BAK overexpression mediates p53-independent apoptosis inducing effects on human gastric cancer cells.
BACKGROUND
BAK (Bcl-2 homologous antagonist/killer) is a novel pro-apoptotic gene of the Bcl-2 family. It has been reported that gastric tumors have reduced BAK levels when compared with the normal mucosa. Moreover, mutations of the BAK gene have been identified in human gastrointestinal cancers, suggesting that a perturbation of BAK-mediated apoptosis may contribute to the pathogenesis of gastric cancer. In this study, we explored the therapeutic effects of gene transfer mediated elevations in BAK expression on human gastric cancer cells in vitro.


METHODS
Eukaryotic expression vector for the BAK gene was constructed and transferred into gastric cancer cell lines, MKN-45 (wild-type p53) and MKN-28 (mutant-type p53). RT-PCR and Western Blotting detected cellular BAK gene expression. Cell growth activities were detected by MTT colorimetry and flow cytometry, while apoptosis was assayed by electronic microscopy and TUNEL. Western Blotting and colorimetry investigated cellular caspase-3 activities.


RESULTS
BAK gene transfer could result in significant BAK overexpression, decreased in vitro growth, cell cycle G0/G1 arrest, and induced apoptosis in gastric cancer cells. In transferred cells, inactive caspase-3 precursor was cleaved into the active subunits p20 and p17, during BAK overexpression-induced apoptosis. In addition, this process occurred equally well in p53 wild-type (MKN-45), or in p53 mutant-type (MKN-28) gastric cancer cells.


CONCLUSIONS
The data presented suggests that overexpression of the BAK gene can lead to apoptosis of gastric cancer cells in vitro, which does not appear to be dependent on p53 status. The action mechanism of BAK mediated apoptosis correlates with activation of caspase-3. This could be served as a potential strategy for further development of gastric cancer therapies.
Tong
Qiang-Song
QS
Department of Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. qs_tong@hotmail.com
Zheng
Li-Duan
LD
Wang
Liang
L
Liu
Jun
J
Qian
Wei
W
D017209:Apoptosis / Q000235:genetics*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000235:genetics; D018014:Gene Transfer Techniques; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D013274:Stomach Neoplasms / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D050998:bcl-2 Homologous Antagonist-Killer Protein
C494772:BAK1 protein, human; D008565:Membrane Proteins; D016159:Tumor Suppressor Protein p53; D050998:bcl-2 Homologous Antagonist-Killer Protein; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases