Induction Of Autophagic Cell Death Research Articles

Nonsteroidal Anti-inflammatory Drugs Sensitize CD44-Overexpressing Cancer Cells to Hsp90 Inhibitor Through Autophagy Activation

Recently, novel therapeutic strategies have been designed with the aim of killing cancer stem-like cells (CSCs), and considerable interest has been generated in the development of specific therapies that target stemness-related marker of CSCs. In this study, nonsteroidal anti-inflammatory drugs (NSAIDs) significantly potentiated Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG)-mediated cytotoxicity through apoptotic and autophagic cell death induction, but COX-2-inhibitory function was not required for NSAID-induced autophagy in CD44-overexpressing human chronic myeloid leukemia K562 (CD44highK562) cells. Importantly, we found that treatment with NSAIDs resulted in a dose-dependent increase in LC3-II level and decrease in p62 level and simultaneous reduction in multiple stemness-related markers including CD44, Oct4, c-Myc, and mutant p53 (mutp53) in CD44highK562 cells, suggesting that NSAIDs could induce autophagy, which might mediate degradation of stemness-related marker proteins. Activation of AMPK and inhibition of Akt/mTOR/p70S6K/4EBP1 participated in NSAID-induced autophagy in CD44highK562 cells. In addition, treatment of CD44highK562 cells with NSAIDs inhibited expression of HSF1/Hsps, which resulted in suppression of 17-AAG-induced activation of Hsp70, leading to reversal of 17-AAG resistance and sensitization of CD44highK562 cells to 17-AAG by NSAIDs. In conclusion, combining NSAIDs with Hsp90 inhibitor may offer one of the most promising strategies for eradication of CD44-overexpressing CSCs.

Cholesterol Biosynthesis Is Required for Cell Death in Response to G9A Inhibitor in Lung Cancer (P05-010-19)

ObjectivesG9A is a eukaryotic histone methyltransferase that contributes to transcriptional silencing of tumor suppressor genes by modulating histone 3 lysine 9 methylation (H3K9m). It has been recently suggested as a promising therapeutic target for breast cancer and neuroblastoma. This study was aimed to investigate the effect of G9A inhibition and its cellular metabolic mechanisms. MethodsUsing OncomineTM, G9A overexpression in lung cancer was assessed. Cell viability upon treatment of G9A inhibitor (BIX01294, BIX) and siG9A was measured by MTT and IncuCyteR assays. Additionally, apoptosis and autophagy were analyzed through western blots. In order to identify targets, transcriptomes using RNA sequencing was conducted upon BIX treatment. Further functional relevance of targets was validated using Chromatin IP and recovery tests. ResultsBIX-mediated inhibition of G9A reduced cell viability of lung cancer cells via induction of autophagy. Through RNA sequencing, we found that G9A inhibition mainly affected cholesterol biosynthesis pathway. BIX directly induced the expression of SREBF2 gene, by lowering H3K9me1 and H3K9me2 at the promoter. A cholesterol biosynthesis inhibitor, 25-HC, partially recovered BIX-induced cell death by attenuating autophagy. Our data suggests that cholesterol metabolism can be a potential therapeutic target by G9A inhibition and its induction of autophagic cell death. ConclusionsOur data suggests that cholesterol metabolism can be a potential therapeutic target by G9A inhibition and its induction of autophagic cell death. Funding SourcesNRF of Korea grant (2018R1D1A1B07051274); BK21 Plus Project (22A20130012143).

Combination of arsenic trioxide and cisplatin synergistically inhibits both hexokinase activity and viability of Ehrlich ascites carcinoma cells.

Hexokinase-2 is overexpressed in several carcinomas including breast cancer to sustain energy for rapidly dividing cells and associates with chemoresistance. However, the impact of chemo drugs (alone or in combination) on hexokinase activity and autophagic cell death is unclear. In this report, we used an in vivo murine adenocarcinoma model to validate the effects of As2 O3 and cisplatin on hexokinase activity and autophagic cancer cell death. We found that the two drugs inhibit hexokinase activity and induce autophagic marker, beclin 1 expression. Interestingly, combining As2 O3 with cisplatin synergistically enhanced these effects and alleviated oxidative stress often encountered in As2 O3 treatment. Altogether, our data provide direct evidence that inhibition of hexokinase activity and induction of autophagic cell death are mediating the antineoplastic effects of As2 O3 and cisplatin. Our findings raise the potential of combining As2 O3 with cisplatin as an approach to augment cisplatin-induced cell death and combat cisplatin chemoresistance in cancer.

