Impacts of Autophagy-Inducing Ingredient of Areca Nut on Tumor Cells

Areca nut (AN) is a popular carcinogen used by about 0.6-1.2 billion people worldwide. Although AN contains apoptosis-inducing ingredients, we previously demonstrated that both AN extract (ANE) and its 30-100 kDa fraction (ANE 30-100K) predominantly induce autophagic cell death in both normal and malignant cells. In this study, we further explored the action mechanism of ANE 30-100K-induced autophagy (AIA) in Jurkat T lymphocytes and carcinoma cell lines including OECM-1 (mouth), CE81T/VGH (esophagus), SCC25 (tongue), and SCC-15 (tongue). The results showed that chemical- and small hairpin RNA (shRNA)-mediated inhibition of AMP-activated protein kinase (AMPK) resulted in the attenuation of AIA in Jurkat T but not in OECM-1 cells. Knockdown of Atg5 and Beclin 1 expressions ameliorated AIA in OECM-1/CE81T/VGH/Jurkat T and OECM-1/SCC25/SCC-15, respectively. Furthermore, ANE 30-100K could activate caspase-3 after inhibition of Beclin 1 expression in OECM-1/SCC25/SCC15 cells. Meanwhile, AMPK was demonstrated to be the upstream activator of the extracellular-regulated kinase (ERK) in Jurkat T cells, and inhibition of MEK attenuated AIA in Jurkat T/OECM-1/CE81T/VGH cells. Finally, we also found that multiple myeloma RPMI8226, lymphoma U937, and SCC15 cells survived from long-term non-cytotoxic ANE 30-100K treatment exhibited stronger resistance against serum deprivation through upregulated autophagy. Collectively, our studies indicate that Beclin-1 and Atg5 but not AMPK are commonly required for AIA, and MEK/ERK pathway is involved in AIA. Meanwhile, it is also suggested that long-term AN usage might increase the resistance of survived tumor cells against serum-limited conditions.     

Phenethyl caffeate benzoxanthene lignan is a derivative of caffeic acid phenethyl ester that induces bystander autophagy in WiDr cells

We recently reported that Phenethyl caffeate benzoxanthene lignan (PCBL), a semisynthetic compound derived from Caffeic Acid Phenethyl Ester (CAPE), induces DNA damage and apoptosis in tumor cells. In this study, we further investigated whether PCBL induces autophagy in WiDr cells. We also analyzed the pathways regulating autophagy and the role of autophagy in PCBL-induced cell death. Our acridine orange staining and LC3 II expression results suggest that PCBL induces autophagosomes in WiDr cells. The levels of LC3 II expression we observed after co-treatment of PCBL with bafilomycin A1 and the reductions in p62 expression we observed after PCBL treatment in WiDr cells demonstrate increased autophagic flux, a reliable indicator of autophagic induction. The increased Beclin 1 expression in PCBL-treated cells and the incapacity of PCBL to induce LC3 II in 3-methyladenine (3-MA)-treated cells we observed suggests that PCBL-induced autophagy is class III PI3-kinase dependent. PCBL did not alter phosphorylation of the mTOR substrate p70 S6 kinase, indicating that PCBL-induced autophagy was not mTOR regulated. Two autophagy related proteins, Atg5 and Atg12, also remained uninduced during PCBL treatment. The increased caspase activity and expression levels of LC3 II and p62 we observed in response to PCBL treatment in primary glioma cells demonstrates that PCBL-induced apoptosis and autophagy were not cell line specific. Pharmacological inhibition of autophagy did not alter the antitumor efficacy of PCBL in WiDr cells. This attests to the bystander nature of PCBL-induced autophagy (in terms of cell death). In toto, these data suggest that PCBL induces a class III kinase dependent, but mTOR independent, bystander mode of autophagy in WiDr cells.     

