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.

Endothelin type B receptor interacts with the 78‐kDa glucose‐regulated protein

“…The infected EA.hy926 cells were selected with 0.5 lgÁmL À1 puromycin. The targeting sequence of the GRP78 shRNA used in the present study was similar to that reported previously [15,16].…”

The mechanism of adenosine-mediated activation of lncRNA MEG3 and its antitumor effects in human hepatoma cells

“…Although MEG3 plays a role in inhibiting tumor growth, no data are available on its function under genotoxic stress-induced apoptosis. In our previous study, we demonstrated that adenosine induces hepatoma cell apoptosis and the mechanism is involved in DNA demethylation and genotoxic effects (15,16). In this study, we treated HepG2 cells with adenosine or 5-Aza-2'-deoxy-cytidine (5-Aza-CdR), a selective DNA methyltransferase inhibitor, and observed the methylation status of MEG3 promoter and MEG3 mRNA expression.…”

“…A total of 1.0x10 5 HCC cells were inoculated into each well of a 6-well plate and treated as described above (16). DNA from the cells was extracted using an EZNA Tissue DNA kit according to the manufacturer's instructions.…”

“…Plasmid identification and cell transfection and bacterial transformation. PcDNA3.1-MEG3 plasmid was constructed as previously described (16). PcDNA3.1-MEG3 plasmids were extracted from E. coli according to the manufacturer's specifications.…”

Mitochondrial calcium: Transport and modulation of cellular processes in homeostasis and cancer (Review)

“…Some of these suppressor proteins directly affect calcium flux; for example, silencing of ER oxidoreductin-1α significantly inhibits calcium release from the ER, inducing cell death (58). A reduction in the Bcl-2/Bcl-2-associated X, apoptosis regulator ratio also leads to ER Ca 2+ depletion, which induces cytosolic Ca 2+ overload and causes the loss of mitochondrial membrane potential (59). IP 3 R-3 is an oncoprotein differentially expressed in colorectal carcinoma that is absent in normal colorectal mucosa (Fig.…”