Influence of copper (I) nicotinate complex and autophagy modulation on doxorubicin-induced cytotoxicity in HCC1806 breast cancer cells

PurposeDoxorubicin is regarded as the most therapeutic active agent available for triple-negative breast cancer (TNBC) treatment. However, the development of drug resistance and toxicity limits its effectiveness. Thus, developing novel strategies for TNBC treatment remains a significant challenge and doxorubicin-based combinations either by metal complexes (Copper I nicotinate complex) or with autophagy modulators could provide novel strategies and alternative strategies contributed to cancer cell death pathways, autophagy and apoptosis. Materials and methodsThe viability of HCC1806 TNBC cells and IC50 values of Doxorubicin (DOX), Torin-1 (TOR), Chloroquine (CQ) and Copper (I) nicotinate complex (CNC) were assessed by MTT assay. ELISA was used for detecting microtubule-associated protein 1 light chain 3 (LC3) level. Real time PCR was used to determine (NBR1) gene expression. Cell cycle analysis and quantitative detection of acid vesicular organelles (AVOs) was performed by flow cytometry. TOR and CQ were used as autophagy modulators for induction and suppression of autophagy, respectively. ResultsThe half-maximal inhibition effect of TOR combination with DOX was revealed to the induction of autophagic cell death and apoptotic cell death. On the other hand, combination of CQ with DOX increased the growth inhibitory effect, induced accumulation of AVOs and suppressed apoptotic cell death. However, combination of CNC with DOX inhibited autophagy and induced cell cycle arrest. ConclusionDoxorubicin drug based combinations either with TOR, CQ or CNC could positively affect DOX effectiveness and reduce DOX doses applied on HCC1806 cells through modulation of autophagy.

MAL-PDT inhibits oral precancerous cells and lesions via autophagic cell death.

Oral cancer is a common cancer with a high mortality rate. While surgery is the most effective treatment for oral cancer, it frequently causes deformity and dysfunction in the orofacial region. In this study, methyl aminolevulinate photodynamic therapy (MAL-PDT) as a prevention tool against progression of precancerous lesion to oral cancer was explored. For in vitro studies, we evaluated the effects of MAL-PDT on viability of DOK oral precancerous cells by XTT, cell morphology by TEM, and intracellular signaling pathways by flow cytometry, Western blotting, and immunofluorescence. For in vivo study, DMBA was used to induce oral precancerous lesions in hamsters followed by MAL-PDT treatment. We measured tumor size and body weight weekly. After sacrifice, buccal pouch lesions were processed for H&E stain and immunohistochemistry analysis. MAL-PDT induced autophagic cell death in DOK oral precancerous cells. The autophagy-related markers LC3II and p62/SQSTM1 and autophagosome formation in DOK cells were increased after MAL-PDT treatment. In vivo, Metvix® -PDT treatment decreased tumor growth and enhanced LC3II expression in hamster buccal pouch tumors induced by DMBA. Our in vitro and in vivo results suggest that MAL-PDT may provide an effective therapy for oral precancerous lesions through induction of autophagic cell death.

The preventive effect of deep sea water on the development of cancerous skin cells through the induction of autophagic cell death in UVB-damaged HaCaT keratinocyte