ERK-mediated autophagy promotes inactivated Sendai virus (HVJ-E)-induced apoptosis in HeLa cells in an Atg3-dependent manner

Background

Apoptosis and autophagy are known to play important roles in cancer development. It has been reported that HVJ-E induces apoptosis in cancer cells, thereby inhibiting the development of tumors. To define the mechanism by which HVJ-E induces cell death, we examined whether HVJ-E activates autophagic and apoptotic signaling pathways in HeLa cells.

Methods

Cells were treated with chloroquine (CQ) and rapamycin to determine whether autophagy is involved in HVJ-E-induced apoptosis. Treatment with the ERK inhibitor, U0126, was used to determine whether autophagy and apoptosis are mediated by the ERK pathway. Activators of the PI3K/Akt/mTOR/p70S6K pathway, 740 Y-P and SC79, were used to characterize its role in HVJ-E-induced autophagy. siRNA against Atg3 was used to knock down the protein and determine whether it plays a role in HVJ-E-induced apoptosis in HeLa cells.

Results

We found that HVJ-E infection inhibited cell viability and induced apoptosis through the mitochondrial pathway, as evidenced by the expression of caspase proteins. This process was promoted by rapamycin treatment and inhibited by CQ treatment. HVJ-E-induced autophagy was further blocked by 740 Y-P, SC79, and U0126, indicating that both the ERK- and the PI3K/Akt/mTOR/p70S6K-pathways were involved. Finally, autophagy-mediated apoptosis induced by HVJ-E was inhibited by siRNA-mediated Atg3 knockdown.

Conclusion

In HeLa cells, HVJ-E infection triggered autophagy through the PI3K/Akt/mTOR/p70S6K pathway in an ERK1/2-dependent manner, and the induction of autophagy promoted apoptosis in an Atg3-dependent manner.

     

Plumbagin induces autophagy and apoptosis of SMMC-7721 cells in vitro and in vivo

Plumbagin (PL), an active naphthoquinone compound, has been demonstrated to be a potential anticancer agent. However, the underlying anticancer mechanism is not fully understood. In this study, the human hepatocellular carcinoma (HCC) SMMC-7721 cell line was studied in an in vitro model. The cell proliferation was inhibited by PL in a dose- and time-dependent manner. Electron microscopy, acridine orange staining, and immunofluorescence were used to evaluate autophagosome formation and LC3 protein expression in PL-treated SMMC-7721 cells. Real-time polymerase chain reaction and Western blot showed that PL treatment suppressed the expression of apoptosis and autophagy factors (LC3, Beclin1, Atg7, and Atg5), which are associated with tumor apoptosis and autophagy in SMMC-7721 cells. In the study of in vitro tumor nude mouse models, PL can inhibit tumor growth. Cell apoptosis and autophagy of the transplanted tumors were evaluated by hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining, and Western blot. In addition, in the in vivo studies of HCC cells, we found that pretreatment with the autophagy inhibitor 3-methyladenine blocked the formation of apoptosis induced by PL. In contrast, administration of the apoptosis inhibitor Z-VAD did not affect PL-induced autophagy. Taken together, our findings strongly suggest that PL is a promising drug with significant antitumor activity in HCC.     

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.     

Autophagy Plays a Critical Role in ChLym-1-Induced Cytotoxicity of Non-Hodgkin’s Lymphoma Cells

Autophagy is a critical mechanism in both cancer therapy resistance and tumor suppression. Monoclonal antibodies have been documented to kill tumor cells via apoptosis, antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In this study, we report for the first time that chLym-1, a chimeric anti-human HLA-DR monoclonal antibody, induces autophagy in Raji Non-Hodgkin’s Lymphoma (NHL) cells. Interestingly, inhibition of autophagy by pharmacological inhibitors (3-methyladenine and NH₄Cl) or genetic approaches (siRNA targeting Atg5) suppresses chLym-1-induced growth inhibition, apoptosis, ADCC and CDC in Raji cells, while induction of autophagy could accelerate cytotoxic effects of chLym-1 on Raji cells. Furthermore, chLym-1-induced autophagy can mediate apoptosis through Caspase 9 activation, demonstrating the tumor-suppressing role of autophagy in antilymphoma effects of chLym-1. Moreover, chLym-1 can activate several upstream signaling pathways of autophagy including Akt/mTOR and extracellular signal-regulated kinase 1/2 (Erk1/2). These results elucidate the critical role of autophagy in cytotoxicity of chLym-1 antibody and suggest a potential therapeutic strategy of NHL therapy by monoclonal antibody chLym-1 in combination with autophagy inducer.     

