The Effect And Its Mechanism Of CP And SP On Human Small Cell Lung Cancer H446 Cell

There were lots of Pueraria lobata(Willd.) Ohwi growing in DABIE Mountain. Radix Puerariae had kinds of pharmacological action. But it hadn't reported that Radix Puerariae had anti-tumor action abroad. In domestic research, Du Deji had observed Radix Puerariae's anti-tumor action against P388 leukemia, W256 cancer, EAC ascitic tumor, S180 tumor carneus, H22 liver cancer in vitro and anti-tumor action against EAC ascitic tumor, S180 tumor carneus, Lewis lung cancer, H22 liver cancer in vivo. However, it hadn't reported that CP and SP could inhibite the human small cell lung cancer proliferation and its mechanism, especially, which had stonger anti-tumor action between CP (pueraria crude extract)and SP(standard of pure puerarin).Nowadays in a great many countries, lung cancer had been the leading cause of tumor-specific death. Lung cancer was a common fatal disease with highest mobidity and mortality rate among tumors in China. The searching for effective drugs to therapy, or those with potential to prevent lung cancer, was of great importance. In recent years, anti-tumor drug derived from plant drawed more and more research attention for their functions of cancer therapy and chemoprevention.The imbalance of cell proliferation, apoptosis and cell cycle leads to tumor. Thus, anti-proliferation, inducing apoptosis and regulating cell cycle would open a new idea and bright prospects in tumor therapy.Therefore, this study was designed to explore the effect and its mechanism of CP and SP on H446 cell through cell culture in vitro in three aspects of anti-proliferation, inducing apoptosis and regulating cell cycle.ObjectivesThe main objective of this research were to observe the influence of CP and SP on H446 cell proliferation viability, apoptosis rate and cell cycle distribution and to explore its mechanism of anti-proliferation, inducing apoptosis and changing cell cycle distribution.Methods1. H446 cell growth inhibiting rate was detected by MTT assay with different concentrations of CP and SP after 24,48,72,96,120 hours. 2. Apoptosis was observed by light microscope analysis of cell morphology.3. Cell apoptosis rate and cell cycle distribution were analyzed by flow cytometry.4. The molecular mechanisms of anti-proliferation, inducing apoptosis and regulating cell cycle in H446 cell were studied at protein expression level of PCNA,Bax,Bcl-2,Fas,cyclinD1,CDK₂,CDK₄and P27 using immunohistochemistry and flow cytometry.Results1. Compared with DMSO control, both CP and SP effectively inhibited H446 tumor cell proliferation. The effects were a dose-dependent and time-dependent fashion.2. The suppression effect of CP was stronger than that of SP in same situation.3. The typical characters of apoptosis in the tested cells were observed after treatment with CP and SP. The quantitative analysis showed the apoptosis rate induced by CP and SP were respectively 14.71% and 2.61%. There was significant difference between CP and control group.4. Distribution of cell cycle showed CP and SP could change the location of the cell cycle. The percentages of G0/G1 phase cell numbers were respectively 79.20% and 72.20%, while those of G2/M phase were respectively 8.94% and 10.50%. There was significant difference compared with control group.5. The dynamics of cell cycle showed the cell proliferation index of CP and SP group decreased with significant difference compared with control group. The cell proliferation index of CP group was lower than that of SP group with significant difference.6. The expression of PCNA protein markedly decreased after treatment with CP on H446 tumor cell compared with control group.7. The expression proportion of Bax/Bcl-2 protein increased after treatment with CP and SP on H446 tumor cell compared with control group with significant difference. And the expression proportion of Bax/Bcl-2 with CP was higher than that with SP. There was significant difference.8. The expression of Fas protein markedly increased after treatment with CP and SP on H446 tumor cell compared with control group with significant difference. And the expression of Fas with CP was higher than that with SP. There was significant difference.9. The expression of CDK₂ protein markedly decreased after treatment with CP and SP on H446 tumor cell compared with control group with significant difference. And H446 tumor cell expressed cyclinD₁ and CDK₄ proteins negatively, with expression of P27 protein positively. The fluorescence index of cyclinD₁ and CDK₄ after treatment with CP were lower than that with SP, while the fluorescence index of P27 after treatment with CP was higher than that with SP. There were significant difference.Conclusions1. Both CP and SP inhibited H446 tumor cell proliferation. The inhibiting effect of CP was stronger than that of SP.2. CP induced H446 tumor cell apoptosis.3. H446 tumor cell treated with CP and SP were remarkably arrested at G0/G1 phase.4. CP inhibited H446 tumor cell proliferation through down-regulation of PCNA protein.5. CP induced H446 tumor cell apoptosis through up-regulation of expression proportion of Bax/Bcl-2.6. CP induced H446 tumor cell apoptosis through up-regulation of Fas protein.7. Both CP and SP changed H446 tumor cell cycle through down-regulation of cyclinD₁,CDK₂,CDK₄ protein and up-regulation of P27 protein. Thus, the synthesis of cyclinD-CDK₄,cyclinE- CDK₂ compounds were prohibited so that H446 tumor cell were accumulated in G0/G1 phase.