title: Enhanced antitumor effects of adenoviral-mediated siRNA against GRP78 gene on adenosine-induced apoptosis in human hepatoma HepG2 cells.
abstract: Our previous studies show that adenosine-induced apoptosis is involved in endoplasmic reticulum stress in HepG2 cells. In this study, we have investigated whether knockdown of GRP78 by short hairpin RNA (shRNA) increases the cytotoxic effects of adenosine in HepG2 cells. The adenovirus vector-delivered shRNA targeting GRP78 (Ad-shGRP78) was constructed and transfected into HepG2 cells. RT-PCR assay was used to determine RNA interference efficiency. Effects of knockdown of GRP78 on adenosine-induced cell viabilities, cell-cycle distribution and apoptosis, as well as relative protein expressions were determined by flow cytometry and/or Western blot analysis. The intracellular Ca2+ concentration was detected by laser scanning confocal microscope. Mitochondrial membrane potential (ΔΨm) was measured by a fluorospectrophotometer. The results revealed that GRP78 mRNA was significantly downregulated by Ad-shGRP78 transfection. Knockdown of GRP78 enhanced HepG2 cell sensitivity to adenosine by modulating G0/G1 arrest and stimulating Bax, Bak, m-calpain, caspase-4 and CHOP protein levels. Knockdown of GRP78 worsened cytosolic Ca2+ overload and ΔΨm loss. Knockdown of caspase-4 by shRNA decreased caspase-3 mRNA expression and cell apoptosis. These findings indicate that GRP 78 plays a protective role in ER stress-induced apoptosis and show that the combination of chemotherapy drug and RNA interference adenoviruses provides a new treatment strategy against malignant tumors.
authors.affiliation: Department of Gastroenterology and Information, Second Affiliated Hospital, Shantou University Medical College, Shantou 515041, China. lingfeiwu@21cn.com.
authors.forename: Ling-Fei
authors.initials: LF
authors.lastname: Wu
authors.affiliation: Department of Gastroenterology and Information, Second Affiliated Hospital, Shantou University Medical College, Shantou 515041, China. yitian7000@163.com.
authors.forename: Yi-Tian
authors.initials: YT
authors.lastname: Guo
authors.affiliation: Department of Gastroenterology and Information, Second Affiliated Hospital, Shantou University Medical College, Shantou 515041, China. zhangqinghua1210@163.com.
authors.forename: Qing-Hua
authors.initials: QH
authors.lastname: Zhang
authors.affiliation: Department of Gastroenterology and Information, Second Affiliated Hospital, Shantou University Medical College, Shantou 515041, China. xiangmengqi2362@126.com.
authors.forename: Meng-Qi
authors.initials: MQ
authors.lastname: Xiang
authors.affiliation: Department of Gastroenterology and Information, Second Affiliated Hospital, Shantou University Medical College, Shantou 515041, China. 07wdeng1@stu.edu.cn.
authors.forename: Wei
authors.initials: W
authors.lastname: Deng
authors.affiliation: Department of Gastroenterology and Information, Second Affiliated Hospital, Shantou University Medical College, Shantou 515041, China. gnmu2002@126.com.
authors.forename: Yan-Qing
authors.initials: YQ
authors.lastname: Ye
authors.affiliation: Department of Gastroenterology and Information, Second Affiliated Hospital, Shantou University Medical College, Shantou 515041, China. puzejin@sina.com.
authors.forename: Ze-Jin
authors.initials: ZJ
authors.lastname: Pu
authors.affiliation: Department of Gastroenterology and Information, Second Affiliated Hospital, Shantou University Medical College, Shantou 515041, China. jlfeng6@tom.com.
authors.forename: Jia-Lin
authors.initials: JL
authors.lastname: Feng
authors.affiliation: Department of Gastroenterology and Information, Second Affiliated Hospital, Shantou University Medical College, Shantou 515041, China. guanyouhuang@hotmail.com.
authors.forename: Guan-You
authors.initials: GY
authors.lastname: Huang
mesh_terms: D000241:Adenosine / Q000633:toxicity*; D000256:Adenoviridae / Q000235:genetics; D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology; D053471:Caspases, Initiator / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D000091342:Endoplasmic Reticulum Chaperone BiP; D059585:G1 Phase Cell Cycle Checkpoints / Q000187:drug effects; D005822:Genetic Vectors / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D051742:Transcription Factor CHOP / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; C497249:DDIT3 protein, human; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051742:Transcription Factor CHOP; C093516:CASP4 protein, human; D053471:Caspases, Initiator; D000241:Adenosine