Rapamycin and Interleukin-1β Impair Brain-derived Neurotrophic Factor-dependent Neuron Survival by Modulating Autophagy

The mammalian target of rapamycin (mTOR) pathway has multiple important physiological functions, including regulation of protein synthesis, cell growth, autophagy, and synaptic plasticity. Activation of mTOR is necessary for the many beneficial effects of brain-derived neurotrophic factor (BDNF), including dendritic translation and memory formation in the hippocampus. At present, however, the role of mTOR in BDNF''s support of survival is not clear. We report that mTOR activation is necessary for BDNF-dependent survival of primary rat hippocampal neurons, as either mTOR inhibition by rapamycin or genetic manipulation of the downstream molecule p70S6K specifically blocked BDNF rescue. Surprisingly, however, BDNF did not promote neuron survival by up-regulating mTOR-dependent protein synthesis or through mTOR-dependent suppression of caspase-3 activation. Instead, activated mTOR was responsible for BDNF''s suppression of autophagic flux. shRNA against the autophagic machinery Atg7 or Atg5 prolonged the survival of neurons co-treated with BDNF and rapamycin, suggesting that suppression of mTOR in BDNF-treated cells resulted in excessive autophagy. Finally, acting as a physiological analog of rapamycin, IL-1β impaired BDNF signaling by way of inhibiting mTOR activation as follows: the cytokine induced caspase-independent neuronal death and accelerated autophagic flux in BDNF-treated cells. These findings reveal a novel mechanism of BDNF neuroprotection; BDNF not only prevents apoptosis through inhibiting caspase activation but also promotes neuron survival through modulation of autophagy. This protection mechanism is vulnerable under chronic inflammation, which deregulates autophagy through impairing mTOR signaling. These results may be relevant to age-related changes observed in neurodegenerative diseases.     

Melatonin ameliorates pressure overload-induced cardiac hypertrophy by attenuating Atg5-dependent autophagy and activating the Akt/mTOR pathway

Cardiac hypertrophy, including hypertension and valvular dysfunction, is a pathological feature of many cardiac diseases that ultimately leads to heart failure. Melatonin confers a protective role against pathological cardiac hypertrophy, but the underlying mechanisms remain elusive. In the present study, we hypothesized that melatonin protects against pressure overload-induced cardiac hypertrophy by attenuating Atg5-dependent autophagy and activating the Akt/mTOR pathway. Male C57BL/6 mice that received adenovirus carrying cardiac-specific Atg5 (under the cTNT promoter; Ad-cTNT-Atg5) underwent transverse aortic constriction (TAC) or sham operation and received an intraperitoneal injection of melatonin (10 mg/kg/d), vehicle or LY294002 (10 mg/kg/d) for 8 weeks. Melatonin treatment for 8 weeks markedly attenuated cardiac hypertrophy and restored impaired cardiac function, as indicated by a decreased HW/BW ratio, reduced cell cross-sectional area and fibrosis, downregulated the mRNA levels of ANP, BNP, and β-MHC and ameliorated adverse effects on the LVEF and LVFS. Melatonin treatment also inhibited apoptosis and alleviated autophagy dysfunction. Furthermore, melatonin inhibited Akt/mTOR pathway activation, while these effects were blocked by LY294002. In addition, the effect of melatonin regulation on TAC-induced autophagy dysfunction was inhibited by LY294002 or cardiac-specific Atg5 overexpression. As expected, Akt/mTOR pathway inhibition or cardiac-specific Atg5 overexpression restrained melatonin alleviation of pressure overload-induced cardiac hypertrophy. These results demonstrated that melatonin ameliorated pressure overload-induced cardiac hypertrophy by attenuating Atg5-dependent autophagy and activating the Akt/mTOR pathway.     

Autophagy is a regulator of TRAIL-induced apoptosis in NSCLC A549 cells

Autophagy, a catabolic process by which cytoplasmic components are degraded in lysosomes, plays an important role in the maintenance of cellular homeostasis. Dysregulation of autophagy is associated with several diseases. However, few studies have addressed the role of autophagy in the lung, and its role in lung diseases remains unclear. In the present study, we examined the effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on autophagy in A549 cells and explored the underlying mechanisms. We showed that TRAIL promoted autophagosome formation, as detected by the levels of LC3-II, and its effect on promoting autophagy was dependent on the expression of the autophagy related genes (ATGs) Atg5, Atg7, and beclin-1. TRAIL-induced ATG expression was attenuated by JNK silencing or treatment with the JNK inhibitor SP600125, indicating the involvement of the JNK pathway. Crosstalk between autophagy and apoptosis was demonstrated by silencing the autophagy related genes Atg5, Atg7, and beclin-1, and the dependence of TRAIL-induced apoptosis on autophagy-related gene expression. Taken together, our results indicate that TRAIL promotes autophagy in A549 cells via a mechanism involving the modulation of ATG expression through the JNK pathway. Inhibition of autophagy enhanced TRAIL-induced cell proliferative inhibition and apoptosis in A549 cells.     

Autophagy and apoptosis act as partners to induce germ cell death after heat stress in mice

Testicular heating suppresses spermatogenesis which is marked by germ cell loss via apoptotic pathways. Recently, it is reported that autophagy also can be induced by heat treatment in somatic cells. In this study, the status of autophagy in germ cells after heat treatment, as well as the partnership between autophagy and apoptosis in these cells was investigated. The results demonstrated that besides initiating apoptotic pathways, heat also induced autophagic pathways in germ cells. Exposure of germ cells to hyperthermia resulted in several specific features of the autophagic process, including autophagosome formation and the conversion of LC3-I to LC3-II. Furthermore, the ubiquitin-like protein conjugation system was implicated as being likely responsible for heat-induced autophagy in germ cells since all genes involving this system were found to be expressed in the testes. In addition, the upstream protein in this system, Atg7 (Autophagy-related gene 7), was found to be expressed in all types of spermatogenic cells, and its expression level was positively correlated with the level of autophagy in germ cells. As a result, Atg7 was selected as the investigative target to further analyze the role of autophagy in heat-induced germ cell death. It was shown that down expression of Atg7 protein resulted in the notable decrease in the level of autophagy in heat-treated germ cells, and this down-regulation of autophagy caused by Atg7 knockdown further reduced the apoptotic rate of germ cells. These results suggest that autophagy plays a positive role in the process of germ cell apoptosis after heat treatment. In conclusion, this study demonstrates that heat triggers autophagy and apoptosis in germ cells. These two mechanisms might act as partners, not antagonist, to induce cell death and lead to eventual destruction of spermatogenesis.     

