylnaphthalide lignan isolated from Justicia procumbens, which has been used as an herbal remedy in Taiwan and China for fever, cancer, and pain. Our previous report shows that JA induced apoptosis of human hepatocellular carcinoma (HCC) Hep 3B cells, leading to the suppression of tumor cell growth in NOD‐SCID mice. Here we reveal that treatment of JA not only increased the expression of autophagic marker microtubule‐associated protein 1 light chain‐II (LC3‐II) by immunoblotting, but also augmented the formation of acidic vesicular organelles by flow cytometry. Administration of autophagy inhibitor bafilomycin A1 (BAF) increased protein expression of LC3‐II and lysosomal marker LAMP2a and decreased colocalization of LC3 and LAMP2a puncta by confocal microscopy, confirming that JA not only induced autophagy but also completed the autophagic flux. JA‐induced autophagy was observed via activation of Raf/MEK/ERK pathway, and was not associated with inhibition of class I PI3K/Akt/mTOR or induction of class III PI3K/Beclin 1 pathway. The novelty of JA is that pre‐treatment of cells with BAF elevated the percentages of cells at sub‐G1 phase and raised the expression of cleaved caspase 3. All together, our data suggest the use of autophagy inhibitor should be taken into consideration to enhance the chemotherapeutic potential of JA for treatment of human HCC.Grant Funding Source: Supported in part by the National Science Council, Taiwan, No. NSC 98‐2313‐B‐003‐002‐MY3