Ultraviolet light (UV) is a major inducer of skin cancer. Therefore, recovery and removal of UV-damaged skin cells are important in the prevention of skin carcinogenesis. Here, we investigated the effect of deep sea water (DSW) in HaCaT keratinocyte exposed by UVB (λ = 290∼320 nm). The result showed that UVB-induced cell death was reinforced by DSW treatment in a hardness-dependent manner. Furthermore, the increase of cell death by DSW was associated with the down-regulation of survivin and RAD51 expressions induced by UVB. Moreover, we confirmed the inhibition of H2 A.X phosphorylation, a marker for double-stranded DNA damage, and the enhancement of LC3-II and SQSTM1/p62 expressions by DSW administration in UVB-radiated HaCaT keratinocyte. The results imply that the enhancement of UVB-induced cell death by DSW is associated with autophagy. Therefore, we further explored the regulation of autophagy-regulating proteins and apoptosis-related factors expression. Phosphorylation of mammalian target of rapamycin (mTOR), ribosomal protein S6, and S6 kinase by UVB radiation were regressed via DSW treatment, underlying the increase of AMP-activated protein kinase (AMPK) phosphorylation. Furthermore, UVB-enhanced nuclear factor κB (NF-κB) and c-Jun N-terminal kinase (JNK) phosphorylations were increased with DSW treatment. Contrastingly, DSW lessened the Ser15 phosphorylation of p53 and cleavage of poly (ADP-ribose) polymerase induced by UVB radiation. Consequently, the results demonstrate that DSW enhances UVB-damaged skin cell clearance through the activation of autophagic cell death underlying the regulation of AMP-activated protein kinase (AMPK)/mTOR signaling as well as NF-κB and JNK phosphorylations. In conclusion, this investigation suggests that DSW is a potent candidate for the prevention of UV-induced skin cancer development.

Induction of autophagic cell death in human ovarian carcinoma cells by Antrodia salmonea through increased reactive oxygen species generation.

We reported in our previously executed studies that the fermented culture broth of Antrodia salmonea (AS), a mushroom used in Taiwanese folk medicine induced reactive oxygen species (ROS)-mediated apoptosis in human ovarian carcinoma cells. In this study, we studied the anticancer efficacies of AS (0-240 μg/ml) by examining the key molecular events implicated in cell death associated with autophagy in SKOV-3 and A2780 human ovarian carcinoma cells and clarified the fundamental molecular mechanisms. Treatment of ovarian carcinoma cells with AS-induced autophagic cell death mediated by increased microtubule-associated protein LC3-II, GFP-LC3 puncta, and acidic vesicular organelle (AVO) formation. These events are linked with the activation of p62/SQSTM1, the inhibition of ATG4B, the expression of ATG7, and the dysregulation of Beclin-1/Bcl-2 (i.e., B-cell lymphoma 2). N-acetylcysteine inhibited AS-induced ROS generation, which in turn constricted AS-induced LC3 conversion, AVO formation, and ATG4B inhibition, indicating ROS-mediated autophagy cell death. In addition, the 3-methyladenine (3-MA) or chloroquine (CQ)-induced autophagy inhibition decreased AS-induced apoptosis. Additionally, apoptosis inhibition by Z-VAD-FMK, a pan-caspase inhibitor, substantially suppressed AS-induced autophagy. Furthermore, AS-inhibited HER-2/ neu and PI3K/AKT signaling pathways which were reversed by autophagy inhibitors 3-MA and CQ. Thus, A. salmonea is a potential chemopreventive agent that is capable of activating ROS-mediated autophagic cell death in ovarian carcinoma cells.

ONS-donor ligand based Pt(II) complexes display extremely high anticancer potency through autophagic cell death pathway