A plant triterpenoid, avicin D, induces autophagy by activation of AMP-activated protein kinase

Avicins, a family of plant triterpene electrophiles, can trigger apoptosis-associated tumor cell death, and suppress chemical-induced carcinogenesis by its anti-inflammatory, anti-mutagenic, and antioxidant properties. Here, we show that tumor cells treated with benzyloxycarbonylvalyl-alanyl-aspartic acid (O-methyl)-fluoro-methylketone, an apoptosis inhibitor, and Bax(-/-)Bak(-/-) apoptosis-resistant cells can still undergo cell death in response to avicin D treatment. We demonstrate that this non-apoptotic cell death is mediated by autophagy, which can be suppressed by chloroquine, an autophagy inhibitor, and by specific knockdown of autophagy-related gene-5 (Atg5) and Atg7. Avicin D decreases cellular ATP levels, stimulates the activation of AMP-activated protein kinase (AMPK), and inhibits mammalian target of rapamycin (mTOR) and S6 kinase activity. Suppression of AMPK by compound C and dominant-negative AMPK decreases avicin D-induced autophagic cell death. Furthermore, avicin D-induced autophagic cell death can be abrogated by knockdown of tuberous sclerosis complex 2 (TSC2), a key mediator linking AMPK to mTOR inhibition, suggesting that AMPK activation is a crucial event targeted by avicin D. These findings indicate the therapeutic potential of avicins by triggering autophagic cell death.     

Ornithine decarboxylase prevents methotrexate-induced apoptosis by reducing intracellular reactive oxygen species production

Methotrexate (MTX), a folate antagonist, was developed for the treatment of malignancies, and is currently used in rheumatoid arthritis (RA) and other chronic inflammatory disorders. It has been proven in short-term and long-term prospective studies that low doses of MTX (0.75 mg/Kg/week) are effective in controlling the inflammatory manifestations of RA. Low-concentrations of MTX achieve apoptosis and clonal deletion of activated peripheral T cells. One of the mechanisms of the anti-inflammatory and immunosuppressive effects may be the production of reactive oxygen species (ROS). However, the drug resistance of MTX in malignancies remains poorly understood. Ornithine decarboxylase (ODC) plays an important role in diverse biological functions, including cell development, differentiation, transformation, growth and apoptosis. In our previous studies, ODC overexpression was shown to prevent TNFα-induced apoptosis via reducing ROS. Here, we also investigated one mechanism of MTX-induced apoptosis and of drug resistance as to the anti-apoptotic effects of ODC during MTX treatment. We found MTX could induce caspase-dependent apoptosis and promote ROS generation together with disrupting the mitochondrial membrane potential (ΔΨ_m) of HL-60 and Jurkat T cells. Putrescine and ROS scavengers could reduce MTX-induced apoptosis, which leads to the loss of ΔΨ_m, through reducing intracellular ROS. Overexpression of ODC in parental cells had the same effects as putrescine and the ROS scavengers. Moreover, ODC overexpression prevented the decline of Bcl-2 that maintains ΔΨ_m, the cytochrome c release and activations of caspase 9 and 3 following MTX treatment. The results demonstrate that MTX-induced apoptosis is ROS-dependent and occurs along a mitochondria-mediated pathway. Overexpressed ODC cells are resistant to MTX-induced apoptosis by reducing intracellular ROS production.     

Inhibition of autophagy enhances apoptosis induced by the PI3K/AKT/mTor inhibitor NVP‐BEZ235 in renal cell carcinoma cells

The PI3K/AKT/mTOR pathway plays a key role in the development of the hypervascular tumor renal cell carcinoma (RCC). NVP‐BEZ235 (NVP), a novel dual PI3K/mTOR inhibitor, showed great antitumor benefit and provided a treatment strategy in RCC. In this study, we test the effect of NVP on survival rate, apoptosis and autophagy in the RCC cell line, 786‐0. We also explore the hypothesis that NVP, in combination with autophagy inhibitors, leads to apoptosis enhancement in 786‐0 cells. The results showed that the PI3K/AKT/mTOR pathway proteins p‐AKT and p‐P70S6K were highly expressed in RCC tissue. We also showed that NVP inhibited cell growth and induced apoptosis and autophagy in RCC cells. The combination treatment of NVP with autophagy inhibitors enhanced the effect of NVP on suppressing 786‐0 growth and induction of apoptosis. This study proposes a novel treatment paradigm where combining PI3K/AKT/mTOR pathway inhibitors and autophagy inhibitors lead to enhanced RCC cell apoptosis. Copyright © 2012 John Wiley & Sons, Ltd.     

