Experimental Study On Enhanced Anti-Tumor Activity Of Co-Expression By Ad-ING4-OSM Double-Gene Combined With Radiotherapy For Human Laryngeal Cancer In Vitro And In Vivo

Objective: To investigate the synergistic enhanced anti-tumor effects ofco-expression by Ad-ING4-OSM double-gene (Ad-ING4-OSM) combined withradiotherapy on human laryngeal cancer Hep-2cells in vitro and in vivo and itsmolecular mechanism.Methods: Recombinant adenoviral vectors were transfected into QBI-293A cellsfor high-tilter amplification. Ad-GFP was used to infect the human laryngeal cancerHep-2cells in order to determine the most suitable dose(MOI). The apoptotic effectcaused by radiotherapy was evaluated by flow cytometry (FCM),dyed by Annexin-V-PE/7-AAD,in order to determine the most suitable radiotherapy dose.In vitro,the humanlaryngeal cancer Hep-2cells were infected with Ad-ING4-OSM alone or combined withradiotherapy,then the expression of ING4/OSM gene were detected by RT-PCR andWestern blot; The influence of cell growth and apoptotic effect were assessed by MTTassay and FCM(dyed by Annexin-V-PE/7-AAD) respectively; The effects on cell cyclewere evaluated by FCM(dyed by PI); Nuclear morphological changes of Hep-2cellswere detected by Hoechst33258; The expression of P21, P27, P53and Survivin weredetected by semi-quantitative RT-PCR.In vivo, the human laryngeal cancer Hep-2cellstransplanted tumor models were constructed in athymic nude mouses. Aftertreated,measure the size and weight of the tumor,then calculate tumor inhibition rate ofeach group to evaluate its anti-tumor effect. The expression of P21, P53, Bax, Bcl-2,Caspase-3, Cox-2, CD34in the solid tumors in nude mouse model and the possiblemolecular mechanism were tested by immunohistochemistry analysis.Results: Recombinant adenoviral vectors could be amplified in QBI-293A cellssuccessfully; The most suitable dose of Ad-GFP used to infect the human laryngeal cancer Hep-2cells was100MOI; The most suitable radiotherapy dose was6Gy;Exogenous ING4and OSM were proved successful transcribed and translated inlaryngeal cancer Hep-2cells by RT-PCR and Western blot; Ad-ING4-OSM combinedwith radiotherapy could significantly inhibit not only laryngeal cancer Hep-2cells butalso transplanted tumor growth, induce apoptosis. These effects were more significant inAd-ING4-OSM combined with radiotherapy group than that of single radiotherapy orAd-ING4-OSM group(P＜0.05),exhibiting synergetic anti-tumor effect; In vitro,Ad-ING4-OSM combined with radiotherapy could inhibit the cell proliferation by G1and G2/M phase arrest, and had more significantly effect on up-regulations of P21, P27,P53and down-regulation of Survivin. In vivo, the immunohistochemisty resultsrevealed that Ad-ING4-OSM combined with radiotherapy had more marked effect onup-regulating the expression of P21, P53, Bax, Caspase-3and down-regulating theexpression of Cox-2, Bcl-2, CD34,compared with single radiotherapy orAd-ING4-OSM group (P＜0.05).Conclusion:1. In vitro,Ad-ING4-OSM combined with radiotherapy had not only suppressedstatistically the human laryngeal cancer Hep-2cells and induced apoptosis whichexcelled obviously radiotherapy and Ad-ING4-OSM single work(P<0.05),but alsoexpressed synergetic effects of enhanced anti-tumor effect. Ad-ING4-OSM combinedwith radiotherapy could inhibit the cell proliferation by G1and G2/M phase arrest,andhad more significantly effect on up-regulations of P21,P27,P53and down-regulation ofSurvivin，which may be important mechanism involved in its synergetic effect insuppressing laryngea cancer.2. In vivo, Ad-ING4-OSM combined with radiotherapy had suppressed statisticallythe human laryngeal cancer Hep-2cells tumor growth in nude mice which excelledobviously radiotherapy and Ad-ING4-OSM single work, expressing synergetic effectsof enhanced anti-tumor effect. Ad-ING4-OSM combined with radiotherapy had moremarked effect on up-regulating the expression of P21, P53, Bax, Caspase-3anddown-regulating the expression of Cox-2, Bcl-2, CD34compared with radiotherapy orAd-ING4-OSM,which may be responsible for its synergetic enhanced anti-tumor effect on human laryngeal cancer Hep-2cells in vivo in athymic nude mouse model.