Studies On The In Vitro Anti-Breast Cancer Effects Of Diterpenoids Extracted From Chloranthaceae And Their Mechanisms Of Action Based On Cytoskeleton

Objective: To investigate the inhibitory effect of diterpenoids extracted from Chloranthaceae on proliferation of breast cancer cells and its mechanism of action base on cytoskeleton.Methods: MTT assay was used to detect 29 monomeric compounds extracted from the Chloranthaceae diterpenoids,and monomeric compounds were selected that can obviously effective inhibit the proliferation of MDA-MB-231 and MDA-MB-468 cells in breast cancer,And further explore its anti-breast cancer effect and the mechanism of action.MTT assay was used to observe the effect of different concentrations of compound S-36 B on the proliferation effect of MDA-MB-231 and MDA-MB-468 cells,so that was selected the appropriate dosage concentration.the MDA-MB-231 cell administration group was set to 1.57umol/L,7.89umol/L,63.09umol/L,MDA-MB-468 cell administration group was set to 1.57umol/L,15.77umol/L,63.09umol/L;The SRB method was used to determine the proliferation of MDA-MB-231 and MDA-MB-468 cells in the compound S-36 B administration group;To observed the effect of each drug-administered group on the ability of cell clone formation by plate colony formation assay;Growth curve experiments were used to observe the inhibition effect of MDA-MB-231 and MDA-MB-468 cell proliferation in different concentrations of compound S-36 B at different time.Phalloidin-FITC method was used to label the skeletal protein F-actin in two breast cancer cells,and the effect of compound s-36 b on its morphological changes was observed under an inverted fluorescence microscope;the Immunofluorescence labeling was used to detect the expression of ?-Tubulin protein in two breast cancer cells under the action of compound S-36B;The protein expression of p-Cofilin,p-LIMK1?Akt?p-Akt(Ser473)?p70S6K? p-p70S6K(Thr389)and ?-Tubulin in MDA-MB-231 and MDA-MB-468 cells were further detected by Western Blot.Results: 1.MTT assay results showed that the various concentrations inhibitory rate of compound S-36 B on MDA-MB-231 cells was 16.87%±0.0439?44.22%±0.0777?75.12%±0.0562?80.16%±0.0703?85.05%±0.0098?85.22%± 0.0132(compared with the blank control group,the concentrations was 1.57umol/L,7.89umol/L,23.66umol/L P>0.05,15.77umol/L,47.32umol/L,63.09umol/L P<0.05);the various concentrations inhibitory rate of compound S-36 B on MDA-MB-468 cells was 13.86%± 0.0508?22.09%±0.0799?47.02%±0.0406?64.74%±0.0810?83.51%±0.0293?85.31%±0.0074(compared with the blank control group,the concentrations was 1.57umol/L,7.89umol/L,23.66umol/L P>0.05,15.77umol/L,47.32umol/L,63.09umol/L P<0.05).2.SRB results showed that the each administration group survival rate of compound S-36 B on MDA-MB-231 cells was that 85.83%±0.0409?48.65%±0.0465?17.81%±0.0071,the concentration of the positive drug paclitaxel was 0.003umol/L,the survival rate was 82.61%±0.0360(P<0.05).each administration group survival rate of compound S-36 B on MDA-MB-468 cells was that 81.70%±0.0667?48.85%±0.0436?17.26%±0.0186,the concentration of the positive drug paclitaxel was 0.003umol/L,the survival rate was 83.01%± 0.0399(P<0.01).3.Experimental results of cell plate clone formation showed that the each administration group cloning rate of compound S-36 B on MDA-MB-231 cells was that 29%±0.0508?1.6%± 0.0102?0?0,the concentration of the positive drug paclitaxel was 0.003umol/L,the cloning rate was 1%±0.0037(P<0.01);the each administration group cloning rate of compound S-36 B on MDA-MB-468 cells was that 19.53%±0.0267?0.73%±0.0024?0.07%±0.0009?0,the concentration of the positive drug paclitaxel was 0.003umol/L,the cloning rate 1.06%±0.0033(P<0.01).4.Cell growth curve experiments showed that compound S-36 B can inhibited the proliferation of MDA-MB-231 and MDA-MB-468 cells,both of which were time-dependent and dose-dependent.5.Experimental results of actin filaments F-actin showed that compared with the blank group,under the different concentrations of compound S-36 B,MDA-MB-231 and MDA-MB-468 cytoskeleton protein F-actin showed gradually disordered arrangement,decreased expression and slowly decreased pseudopodia.6.The results of cellular immunofluorescence experiments showed that compared with the blank group,the distribution of ?-Tubulin protein around the nucleus gradually decreased and the fluorescence intensity decreased with the increase of the dosing concentration of MDA-MB-231 and MDA-MB-468 cells..7.Western blot results showed that under the action of compound s-36 b,p-Cofilin,p-LIMK1,Akt and p70S6 K proteins were significantly down-regulated,while p-Akt(Ser473),p-p70s6k(Thr389)and ?-tubulin showed no significant difference.Conclusions: 1.Compound S-36 B shows significant inhibition on the proliferation of MDA-MB-231 cells and MDA-MB-468 cells in vitro.2.Inhibition on proliferation of breast cancer cells induced by compound S-36 B may involve the phosphorylation of LIMK1/Cofilin signaling pathway activity,which can further affect the cytoskeleton architecture by down-regulating the phosphorylation level of LIMK1/Cofilin protein.