The Anticancer Effect And Mechanisms Of ACM In Oral Squamous Carcinoma KB Cells

Objective:To investigate the in vitro antitumor effect of ACM on the oral squamous carcinoma KB cells and its mechanisms.Methods:1.The in vitro inhibitory activities of ACM on the growth of eight human cancer cell lines were evaluated by MTT assay.2.The morphology of KB cells was observed by DAPI staining,and the effects of ACM on ROS level,cell apoptosis and cell cycle were detected by flow cytometry.3.The expression of different apoptosis-associated proteins and cell cycle regulatory proteins was examined by western blot assay.4.The expression of cell apoptosis genes such as c-Jun,Bim and Puma was detected by Real-time PCR.The nuclear localization of c-Jun was observed by immunofluorescence staining.Results:1.The results of MTT assay showed that ACM exhibited the significant cytotoxicity against HT-29,HepG2,MCF-7,A549,HeLa and KB cells,with the IC₅₀ values of5.40±1.10,3.23±0.56,2.11±1.15,2.57±0.29,3.43±0.44l and 4.32±1.05?g/ml at 48 h,respectively.Furthermore,ACM inhibited the proliferation of KB cells in a time-and dose-dependent manner,and the IC₅₀ values were 18.1±0.44,4.32±1.05 and3.94±0.55?g/ml at 24 h,48 h and 72 h,respectively.2.The results of DAPI staining showed the chromatin condensation and nucleus broke into pieces in KB cells treated with ACM for 48 hours.The flow cytometry analysis indicated that the apoptotic rates and ROS level of KB cells increased significantly in a dose-dependent manner,suggesting that ACM could actually induce apoptosis in KB cells.The analysis of cell cycle indicated that the cell cycle was blocked at G2/M phase.3.The western blotting analysis showed that ACM activated the proteins of pro-caspase-3 and pro-caspase-9,while Bcl-2 was down-regulated Bax and Cyt c was up-regulated in KB cells.Meanwhile,ACM increased the expression of P21 and suppressed the expression of cyclin B1,mitotic phosphatase cdc25 and mitotic kinase cdc2.4.Our investigation also found that the JNK pathway was activated by ACM in a dose-dependent manner.The protein level of its downstream molecule c-Jun was also decreased.Additionally,ACM induced the nuclear localization and transcription of c-Jun.Furthermore,we found the JNK inhibitor?SP600125?could significantly reverse ACM-induced apoptosis and G2/M arrest.Conclusions:ACM can significantly inhibit the growth of human oral squamous carcinoma KB cells.The mechanisms might be involved in the activation of ROS-JNK pathway,promotion of the nuclear localization of c-Jun,as well as regulation of the key apoptosis-related proteins and cell cycle arrest-related proteins to eventually trigger apoptosis and G2/M phase arrest.