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Infection with cagA-positive Helicobacter pylori is associated with a higher risk of gastric cancer. The cagA gene product, CagA, is translocated into gastric epithelial cells and perturbs host cellular biological functions. Etoposide, a topoisomerase II inhibitor widely used to couple DNA damage to apoptosis, is a common cytotoxic agent used for advanced gastric cancer. We investigate the effect of CagA on etoposide-induced apoptosis in gastric cancer cells to elucidate whether CagA play a role in gastric carcinogenesis via impairing DNA damage-dependent apoptosis. AGS cell lines stably expressing CagA isolated from H. pylori 26695 strain were established. In the presence of etoposide, viability of parental AGS cells was decreased in a time-and dose-dependent manner, whereas CagA-expressing AGS cells were less susceptible to etoposide induced cell-killing effect. Suppression of etoposide-induced apoptosis was shown in CagA-expressing but not in parental AGS cells by DNA fragmentation, cell cycle, and annexin-V assays. This inhibitory effect of etoposide-induced apoptosis conferred by CagA was also demonstrated in SCM1 and MKN45 gastric cancer cell lines, with two additional chemotherapeutics, 5-FU and cisplatin. The effect of Akt activation on inhibition of etoposide-induced cytotoxicity by CagA was also evaluated. CagA expression and etoposide administration activate Akt in a dose-dependent manner. Enhancement of etoposide cytotoxicity by a PI-3-kinase inhibitor, LY294002, was evident in parental but was attenuated in CagA-expressing AGS cells. CagA may activate Akt, either in the absence or presence of etoposide, potentially contributing to gastric carcinogenesis associated with H. pylori infection and therapeutic resistance by impairing DNA damage-dependent apoptosis. more...

// Kai-Lin Yang 1, 2, 3 , Cheng-Chung Huang 1 , Mau-Shin Chi 1 , Hsin-Chien Chiang 1 , Yu-Shan Wang 1 , Chien-Chung Hsia 4 , Gabor Andocs 5 , Hsin-Ell Wang 2 , Kwan-Hwa Chi 1, 2, 6 1 Department of Radiation Therapy and Oncology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan 2 Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, Taiwan 3 School of Medicine, Fu Jen Catholic University, New Taipei, Taiwan 4 Institute of Nuclear Energy Research, Taoyuan, Taiwan 5 Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Japan 6 Institute of Veterinary Clinical Science, School of Veterinary Medicine, National Taiwan University, Taipei, Taiwan Correspondence to: Kwan-Hwa Chi, email: M006565@ms.skh.org.tw Hsin-Ell Wang, email: hewang@ym.edu.tw Keywords: modulated electro-hyperthermia, conventional capacitive coupling hyperthermia, water bath, cytotoxicity, cell membrane Received: May 03, 2016 Accepted: August 11, 2016 Published: August 20, 2016 ABSTRACT Radiofrequency-induced hyperthermia (HT) treatments for cancer include conventional capacitive coupling hyperthermia (cCHT) and modulated electro-hyperthermia (mEHT). In this study, we directly compared these methods with regard to in vitro cytotoxicity and mechanisms of action under isothermal conditions. Hepatoma (HepG2) cells were exposed to HT treatment (42°C for 30 min) using mEHT, cCHT or a water bath. mEHT produced a much higher apoptosis rate (43.1% ± 5.8%) than cCHT (10.0% ± 0.6%), the water bath (8.4% ± 1.7%) or a 37°C control (6.6% ± 1.1%). The apoptosis-inducing effect of mEHT at 42°C was similar to that achieved with a water bath at 46°C. mEHT also increased expression of caspase-3, 8 and 9. All three hyperthermia methods increased intracellular heat shock protein 70 (Hsp70) levels, but only mEHT greatly increased the release of Hsp70 from cells. Calreticulin and E-cadherin levels in the cell membrane also increased after mEHT treatment, but not after cCHT or water bath. These results suggest that mEHT selectively deposits energy on the cell membrane and may be a useful treatment modality that targets cancer cell membranes. more...

// Kwan-Hwa Chi 1, 2, 4 , Yu-Shan Wang 1, 3 , Yi-Chun Huang 3 , Hsin-Chien Chiang 3 , Mau-Shin Chi 1 , Chau-Hwa Chi 4 , Hsin-Ell Wang 2 , Shang-Jyh Kao 5 1 Department of Radiation Therapy and Oncology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan 2 Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, Taiwan 3 Department of Research and Development, JohnPro Biotech Inc., Taipei, Taiwan 4 Institute of Veterinary Clinical Science, National Taiwan University, Taipei, Taiwan 5 Division of Pulmonary Medicine, Department of Internal Medicine, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan Correspondence to: Kwan-Hwa Chi, email: M006565@ms.skh.org.tw Shang-Jyh Kao, email: M001002@ms.skh.org.tw Keywords: autophagy, rapamycin, chloroquine, chemosensitization, synthetic lethality Received: January 25, 2016 Accepted: July 09, 2016 Published: July 28, 2016 ABSTRACT While combined chemotherapy (CT) with an autophagy inducer and an autophagy inhibitor appears paradoxical, it may provide a more effective perturbation of autophagy pathways. We used two dissimilar cell lines to test the hypothesis that autophagy is the common denominator of cell fate after CT. HA22T cells are characterized by CT-induced apoptosis and use autophagy to prevent cell death, while Huh7.5.1 cells exhibit sustained autophagic morphology after CT. Combined CT and rapamycin treatment resulted in a better combination index (CI) in Huh7.5.1 cells than combined CT and chloroquine, while the reverse was true in HA22T cells. The combination of 3 drugs (triplet drug treatment) had the best CI. After triplet drug treatment, HA22T cells switched from protective autophagy to mitochondrial membrane permeabilization and endoplasmic reticulum stress response-induced apoptosis, while Huh7.5.1 cells intensified autophagic lethality. Most importantly, both cell lines showed activation of Akt after CT, while the triplet combination blocked Akt activation through inhibition of phospholipid lipase D activity. This novel finding warrants further investigation as a broad chemosensitization strategy. more...

Glioblastoma (GBM) cells are characterized by high phagocytosis, lipogenesis, exocytosis activities, low autophagy capacity and high lysosomal demand are necessary for survival and invasion. The lysosome stands at the cross roads of lipid biosynthesis, transporting, sorting between exogenous and endogenous cholesterol. We hypothesized that three already approved drugs, the autophagy inducer, sirolimus (rapamycin, Rapa), the autophagy inhibitor, chloroquine (CQ), and DNA alkylating chemotherapy, temozolomide (TMZ) could synergize against GBM. This repurposed triple therapy combination induced GBM apoptosis in vitro and inhibited GBM xenograft growth in vivo. Cytotoxicity is caused by induction of lysosomal membrane permeabilization and release of hydrolases, and may be rescued by cholesterol supplementation. Triple treatment inhibits lysosomal function, prevents cholesterol extraction from low density lipoprotein (LDL), and causes clumping of lysosome associated membrane protein-1 (LAMP-1) and lipid droplets (LD) accumulation. Co-treatment of the cell lines with inhibitor of caspases and cathepsin B only partially reverse of cytotoxicities, while N-acetyl cysteine (NAC) can be more effective. A combination of reactive oxygen species (ROS) generation from cholesterol depletion are the early event of underling mechanism. Cholesterol repletion abolished the ROS production and reversed the cytotoxicity from QRT treatment. The shortage of free cholesterol destabilizes lysosomal membranes converting aborted autophagy to apoptosis through either direct mitochondria damage or cathepsin B release. This promising anti-GBM triple therapy combination severely decreases mitochondrial function, induces lysosome-dependent apoptotic cell death, and is now poised for further clinical testing and validation. more...

// Ching-Min Lin 1, * , Yu-Ling Lin 2, 3, * , Shu-Yi Ho 3 , Pin-Rong Chen 1 , Yi-Hsuan Tsai 1 , Chen-Han Chung 1 , Chia-Hsiang Hwang 4 , Nu-Man Tsai 5, 6, * , Shey-Cherng Tzou 1, 3 , Chun-Yen Ke 1 , Jung Chang 3 , Yi-Lin Chan 7 , Yu-Shan Wang 8 , Kwan-Hwa Chi 8, * and Kuang-Wen Liao 1, 3, 9, * 1 Institute of Molecular Medicine and Bioengineering, National Chiao Tung University, Hsinchu, Taiwan 2 Center for Bioinformatics Research, National Chiao Tung University, Hsinchu, Taiwan 3 Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan 4 Yung-Shin Pharmaceutical Industry Co., Ltd., Taichung, Taiwan 5 Department of Medical and Laboratory Biotechnology, Chung Shan Medical University, Taichung, Taiwan 6 Clinical Laboratory, Chung Shan Medical University Hospital, Taichung, Taiwan 7 Department of Life Science, Chinese Culture University, Taichung, Taiwan 8 Department of Radiation Therapy and Oncology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan 9 Graduate Institut of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan * These authors contributed equally to this work Correspondence to: Kuang-Wen Liao, email: liaonms@mail.nctu.edu.tw Keywords: biflavonoid, angiogenesis, metastasis, actin polymerization, cAMP response element-binding protein (CREB) Received: February 02, 2016 Accepted: June 29, 2016 Published: July 30, 2016 ABSTRACT 7,7”-Dimethoxyagastisflavone (DMGF), a biflavonoid isolated from Taxus x media cv. Hicksii, induces apoptotic and autophagic cell death. However, whether DMGF suppresses tumor metastasis is unclear. The aim of this study was to investigate the anti-metastatic activities of DMGF on the metastatic processes of melanoma cells in vivo and in vitro . A transwell assay showed that DMGF could effectively attenuate the motility of B16F10 cells, and the results of real-time PCR revealed that DMGF also suppressed the expressions of matrix metalloproteinase-2 (MMP-2). Moreover, DMGF did not influence tube formation but inhibited the migration of endothelial cells. Furthermore, animal models were used to monitor the effects of DMGF on tumor metastasis, and all models showed that DMGF significantly suppressed the metastatic behaviors of B16F10 cells, including intravasation, colonization, and invasion of the lymphatic duct. In addition, DMGF could also reduce the densities of the blood vessels in the tumor area in vivo. Further investigation of the molecular mechanisms of anti-metastatic activity revealed that DMGF can down-regulate the levels of key modulators of the Cdc42/Rac1 pathway to interfere in F-actin polymerization and suppress the formation of lamellipodia by reducing the phosphorylation of CREB. These data suggested that DMGF presents anti-metastatic activities in B16F10 melanoma cells. Here, we demonstrated that DMGF can inhibit the metastasis of highly invasive melanoma cancer cells through the down-regulation of F-actin polymerization. Considering these findings, DMGF may be further developed to serve as a chemoprevention drug for patients with metastatic melanoma. more...