ABSTRACT
This study examined the synergetic effect of class IA Phosphoinositide 3-kinases catalytic subunit p110β knockdown in conjunction with oxaliplatin treatment on colon cancer cells.Down-regulation of p110β by siRA interference and oxaliplatin treatment were applied in colon cancer cell lines HT29,SW620 and HCT116.MTT assay was used to measure the inhibitory effect of p110 knockdown on the proliferation of colon cancer cell lines.SubG1 assay and Annexin-V FITC/PI double-labeling cytometry were applied to detect cell apoptosis.And cell cycle was evaluated by using PI staining and flow cytometry.The expression of caspase 3,cleaved PARP,p-Akt,T-Akt and p 110β was dctermined by western blotting.The results suggested that down-regulation of p110β expression by siRNA obviously reduced cell number via accumulation in G0-G1 phase of the cell cycle in the absence of notablely increased apoptosis in colon cancer cell lines HT29 and SW620 (S phase arrest in -HCT116).Moreover,inhibition of p110β expression increased oxaliplatin-induced cell apoptosis and cell cycle arrest in HT29,HCT116 and SW620 cell lines.In addition,increases of cleaved caspase-3 and cleaved PARP induced by oxaliplatin treatment were determined by immunoblotting in p110β knockdown group compared with normal control group and wild-type group.It is concluded that down-regulated expression of p110β could inhibit colon cancer cells proliferation and result in increased chemosensitivity of colorectal cancer cells to oxaliplatin through augmentation of oxaliplatin-induced cell apoptosis and cell cycle arrest.
ABSTRACT
In order to examine the effect of GRIM 19 on colon cancer cell SW480, the recombinant adenovirus carrying GRIM19 gene was constructed and transfected into SW480 cells. GRIMI9 cDNA was amplified by PCR with the template pcxn2-GRlMl9 and cloned into the shuttle plasmid pAdTrack-CMV. The plasmid pAdTrack-CMV-GRIM19 was linearized by PmeI and homologously recombined with bone plasmid pAdEasy-1 in BJ5183, followed by identification by enzyme diges- tion. After transfection of linearized pAd-GRIM19 with PacI into HEK293 cells, Ad-GRIMI9 was obtained and amplified by 3 circles. SW480 cells were infected with Ad-GRIM19. The apoptosis rate was detected by flow cytometry. Agarose electrophoresis revealed the bands of recombinant plasmids identified by enzyme digestion were in the right range corresponding with expectation. Under the fluorescent microscopy, the package of Ad-GRIM19 in HEK293 cells and the expression of Ad-GRIM19 in SW480 cells were observed. The transfection of Ad-GRIM19 into SW480 cells in-creased the apoptosis rate of SW480 cells as compared with controls. It was concluded that Ad-GRIM19 was successfully constructed and the overexpression of GRIM19 in colon cancer cell lines could promote the apoptotic cell death.