| ObjectiveTao’s studies suggest that, apoptosis occurs only after the two events have taken place that mitotic spindle assembly checkpoint (SAC) caused M-arrest and M-slippage in cell cycle regulation. CDK1 inhibitors can accelerate the M-arrested cell forward to M-slippage, and to enter the irreversible apoptotic pathway. Evodiamine is the main active substances of traditional Chinese medicine Evodia. The role of our group preliminary study confirmed that:Evodiamine has a significant role in inducing tumor cell apoptosis, and tumor cell apoptosis induced in G2/M phase, with the existence of M-arrest. In addition, evodiamine joint indirubin and its derivatives or RO3306 (of CDK1 inhibitors) does have a synergistic killing effect. On this basis, the M-arrest and M-slippage molecular mechanisms of the combined effects of evodiamine and CDK1 inhibitors is to further explore in this study. Shed light on this theory provide a scientific basis for the clinical treatment of the tumor. It lays the foundation to further search for more Chinese medicines for cell cycle and to develop the theory of the combination of traditional Chinese medicine point of the study to the mechanism of drug action. It has a far-reaching impact on traditional Chinese medicine modernization.MethodsExpression of cyclinB1, cyclinE, bax, bal-2 and other proteins is detected by Western Blot to observed how evodiamine impact on human hepatoma cell line HepG2. FCM method detects the cell cycle caused by the drug effects after different times, in order to determine the time of the M-slippage occurred at.Expression of cyclinB1, cyclinE, bax, bal-2 and other proteins is detected by Western Blot to observed the combined effects of Evodiamine and CDK1 inhibitors. FCM method detects the cell cycle caused by the drugs in combination after different times, in order to determine the time of the M-slippage occurred at after the drugs in combination.Constructing mouse liver tumor model, divided into model group, paclitaxel group, the Evodia group, Qingdai group, Evodia plus Qingdai group to compare the efficacy and to explore the way of dosing, time, dosage, how to achieve maximum synergies.Result1. Results of Evodiamine effects on HepG2 cells:The FCM assays results shows, with 2μmol·L-1 evodiamine interference, in 6-15 h, the percentage content of the G2/M phase cells gradually increased and reached a peak in 15h, in 15-24 h, G2/M phase.cell percentage decreased gradually, G1 and S phase cell percentage content start to rise, and the apoptosis rate began to increase too.2. Results of paclitaxel combined with CDK1 inhibitor impact on HepG2 cells:MTT assay showed that the combination of PTX and RO3306 can significantly enhance the tumor inhibitory rate, of which value of q in the dose of PTX 0.2μmol·L-1 and RO3306 2μmol·L-1 combined is greater than 1.15, showing that both the Joints synergies. FCM results showed that PTX enables cell cycle arrest in G2/M phase. G1 phase cells decreased with increasing PTX concentration, but the G2/M phase cells and apoptotic rates are increased with concentration-dependent. RO3306 have a certain amount of S phase or G2/M phase arrest and low rate of apoptosis. HepG2 cell apoptosis rate after PTX and RO3306 joint is greater than the two alone, of the value of q is greater than 1.15. In the combined group, with PTX concentration increases, the G1 phase cells reduced, while the G2/M phase cells and apoptotic rate increase, but the concentration dependence of the rate of apoptosis is not obvious. Western blot showed that compared with the control group, 0.2μmol·L-1 paclitaxel alone after 18h, the expression of bcl-2 protein levels decreased significantly, while bax protein levels increase. CyclinB1 protein increase and cyclinE protein levels were significantly decreased.2μmolL·1 RO3306 alone 6h, bcl-2 protein level was significantly lowered and bax protein levels raise, suggesting that RO3306 induce to apoptosis. Cyclin B1 protein did not change obviously, while the CyclinE increased. Compared with the control group, paclitaxel group and RO3306 group, in the combination group, bcl-2 protein levels were significantly decreased, and bax protein levels were significantly increased, indicating that the combined group induces the strongest apoptotic effect. In addiction, cyclinBl protein expression levels are significantly down, while CyclinE expression levels are not different to the control group, but significantly more than the paclitaxel group.3. Results of the evodiamine joint CDK1 inhibitor effects on HepG2 cells:FCM results showed that the EVO arrest cell cycle in G2/M phase, with the EVO concentration, G1 phase cells decreased, while G2/M phase cells and apoptotic rate increase. The RO3306 have a certain role in S phase or G2/M phase arrest, but a lower rate of apoptosis. The apoptotic rate of the combine is greater than the two alone, of the q values of greater than 1.15, indicating the synergy. In the combination group, with the EVO concentration, G1 phase cells decreased, while G2/M phase cells and apoptotic rate increase.4. Results of animal experiments:Experiment 1:Experiment 2:the inhibition rate of CTX group, the joint sequential group, the joint Anti-sequential group were 85.5%、80.5%、55.3%. Compared with model group, the tumor quality of all the treatment groups is lighter, of both the cyclophosphamide group and the joint sequential group are significantly different(P<0.05), But between the joint anti-sequential group and model group showed no significant difference (P>0.05).Experiment 2:the inhibition rate of CTX group, the Evodia group, the indigo group, the joint sequential group, the joint Anti-sequential group were 96.0%、68.7%、28.5%、65.4%、 69.5%. Compared with model group, the tumor quality of all the treatment groups is lighter, of the cyclophosphamide group is significantly different(P<0.05) and among other groups showed no significant difference (P>0.05). However, the p values of the Evodia group, the joint sequential group and joint anti-sequential group is 0.083,0.095 and 0.084. This suggests that increasing the number of samples may reach significant difference.ConclusionEvodiamine induces HepG2 cells to realize the M-arrest time is about 15h, to enter the irreversible apoptosis time is about 21h. Evodiamine-induced apoptosis of HepG2 cells is related to M-arrest and M-slippage events. Evodiamine joint CDK1 inhibitor RO3306 can collaborate in HepG2 cells. The mechanism is Evodiamine to achieve cells M-arrest first, and then by adding RO3306, cells to accelerate M-slippage, thus synergistic inhibition of cell growth. Animal experiments confirmed that Chinese medicine Evodia joint Qingdai can inhibit liver tumor growth, and sequential combination therapy had a synergistic role. This study is to explore new research methods for clarifying the pharmacology of traditional Chinese medicine, and to provide a scientific basis and reference for the Chinese medicine clinical efficacy and the development of new anti-cancer traditional Chinese medicine. |
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