The Effects And Studies On The Mechanism Of Petroleum Ether Extract Of Lamiophlomis Rotate On Proliferation Of Human Tongue Squamous Cell Carcinoma Cell Line Tca8113

Objective: 1.To explores the effect of petroleum ether extract of Lamiophlomis rotate on proliferation of Tca8113 cell line.2. To explore the effects of colony formation ability of Petroleum Ether Extract of Lamiophlomis rotate on proliferation of Tca8113 cell line.3. To explore the inducing apoptosis effect of petroleum ether extract of Lamiophlomis rotate on proliferation of Tca8113 cell line.4. To explore cytotoxicity of mouse fibroblast cell line L929 by petroleum ether extract of Lamiophlomis rotate.5. To explore cell cycle distribution of Tca8113 cell line by petroleum ether extract of Lamiophlomis rotate.6. To explore the effect of petroleum ether extract of Lamiophlomis rotate on expression of BAX, Bcl-2 and Casepase 3 protein in Tca8113 cell line.Methods: SRB assay, growth curve, cell clone formation rate and flow cytometry were used to evaluate growth and vitality of Tca8113 cell line, both treated with different concentrations of PEELR with bleomycin(BLM,10 μg/m L) as the drug control group and no treatment group as the blank control group SRB assay was used to explore cytotoxicity of mouse fibroblast cell line L929 by petroleum ether extract of Lamiophlomis rotate(PEELR). Morphologic change of cells, cell number and proportion of apoptosis cells and necrosis cells were observed at the same field of different concentrations of PEELR under Hoechst33258/PI Staining of Tca8113 cell line. Cell cycle analysis was used to see the situation of cycle arrest. Western bolt was used to explore the effect of petroleum ether extract of Lamiophlomis rotate on expression of BAX, Bcl-2 and Casepase 3 protein in Tca8113 cell line.Results: 1. SRB assay and growth curve of Tca8113 cell line showed that the IC50 of Tca8113 cell line under different concentrations of PEELR was 66.388μg/m L; Apoptotic cells was directly proportional to the drug dose; There were no significant differences between low concentration of PEELR(25 μg/m L and 50 μg/m L)and control group(p>0.05). the effect of long-term with PEELR alos showed no significant differences between middle concentration of PEELR(100 μg/m L), high concentration of PEELR(200 μg/m L and 400 μg/m L)and BLM(10 μg/m L)group(p>0.05).2. Significant negative correlation was observed between cell clone formation rate and the concentration of PEELR by cell clone formation. Compared with control group, PEELR have a significant inhibition in colony formation ability of Tca8113 cell line(p<0.05). There was also no significant differences between middle concentration of PEELR(100 μg/m L), high concentration of PEELR(200 μg/m L)and BLM(10 μg/m L)group in colony formation ability of Tca8113 cell line(p>0.05).3. Through Annexin V /PI staining were used to detect the early apoptotic cells by flow cytometry have demonstrated that PEELR has an apparently capability to induce apoptosis, and its’ functions was correlated with drug concentration. Compared with control group, PEELR has a significant different in the apoptotic inducing ability of Tca8113 cell line(p<0.01); but it(50 μg/m L and 100 μg/m L) has no difference with the BLM(10 μg/m L)group(p>0.05). There was an unique significant difference between PEELR and the control group in the proportion of apoptosis cells and necrosis cells(p<0.01). There was a significant difference between PEELR and the BLM(10 μg/m L)group in the proportion of apoptosis cells and necrosis cells(p<0.05); But there was no difference between the different concentrations of PEELR.4. SRB assay of L929 cell line showed that the IC50 of L929 cell line under different concentrations of PEELR was 294.119μg/m L. The inheritance of different concentrations of PEELR and the BLM(10 μg/m L)on the L929 cell line(24 h); There were no significant differences between low concentration of PEELR( 25 μg/m L and 50 μg/m L), middle concentration of PEELR(100 μg/m L), control group and the BLM(10 μg/m L)group(p>0.05). Apoptotic cells was directly proportional to the high concentration of PEELR(200 μg/m L and 400 μg/m L).5. Significant negative correlation was observed between the concentrations of PEELR and cell number by Hoechst33258/PI staining of Tca8113 cell line; but significant positive correlation was observed with the proportion of apoptosis cells and necrosis cells. The PEELR’s normal cells have no significant difference compared with control groups’. Nuclear shrinkage was observered in the PEELR’s apoptosis cells. The proportion of apoptosis cells was more than the proportion of necrosis cells in PEELR group(p<0.01).6. There was a significant negative correlation between the concentrations of PEELR and S phase fraction of Tca8113 cell line; so did the proliferation index of Tca8113 cell line.7. Western blot resluts showed that: The Expression of BAX and Casepase-3 were directly proportional to the concentration of PEELR. There was a significant negative correlation between the concentrations of PEELR and the expression of Bcl-2 of Tca8113 cell line. All of the PEELR could decrease the Bcl-2/ Bax expression ratio, and have a significant differences between each other( p <0.01). There was a significant negative correlation between the concentrations of PEELR and the Bcl-2/ Bax expression ratio of Tca8113 cell line. Only high concentration of PEELR could reduce the Bcl-2/ Bax expression ratio smaller than 1.Conclusion: PEELR may be a better choice to inhibit the growth of Tca8113 cell line for it has a significant inhibition in proliferation ability and an apparently capability to induce apoptosis of Tca8113 cell line with smaller cytotoxicity to mouse fibroblast cell line L929.that might partly be related to the inhibition of the mitochondrial control of apoptosis pathways and the level of BAX, Casepase-3 as well as to the increase of the level of PEELR, but downregulated Bcl-2 expression.The growth and vitality of Tca8113 cell line were decreased with the increase of the concentrations of PEELR, the effects on proliferation in Tca8113 cell line were equal to(P>0.05) to bleomycin(10 μg/m L),when PEELR at a concentration of 100 μg/m L. Meanwhile, the effects on proliferation in L929 cell line were equal to(P>0.05) bleomycin(10 μg/m L). Both of them were almost the same as control group.