Abstract: Objective: To obtain the conditionally replicative adenovirus with triple-targeting Smac overexpression using gene recombination technology,and to explore its effects on the apoptosis and cell cycle progression of MDA-MB-231 cells. Methods: The triple-targeting Smac overexpression vector pShuttle-Egr1-Smac-HRE-hTERT-E1A-E1Bp-E1B55K was constructed by gene recombination technology,which was recombined with the skeleton vector pAdEasy in the BJ5183 bacteria (AdEasy-1+) to obtain the conditionally replicative adenovirus CRAd.pE-Smac.After the MDA-MB-231 cells were infected with CRAd.pE-Smac,the cancer cells were mimiced into hypoxic status with chemical reagent CoCl₂,then control group,CRAd.pE-Smac group,hypoxia group and CRAd.pE-Smac+hypoxia group were set up;the cells were irradiated with 4 Gy X-rays,and each group was divided into non-irradiation group and irradiation group. The Smac protein expression was detected by Western blotting assay,the apoptotic rates and the percentages of cells at different phases were detected by flow cytometry. Results: The Western blotting results showed that the Smac protein expressions were increased after infection of CRAd.pE-Smac, hypoxia and 4 Gy irradiation,especially in CRAd.pE-Smac +hypoxia+4 Gy irradiation group.The FCM results showed that the apoptotic rates in CARd.pE-Smac,hypoxia,CARd.pE-Smac + hypoxia group were increased compared with control group (P<0.05 or P<0.01),and the apoptotic rates of cells irradiated with 4 Gy were significantly increased compared with the unirradiated cells (P<0.05 or P<0.01),especially in CRAd.pE-Smac + hypoxia + 4 Gy irradiation group; the percentages of the cells at S and G₂/M phases in irradiation groups were significantly increased (P<0.05 or P<0.01),which had the similar regularity with the apoptotic change. Conclusion: After the MDA-MB-231 cells are infected with the conditionally replicative adenovirus CRAd.pE-Smac and treated with hypoxia and irradiation,the triple-targeting Smac overexpression can be achieved,and it has the role of promoting the cancer cell apoptosis and inducing the G₂/M arrest.

Key words: hypoxia, second mitochondria-derived activator of caspase, apoptosis, breast neoplasms, radiation