The current study unveils ONS-donor ligand based Pt(II) complexes with unusual anticancer potency showing higher anticancer effect as compared to cisplatin. This series of Pt(II)(R-salicylaldimine)Cl (C1a-C4a) (R = 5-H, 5-CH3, F, 3-CH3O) complexes were prepared in single step in good isolated yields from commercially available materials. The chloride ancillary ligand of “a” series (C1a-C4a) was replaced with 4-picoline and “b” series of four complexes Pt(II)(R-salicylaldimine)(4-picoline)BF4 (C1b-C4b) (R = 5-H, 5-CH3, F, 3-CH3O) was obtained. All these complexes were characterized by different structure elucidation techniques. Among these, the structures of C1a, C2a, C2b and C3b were determined in solid state by single crystal X-ray analysis. We found quick aquation of “a” series of complexes in DMSO/water mixture that was well investigated by 1H NMNR, LCMS and ESI-MS, while “b” series of these complexes was quite stable over a month as described by the 1H NMNR in DMSO/D2O mixture. This ONS-donor ligand based class of Pt(II) complexes showed unusual anticancer potency in non-small cell lung cancer A549, colorectal cancer HT-29 and triple negative breast cancer MDA-MB-231 cells. These Pt(II) complexes induced PARP cleavage and significantly inhibited colony formation ability of cancer cells. Mechanistically, we found reduced aggressive growth of cancer cells by the induction of autophagic cell death via LC3-I/LC3-II expression and recruitment of LC3B to autophagosomal membrane. These complexes induced p21 expression, that suggested their potentials to suppress cell cycle progression. Significant activation of Caspase3/7-dependent apoptotic signaling was observed in cancer cells treated with these Pt(II) complexes. Morphological changes of cancer cells suggested their potentials to modulate epithelial-mesenchymal-transition (EMT) like features of cancer cells. Gel electrophoresis study revealed their interaction with plasmid DNA. Similarly, strong growth retardation effect and filamentous morphology was observed in Escherichia coli (E. coli). These ONS-donor Pt(II) complexes possessed strong anticancer effect in multiple human cancer cells via activation of multiple pathways for apoptotic and autophagic cell death.

Cold PSM, but not TRAIL, triggers autophagic cell death: A therapeutic advantage of PSM over TRAIL.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and cold plasma-stimulated medium (PSM) are promising novel anticancer tools due to their strong anticancer activities and high tumor-selectivity. The present study demonstrated that PSM and TRAIL may trigger autophagy in human malignant melanoma and osteosarcoma cells. Live-cell imaging revealed that even under nutritional and stress-free conditions, these cells possessed a substantial level of autophagosomes, which were localized in the cytoplasm separately from tubular mitochondria. In response to cytotoxic levels of PSM, the mitochondria became highly fragmented, and aggregated and colocalized with the autophagosomes. The cytotoxic effects of PSM were suppressed in response to various pharmacological autophagy inhibitors, including 3-methyladenine (3-MA) and bafilomycin A1, thus indicating the induction of autophagic cell death (ACD). Lethal levels of PSM also resulted in non-apoptotic, non-autophagic cell death in a reactive oxygen species-dependent manner under certain circumstances. Furthermore, TRAIL exhibited only a modest cytotoxicity toward these tumor cells, and did not induce ACD and mitochondrial aberration. The combined use of TRAIL and subtoxic concentrations of 3-MA resulted in decreased basal autophagy, increased mitochondrial aberration, colocalization with autophagosomes and apoptosis. These results indicated that PSM may induce ACD, whereas TRAIL may trigger cytoprotective autophagy that compromises apoptosis. To the best of our knowledge, the present study is the first to demonstrate that PSM can induce ACD in human cancer cells. These findings provide a rationale for the advantage of PSM over TRAIL in the destruction of apoptosis-resistant melanoma and osteosarcoma cells.

ER stress-mediated autophagic cell death induction through methylated β-cyclodextrins-threaded acid-labile polyrotaxanes

Autophagy plays a pivotal role in the development and prevention of numerous diseases, and the induction of autophagy is regarded as a potential therapeutic approach for intractable diseases. In this study, the induction of autophagy by methylated β-cyclodextrins (Me-β-CDs)-threaded acid-labile polyrotaxane (Me-PRX) that can release the threaded Me-β-CDs in response to acidic pH in lysosomes was investigated. We hypothesized that the Me-β-CDs released from the Me-PRX interact with the membrane of organelles and cause autophagy. The Me-PRX preferentially accumulated in endoplasmic reticulum (ER) and caused ER stress, which was confirmed by gene expression analysis and the expression of an ER stress-marker protein. Accompanying the ER stress, cells treated with Me-PRX showed autophagy, which was not observed in cells treated with non-labile Me-PRX, other chemically modified PRXs, or free Me-β-CD. Furthermore, the Me-PRX treatment induced autophagic cell death and caused cell death even in apoptosis-resistant cells. Overall, this study demonstrates that the acid-labile Me-PRX induces ER stress-mediated autophagic cell death, and the Me-PRX would be a promising candidate to induce effective cell death in apoptosis-resistant malignant tumors.

