The Inhibition And The Mechanisms Of Total Alkaloid Of Menispermum On Hela Cells

Objective: To study the effects of Total Alkaloid of Menispermum (TAM) on apoptosis, cell cycle and invasive ablity to the Hela cell. To investigate the antitumor mechanisms of TAM. To produce the scientific evidence for developing and manufacturing new antitumor drugs.Methods: 1 The inhibitory effect on cell growth of Hela was measured by MTT assay in treated or untreated groups (3.125, 6.25, 12.5, 25, 50μg/ml TAM and control) for three different treatment times (24h, 48h and 72h).2 Apoptosis and cell cycle were measured by FCM in four experimental groups (0, 4, 16, 40μg/ml TAM) for 48h.3 Adopting Wright and Giemse's staining to observe the morphology of Hela cells which treated with 40μg/ml TAM.4 Using invasion experiment to detect the Hela cells'invasive abilities which treated with 40μg/ml TAM.5 The protein expressional levels of P-ERK, ERK, C-myc and Cyclin D1 in Hela cells untreated or treated with 4, 16, 40μg/ml TAM for 24h were measured by Western blot.6 Expression of anti-apoptotic gene bcl-2, apoptotic gene bax and MMP-9 in Hela cells of four experimental groups (0, 4, 16, 40μg/ml TAM for 24h), were observed by revers transcription PCR (RT-PCR).7 The protein expression of P-ERK, ERK, Bcl-2 and Bax in Hela cells treated with 40μg/ml TAM for 24h observed by laser scanning microscopes (LSM).Results: 1 TAM could significantly inhibit the growth of Hela cells in vitro (p<0.05). The growth inhibition was in dose-dependent. The greatest inhibit rate was 85.37% observed in the treatment group of Hela cells by 50μg/ml TAM for 72 hours.2 The results of FCM showed that TAM can induce apoptosis and cell cycle arrest of Hela cells. After treated by TAM for 48h, the apoptosis rate of Hela cells increased markely with a dose-dependent manner (p<0.05). Meanwhile, after treated with TAM, the cell cycles of Hela cells were changed, the cells in G₀/G₁ phase were increased and cells in S phase were decreased.3 Wright and Giemsa's staining revealed that the Hela cells treated by TAM had had proapoptotic morphology.4 The results of invasion experiment showed that the invasive ability of Hela cells treated with TAM had decreased remarkable (p<0.05), compared with the contral group.5 The results of western blotting revealed that TAM had down- regulated the level of phopholated ERK, C-myc and CyclinD1 expression in Hela cells in treated group with 4, 16, 40μg/ml TAM for 24 hours, compared with the control group (untreated) (p<0.05),but the level of ERK expression didn't change significantly. 6 TAM inhibited the mRNA expression of the bcl-2 and MMP- 9, increased the mRNA expression of apoptotic gene bax in Hela cells treated by 4, 16, 40μg/ml periplocin for 24 hours compared with untreated cells (p<0.05).7 LSM images show that after treated with 40μg/ml TAM, the fluorescence intensity of P-ERK, Bcl-2 were significantly weaker, and fluorescence intensity of Bax was more increased than that in control group. but fluorescence intensity of ERK was no significant change.Conclusion: 1 TAM could inhibit significantly proliferation of the human cervix cancer Hela cells in vitro in dose-dependent and time-dependent manner.2 In the Hela cells, TAM could repress the ERK1/2 signaling pathway through downregulating the expression of P-ERK.3 TAM could inhibit significantly the expression of bcl-2 ,MMP-9 mRNA, increase the expression of bax mRNA, affect the expression of C-myc and CyclinD1, accordingly, the treatment of TAM induced apoptosis , arrested the cell cycle in G₀/G₁ phase and debilitated invasion ability of Hela cells in vitro.