RNAi knockdown of the Akt1 gene increases the chemosensitivity of gastric cancer cells to cisplatin both in vitro and in vivo.

AIM: To examine the in vitro and in vivo effects of a combined treatment of cis-d iamminedichloroplatinum(II) (cisplatin) with downregulation of Akt1 expression in gastric cancer cells. MATERIALS AND METHODS: Lentivirus-mediated RNA interference (RNAi) was used to silence the Akt1 gene. pGCSIL-Akt1 small hairpin RNA (shRNA) was stably transfected into gastric cancer cells (SGC7901 and BGC823). Next, the effects of Akt1 downregulation on the growth and apoptosis of SGC7901 (BGC823) cells in the presence or absence of cisplatin were investigated by real-time polymerase chain reaction (RT-PCR), Western blot analysis, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-d-iphenyltetrazolium bromide) assay, Hoechst assay, flow cytometric analysis of annexin V-FITC/PI staining, and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling). Finally, the effects of downregulation of Akt1 expression on the sensitivity of SGC7901 cells in a tumor xenograft model of cisplatin were also determined. RESULT: Akt1 silencing reduced gastric cancer proliferation and increased cell apoptosis both in vitro and in vivo. The chemosensitivity of SGC7901 (BGC823) cells to cisplatin increased significantly following the downregulation of Akt1 expression, which might be associated with the inactivation of the PI3K/Akt1 signaling pathway, followed by the induced expression of the pro-apoptotic protein Bax and a concomitant decrease of Bcl-2 expression. CONCLUSION: This study confirmed that downregulation of Akt1 reduced chemotherapy tolerance of gastric cancer cells to cisplatin treatment. Thus, Akt1 silencing and cisplatin appear to be an effective combination treatment strategy for gastric cancer.

LY294002 potentiates the anti-cancer effect of oxaliplatin for gastric cancer via death receptor pathway.

AIM: To examine the effects of combined treatment of oxaliplatin and phosphatidylinositol 3'-kinase inhibitor, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) for gastric cancer. METHODS: Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptotic cells were detected by flow cytometric analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. Western blotting and immuno-precipitation were used to examine protein expression and recruitment, respectively. Nuclear factor κB (NFκB) binding activities were investigated using electrophoretic mobility shift assay. Nude mice were used to investigate tumor growth. RESULTS: Treatment with combined oxaliplatin and LY294002 resulted in increased cell growth inhibition and cell apoptosis in vitro, and increased tumor growth inhibition and cell death in the tumor mass in vivo. In MKN45 and AGS cells, oxaliplatin treatment promoted both protein kinase B (Akt) and NFκB activation, while pretreatment with LY294002 significantly attenuated oxaliplatin-induced Akt activity and NFκB binding. LY294002 promoted oxaliplatin-induced Fas ligand (FasL) expression, Fas-associated death domain protein recruitment, caspase-8, Bid, and caspase-3 activation, and the short form of cellular caspase-8/FLICE-inhibitory protein (c-FLIP(S)) inhibition. In vivo, LY294002 inhibited oxaliplatin-induced activation of Akt and NFκB, and increased oxaliplatin-induced expression of FasL, inhibition of c-FLIP(S), and activation of caspase-8, Bid, and caspase-3. CONCLUSION: Combination of oxaliplatin and LY294002 was therapeutically promising for gastric cancer treatment. The enhanced sensitivity of the combined treatment was associated with the activation of the death receptor pathway.

[The expression and role of PTEN in doxorubicin induced gastric cancer cell apoptosis].

OBJECTIVE: To study the expression of PTEN and its significance in doxorubicin-treated gastric cancer cells. METHODS: (1) Gastric cancer BGC-823 cells were treated with doxorubicin. Cell proliferation and apoptosis were evaluated by MTT and flow cytometry. The expression of PTEN at the mRNA and protein level were determined by RT-PCT and Western blot, respectively. (2) The gastric cancer xenografts model was constructed. The apoptosis of gastric cancer xenografts cells was determined by TUNEL. The expression of PTEN at the mRNA and protein level were detected using RT-PCR and Western blot, respectively. (3)BGC-823 cells were transfected with PTEN siRNA before addition of doxorubicin. The proliferation and apoptosis of these cells as well as the expression level of PTEN protein were determined. RESULTS: (1) After administration of doxorubicin, the proliferation of BGC-823 cells was inhibited in a time-dependent manner. (2) Doxorubicin significantly induced apoptosis of BGC-823 cells. (3) Doxorubicin treated BGC-823 cells showed a significant increase in the expression of PTEN at the mRNA and protein level in a time-dependent manner. TUNEL assay also showed a significant increase of apoptosis rate in gastric cancer xenografts treated with doxorubicin compared with control group [(28.11 ± 1.05)% vs (2.78 ± 1.63)%]. The expression of PTEN at the mRNA and protein level in the gastric cancer xenografts were significantly increased after administration of doxorubicin (0.5667 ± 0.0043 vs 0.2217 ± 0.0063, 0.14 ± 0.26 vs 0.04 ± 0.15, P < 0.05). (4) After treated with doxorubicin, the expression of PTEN in siRNA-transfected BGC-823 cells was significantly higher than that in non-transfected BGC-823 cells (P < 0.0001). The apoptosis of PTEN siRNA-transfected BGC-823 cells was significantly decreased compared with non-transfected BGC-823 cells [(10.35 ± 1.04)% vs (31.37 ± 3.58)%, P < 0.05]. CONCLUSION: Doxorubicin can effectively inhibit the growth and induce the apoptosis of BGC-823 gastric cancer cells. Increasing PTEN protein may be one of the main mechanism involved in this effect.