Glycyrrhizic acid induces human MDA-MB-231 breast cancer cell death and autophagy via the ROS-mitochondrial pathway.

Glycyrrhizic acid (GA), the main component of licorice root extracts, has been shown to suppress cell proliferation and induce apoptosis in various types of cancers. However, the molecular mechanism of its anticancer activity remains poorly understood and warrants further investigation. MDA-MB‑231 cells were treated with various concentrations of GA and the cytotoxic effects of GA were determined using the CCK-8 assay. Apoptosis and cell cycle status were detected by flow cytometry. Reactive oxygen species (ROS) levels and mitochondrial membrane potential (∆Ψm) were assessed using DCFDA, MitoSOX and JC-1 staining. Western blot analysis was used to quantify the expression of autophagy-related proteins. In the present study, induction of autophagic cell death was observed in GA-treated MDA-MB‑231 cells. Downregulation of p62-and beclin1-associated proteins occurred after GA treatment, and, the conversion of LC3 and increased ROS without significant changes in caspase‑associated proteins and intracellular responses were detected. Furthermore, loss of mitochondria and its membrane potential in cells demonstrated that mitochondria were involved in the GA-regulated MDA-MB-231 cell death. The addition of a pan-caspase inhibitor (z-VAD-fmk) did not suppress the GA-induced apoptotic effect, and GA-induced apoptosis was not accompanied by processing of procaspase-8,-9and-3. However, GA triggered the translocation of the apoptosis-inducing factor (AIF) from the mitochondria into the nucleus. In contrast, GA-induced LC3 conversion was significantly inhibited by 3methlyadenine (3MA), an inhibitor of PI3K‑regulated autophagy. Therefore, these results suggest that enhancement of both AIF-andLC3-dependent GA-derived ROS generation plays an important role in the inhibition of the growth of MDA-MB-231 human breast cancer cells.

Combing oncolytic adenovirus expressing Beclin-1 with chemotherapy agent doxorubicin synergistically enhances cytotoxicity in human CML cells in vitro.

Cancer virotherapy provides a new strategy to treat cancer that can directly kill cancer cells by oncolysis. Insertion of therapeutic genes into the genome of a modified adenovirus, thereby creating a so-called gene-virotherapy that shares the advantages of gene therapy and virotherapy. In this study we investigated whether a strategy that combines the oncolytic effects of an adenoviral vector with the simultaneous expression of the autophagy gene Beclin-1 offered a therapeutic advantage for chronic myeloid leukemia (CML) cells with resistance to chemotherapy and evaluated the synergistic effects of SG511-BECN and doxorubicin (Dox) in human CML cells in vitro. Oncolytic virus SG511-BECN was constructed through introducing the Beclin-1 gene into the oncolytic adenoviral backbone. SG511-BECN displayed significantly improved antileukemia activity on multidrug-resistant CML cell line K562/A02, which was mediated via induction of autophagic cell death. Furthermore, Dox could synergize with SG511-BECN to kill the CML cells by improving the infectious efficiency of the oncolytic adenovirus without causing significant damage to normal human mononuclear cells. The results demonstrate that targeting the autophagic cell death pathway and combination of a chemotherapy agent with oncolytic adenovirus may be a novel strategy for the treatment of leukemia with chemotherapy resistance.

Traditional Chinese medicine Danggui Buxue Tang inhibits colorectal cancer growth through induction of autophagic cell death.

PurposeThe induction of autophagic cell death is an important process in the development of anticancer therapeutics. We aimed to evaluate the activity of the ancient Chinese decoction Danggui Buxue Tang (DBT) against colorectal cancer (CRC) and the associated autophagy-related mechanism.Materials and methodsCT26 CRC cells were implanted into syngeneic BALB/c mice for the tumor growth assay. DBT extracts and DBT-PD (polysaccharide-depleted) fractions were orally administered. The toxicity profiles of the extracts were analyzed using measurements of body weight, hemogram, and biochemical parameters. The morphology of tissue sections was observed using light and transmission electron microscopy. Western blotting and small interference RNA assays were used to determine the mechanism.ResultsDBT-PD and DBT, which contained an equal amount of DBT-PD, inhibited CT26 syngeneic tumor growth. In the tumor specimen, the expression of microtubule-associated proteins 1A/1B light chain 3B (LC3B) was upregulated by DBT-PD and DBT. The development of autophagosomes was observed via transmission electron microscopy in tumors treated with DBT-PD and DBT. In vitro experiments for mechanism clarification demonstrated that DBT-PD could induce autophagic death in CT26 cells accompanied by LC3B lipidation, downregulation of phospho-p70s6k, and upregulation of Atg7. RNA interference of Atg7, but not Atg5, partially reversed the effect of DBT-PD on LC3B lipidation and expression of phospho-p70s6k and Atg7. The changes in ultrastructural morphology and LC3B expression induced by DBT-PD were also partially blocked by the knockdown of Atg7 mRNA.ConclusionDBT induced autophagic death of colorectal cancer cells through the upregulation of Atg7 and modulation of the mTOR/p70s6k signaling pathway.

Survivin inhibitor YM155 inhibits proliferation of hepatocellular carcinoma cells by inducing autophagy

Objective To investigate the effects of YM155, a small molecule Survivin inhibitor, on the proliferation of hepatocellular carcinoma (HCC) cells and to explore its molecular mechanism. Methods After HCC cell line SK-Hep-1 was treated with different concentrations of YM155 (0-60 nmol/L), cell proliferation rate of 24, 48 h was assessed by methyl thiazol tetrazolium (MTT), dead cells of 60 h were examined by trypan blue exclusion. SK-Hep-1 cells were treated with 20 nmol/L YM155 for 12 h and 24 h, the expression of Beclin 1, LC3 Ⅰ/Ⅱ, B cell lymphoma/leukemia-2 (bcl-2) and myeloid cell leukemia-1 (Mcl-1) were detected by Western blotting. Results MTT showed that YM155 (0-60 nmol/L)inhibit the proliferation of HCC cells in time and concentration-dependent manner. After treatment with 2 nmol/L and 4 nmol/L YM155 for 60 h, trypan blue exclusion assay showed, dead cells rates were 45% and 90% respectively (P=0.025) relative to the control group [dimethylsurfoxide (DMSO)]; Western blotting results showed that, after SK-Hep-1 cells were treated with 20 nmol/L YM155 for 12 h and 24 h, Beclin 1 and LC3 Ⅱ protein expression levels were increased, while Mcl-1 expession was downregulated, but bcl-2 expression was not altered. Conclusion YM155 significantly suppresses HCC cell proliferation and induced autophagy. Anti-cancer activities of YM155 may be associated with downregulation of Mcl-1 and induction of autophagic cell death. Key words: YM155; Hepatocellular carcinoma; Autophagy; Myeloid cell leukemia-1 gene

Memantine Induces NMDAR1-Mediated Autophagic Cell Death in Malignant Glioma Cells.

ObjectiveAutophagy is one of the key responses of cells to programmed cell death. Memantine, an approved anti-dementia drug, has an antiproliferative effect on cancer cells but the mechanism is poorly understood. The aim of the present study was to test the possibility of induction of autophagic cell death by memantine in glioma cell lines.MethodsGlioma cell lines (T-98 G and U-251 MG) were used for this study.ResultsThe antiproliferative effect of memantine was shown on T-98 G cells, which expressed N-methyl-D-aspartate 1 receptor (NMDAR1). Memantine increased the autophagic-related proteins as the conversion ratio of light chain protein 3-II (LC3-II)-/LC3-I and the expression of beclin-1. Memantine also increased formation of autophagic vacuoles observed under a transmission electron microscope. Transfection of small interfering RNA (siRNA) to knock down NMDAR1 in the glioma cells induced resistance to memantine and decreased the LC3-II/LC3-I ratio in T-98 G cells.ConclusionOur study demonstrates that in glioma cells, memantine inhibits proliferation and induces autophagy mediated by NMDAR1.