Coordinate Autophagy and mTOR Pathway Inhibition Enhances Cell Death in Melanoma

The phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway promotes melanoma tumor growth and survival while suppressing autophagy, a catabolic process through which cells collect and recycle cellular components to sustain energy homeostasis in starvation. Conversely, inhibitors of the PI3K/AKT/mTOR pathway, in particular the mTOR inhibitor temsirolimus (CCI-779), induce autophagy, which can promote tumor survival and thus, these agents potentially limit their own efficacy. We hypothesized that inhibition of autophagy in combination with mTOR inhibition would block this tumor survival mechanism and hence improve the cytotoxicity of mTOR inhibitors in melanoma. Here we found that melanoma cell lines of multiple genotypes exhibit high basal levels of autophagy. Knockdown of expression of the essential autophagy gene product ATG7 resulted in cell death, indicating that survival of melanoma cells is autophagy-dependent. We also found that the lysosomotropic agent and autophagy inhibitor hydroxychloroquine (HCQ) synergizes with CCI-779 and led to melanoma cell death via apoptosis. Combination treatment with CCI-779 and HCQ suppressed melanoma growth and induced cell death both in 3-dimensional (3D) spheroid cultures and in tumor xenografts. These data suggest that coordinate inhibition of the mTOR and autophagy pathways promotes apoptosis and could be a new therapeutic paradigm for the treatment of melanoma.     

Retracted: Long noncoding RNA HAGLROS regulates apoptosis and autophagy in colorectal cancer cells via sponging miR-100 to target ATG5 expression

The aim of this study was to explore the relationship between the expression of HOXD antisense growth-associated long noncoding RNA (HAGLROS) and prognosis of patients with colorectal cancer (CRC), as well as the roles and regulatory mechanism of HAGLROS in CRC development. The HAGLROS expression in CRC tissues and cells was detected. The correlation between HAGLROS expression and survival time of CRC patients was investigated. Moreover, HAGLROS was overexpressed and suppressed in HCT-116 cells, followed by detection of cell viability, apoptosis, and the expression of apoptosis-related proteins and autophagy markers. Furthermore, the association between HAGLROS and miR-100 and the potential targets of miR-100 were investigated. Besides, the regulatory relationship between HAGLROS and PI3K/AKT/mTOR pathway was elucidated. The results showed that HAGLROS was highly expressed in CRC tissues and cells. Highly expression of HAGLROS correlated with a shorter survival time of CRC patients. Moreover, knockdown of HAGLROS in HCT-116 cells induced apoptosis by increasing the expression of Bax/Bcl-2 ratio, cleaved-caspase-3, and cleaved-caspase-9, and inhibited autophagy by decreasing the expression of LC3II/LC3I and Beclin-1 and increasing P62 expression. Furthermore, HAGLROS negatively regulated the expression of miR-100, and HAGLROS controlled HCT-116 cell apoptosis and autophagy through negatively regulation of miR-100. Autophagy related 5 (ATG5) was verified as a functional target of miR-100 and miR-100 regulated HCT-116 cell apoptosis and autophagy through targeting ATG5. Besides, HAGLROS overexpression activated phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway. In conclusion, a highly expression of HAGLROS correlated with shorter survival time of CRC patients. Downregulation of HAGLROS may induce apoptosis and inhibit autophagy in CRC cells by regulation of miR-100/ATG5 axis and PI3K/AKT/mTOR pathway.     

Autophagy pathways activated in response to PDT contribute to cell resistance against ROS damage

Reactive oxygen species (ROS) concurrently instigate apoptosis and autophagy pathways, but the link between these processes remains unclear. Because cytotoxic ROS formation is exploited in anticancer therapy, such as in photodynamic therapy (PDT), a better understanding of the complex interplay between autophagy and apoptosis is urgently required. Previously, we reported that ROS generated by PDT with an endoplasmic reticulum (ER)-associated sensitizer leads to loss of ER-Ca²⁺ homeostasis, ER stress and apoptosis. Here we show that PDT prompted Akt-mTOR (mammalian target of rapamycin) pathway down-regulation and stimulated macroautophagy (MA) in cancer and normal cells. Overexpression of the antioxidant enzyme glutathione peroxidase-4 reversed mTOR down-regulation and blocked MA progression and apoptosis. Attenuating MA using Atg5 knockdown or 3-methyladenine, reduced clearance of oxidatively damaged proteins and increased apoptosis, thus revealing a cytoprotective role of MA in PDT. Paradoxically, genetic loss of MA improved clearance of oxidized proteins and reduced photokilling. We found that up-regulation of chaperone-mediated autophagy (CMA) in unstressed Atg5^−/− cells compensated for MA loss and increased cellular resistance to PDT. CMA-deficient cells were significantly sensitized to photokilling but were protected against the ER stressor thapsigargin. These results disclose a stress-specific recruitment of autophagy pathways with cytoprotective function and unravel CMA as the dominant defence mechanism against PDT.     

Autophagy mediates cytotoxicity of human colorectal cancer cells treated with garcinielliptone FC

The tautomeric pair of garcinielliptone FC (GFC) is a novel tautomeric pair of polyprenyl benzophenonoid isolated from the pericarps of Garcinia subelliptica Merr. (G. subelliptica, Clusiaceae), a tree with abundant sources of polyphenols. Our previous report demonstrated that GFC induced apoptosis on various types of human cancer cell lines including chemoresistant human colorectal cancer HT‐29 cells. In the present study, we observed that many autophagy‐related genes in GFC‐treated HT‐29 cells were up‐ and down‐regulated using a cDNA microarray containing oncogenes and kinase genes. GFC‐induced autophagy of HT‐29 cells was confirmed by observing the formation of acidic vesicular organelles, LC3 puncta, and double‐membrane autophagic vesicles using flow cytometry, confocal microscopy, and transmission electron microscopy, respectively. Inhibition of AKT/mTOR/P70S6K signaling as well as formation of Atg5‐Atg12 and PI3K/Beclin‐1 complexes were observed using Western blot. Administration of autophagy inhibitor (3‐methyladenine and shRNA Atg5) and apoptosis inhibitor Z‐VAD showed that the GFC‐induced autophagy was cytotoxic form and GFC‐induced apoptosis enhanced GFC‐induced autophagy. Our data suggest the involvement of autophagy and apoptosis in GFC‐induced anticancer mechanisms of human colorectal cancer.     

G15, a GPR30 antagonist,induces apoptosis and autophagy in human oral squamous carcinoma cells

As GPR30 has been implicated in mediating cancer cell proliferation, this study aimed to examine the antitumor effect of the GPR30 antagonist G15 in human oral squamous cell carcinoma (OSCC). G15 induced dose-dependent cytotoxicity, apoptosis and G2/M cell cycle arrest in a panel of OSCC cells. The results showed that G15 could inhibit the growth of the oral cancer cells with IC₅₀ value 11.2 μM for SCC4, 15.6 μM for SCC9, and 7.8 μM for HSC-3, respectively. Flow cytometric analysis and Comet assay indicated that G15 suppressed the viability of SCC4 and HSC-3 cells by inducing apoptosis and G2/M arrest. In addition, G15 down regulated the expression of Akt, cell cycle-related proteins, and mitogen-activated protein kinases, but increased the levels of LC3B-II and the accumulation of autophagosomes. Inhibition of autophagy by chloroquine does not affect the G15-induced apoptosis in SCC4 cells. Mechanistic evidence indicated that the antiproliferative effect was mediated through the downregulation of cdc2, cdc25c and NF-κB expression. Taken together, our findings suggest the potential of G15 in treating OSCC.