Ubenimex enhances the radiosensitivity of renal cell carcinoma cells by inducing autophagic cell death.

Renal cell carcinoma (RCC) is resistant to standard radiotherapy. Ubenimex, an aminopeptidase N inhibitor, is widely used as an adjunct therapy after surgery to enhance the function of immunocompetent cells and confer antitumor effects. Our previous study demonstrated that ubenimex induces autophagic cell death in RCC cells. Recently, the molecular mechanism of autophagy induction has been associated with radiosensitivity in RCC cells. In the present study, the ability of ubenimex to enhance RCC cell sensitivity to radiation via the induction of autophagic cell death was determined, and the mechanism of action of this effect was investigated. The 786-O and OS-RC-2 human RCC cell lines were treated with 0.5 mg/ml ubenimex and different doses of irradiation (IR). The cell viability was measured using a colony-formation assay and flow cytometry. Acridine orange (AO)-ethidium bromide (EB) staining was assessed by fluorescence microscopy as an indicator of autophagic cell death. Protein expression was assessed by western blotting. Autophagosomes were evaluated using transmission electron microscopy. RCC cells were used to evaluate the sensitivity to radiation using clonogenic survival and lactate dehydrogenase assays. Furthermore, these parameters were also tested at physiological oxygen levels. The AO-EB staining and flow cytometry of the OS-RC-2 cells indicated that the combined treatment significantly enhanced autophagic cell death compared with ubenimex or IR alone. Therefore, treatment with ubenimex did not significantly alter cell cycle progression but increased cell death when combined with radiation. An Akt agonist could significantly weaken this effect, indicating that ubenimex may act as an Akt inhibitor. Furthermore, the western blot analysis indicated that the combined treatment inhibited the Akt signaling pathway compared with ubenimex treatment or IR alone. Ubenimex may enhance RCC cell sensitivity to radiation by inducing cell autophagy. This induction changes the role of autophagy from protective to lethal in vitro, and this switch is associated with the inhibition of the Akt signaling pathway.

Erratum to: Citrus unshiu leaf extract containing phytol as a major compound induces autophagic cell death in human gastric adenocarcinoma AGS cells

The pharmaceutical potential of the methanolic extract of Citrus unshiu leaves (MECL) was assessed through analysis of its inhibitory effect on cancer cells. The antiproliferative activities of the leaves were evaluated using several cancer cell lines and considerable cytotoxicity was observed in human gastric adenocarcinoma AGS cells. Inhibition of AGS cell viability was both time- and dose-dependent, and MECL induced non-apoptotic cell death. AGS cells treated with MECL increased the formation of acidic vesicular organelles and GFP-LC3 puncta. Pretreatment with an autophagy inhibitor, 3-methyladenine, inhibited MECL-induced cell death. These results indicated that the mechanism underlying the anticancer effects of MECL in AGS cells could be via the induction of autophagic cell death. The major compounds of MECL were identified as phytol, 4-ethenyl-2-methoxyphenol, hexadecanoic acid, 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one, and vitamin E using gas chromatography–mass spectrometry. These results indicate that C. unshiu leaves can be exploited for numerous pharmaceutical applications as a source of anticancer ingredients.

Synergistic chemopreventive effects of curcumin and berberine on human breast cancer cells through induction of apoptosis and autophagic cell death.

Curcumin (CUR) and berberine (BBR) are renowned natural compounds that exhibit potent anticancer activities through distinct molecular mechanisms. However, the anticancer capacity of either CUR or BBR is limited. This prompted us to investigate the chemopreventive potential of co-treatment of CUR and BBR against breast cancers. The results showed that CUR and BBR in combination synergistically inhibited the growth of both MCF-7 and MDA-MB-231 breast cancer cells than the compounds used alone. Further study confirmed that synergistic anti-breast cancer activities of co-treatment of these two compounds was through inducing more apoptosis and autophagic cell death (ACD). The co-treatment-induced apoptosis was caspase-dependent and through activating ERK pathways. Our data also demonstrated that co-treatment of CUR and BBR strongly up-regulated phosphorylation of JNK and Beclin1, and decreased phosphorylated Bcl-2. Inhibition of JNK by SP600125 markedly decreased LC3-II and Beclin1, restored phosphorylated Bcl-2, and reduced the cytotoxicity induced by the two compounds in combination. These results strongly suggested that JNK/Bcl-2/Beclin1 pathway played a key role in the induction of ACD in breast cancer cells by co-treatment of CUR and BBR. This study provides an insight into the potential application of curcumin and berberine in combination for the chemoprevention and treatment of breast cancers.

TSG101 Silencing Suppresses Hepatocellular Carcinoma Cell Growth by Inducing Cell Cycle Arrest and Autophagic Cell Death.

BackgroundThe tumor susceptibility gene 101 (TSG101) was originally identified as a tumor-suppressor gene that mediates many molecular and biological processes, such as ubiquitination, endosomal trafficking, cell survival, and virus budding, but its role in hepatocellular carcinoma (HCC) is currently unknown.Material/MethodsWe assessed the expression of TSG101 in HCC and paracancerous tissues using qPCR. Then, we used the TSG101-specific siRNA mix to disrupt the expression of TSG101 to investigate the subsequent effect on human hepatoma-7 (Huh7) cells. Western blot was used to detect the protein expression of TSG101 and other molecules. Cell growth assay was performed using CCK8. Transwell assay was used to investigate the migration and invasion ability of Huh7 cells after transfection with of TSG101 siRNA. Flow cytometry was used to estimate the effect of TSG101 knockdown on cell cycle and apoptosis. Confocal laser scanning microscopy was used to observe the actin filaments change and the formation of autophagy.ResultsTSG101 was over-expressed in HCC tissues. TSG101 silence was able to suppress Huh7 cell proliferation, migration, and invasion. Furthermore, silencing of TSG101 could induce cell cycle arrest at G1 phase and inhibit the expression of cyclin A and cyclin D, while up-regulating the expression of CDK2. The mechanism might be induction of autophagic cell death and inactivation of Akt and ERK1/2.ConclusionsTSG101 plays an important role in the development of HCC and may be a target for molecular therapy.

6-Shogaol Inhibits Breast Cancer Cells and Stem Cell-Like Spheroids by Modulation of Notch Signaling Pathway and Induction of Autophagic Cell Death.

Cancer stem cells (CSCs) pose a serious obstacle to cancer therapy as they can be responsible for poor prognosis and tumour relapse. In this study, we have investigated inhibitory activity of the ginger-derived compound 6-shogaol against breast cancer cells both in monolayer and in cancer-stem cell-like spheroid culture. The spheroids were generated from adherent breast cancer cells. 6-shogaol was effective in killing both breast cancer monolayer cells and spheroids at doses that were not toxic to noncancerous cells. The percentages of CD44+CD24-/low cells and the secondary sphere content were reduced drastically upon treatment with 6-shogaol confirming its action on CSCs. Treatment with 6-shogaol caused cytoplasmic vacuole formation and cleavage of microtubule associated protein Light Chain3 (LC3) in both monolayer and spheroid culture indicating that it induced autophagy. Kinetic analysis of the LC3 expression and a combination treatment with chloroquine revealed that the autophagic flux instigated cell death in 6-shogaol treated breast cancer cells in contrast to the autophagy inhibitor chloroquine. Furthermore, 6-shogaol-induced cell death got suppressed in the presence of chloroquine and a very low level of apoptosis was exhibited even after prolonged treatment of the compound, suggesting that autophagy is the major mode of cell death induced by 6-shogaol in breast cancer cells. 6-shogaol reduced the expression levels of Cleaved Notch1 and its target proteins Hes1 and Cyclin D1 in spheroids, and the reduction was further pronounced in the presence of a γ-secretase inhibitor. Secondary sphere formation in the presence of the inhibitor was also further reduced by 6-shogaol. Together, these results indicate that the inhibitory action of 6-shogaol on spheroid growth and sustainability is conferred through γ-secretase mediated down-regulation of Notch signaling. The efficacy of 6-shogaol in monolayer and cancer stem cell-like spheroids raise hope for its therapeutic benefit in breast cancer treatment.