The Effects Of Apigenin On Cell Growth And Toxicity In Combination With Cisplatin In Non-small Cell Lung Cancer Cells

In this study,the anti-proliferation activities of apigenin and cisplatin on 6 types of non-small cell lung cancer(NSCLC)cell lines(A549,SPC-A-1,H1299,H1650,LTEP-a2 and H460)with alone and allied manners of administration were detected with MTT assay.According to the results of apigenin treating A549,SPC-A-1,H1299,H1650,LTEP-a2 and H460 cells for different time(24 h,48 h and 72 h),the IC50 were respectively 55.17,34.58,26.41 ?M;47.37,33.28,23.94 ?M;67.72,34.11,22.87 ?M;130.98,50.07,34.36 ?M;125.25,55.11,28.00 ?M and 60.26,37.19,20.85 ?M.The results showed that apigenin significantly inhibited the proliferation of the NSCLC cells in dose and time dependences.Compared with cisplatin alone for NSCLC cells,apigenin increased the sensitivity of the NSCLS cells to cisplatin.Then,cell counting,plate clone formation experiment,Hoechst 33258 staining and flow cytometry were utilized respectively to detect the effects of apigenin on cell proliferation,apoptosis,the influence of the cycle distribution,etc in NSCLC cell line H460.The results showed that when the different concentrations(0,20,40,80 ?M)of apigenin were used on H460 cell line for different time(24,48,72 h),the inhibition rates of cell proliferation were in dose and time dependent manners.As the increasing concentration of apigenin,the longer and more obvious cell growth inhibition of apigenin behaved on H460 cell line.Also,the results of cell plate cloning experiments showed the consistent influence.Hoechst 33258 staining experiment results indicated that compared with negative control,H460 cell line cultured different concentrations of apigenin(0,20,40,80 ?M)for different time(6 h,12 h)appeared different degrees of apoptotic morphological changes,such as shrinking nucleus,uncompleted cell membrane,cell refraction variation and some nuclear fragments.Under inverted fluorescence microscope with ultraviolet excitation,tumor cells were stainned blue,with deeper blue color of dead cells.AnnexinV-FITC/PI double staining detection was used to detec the apoptotic rate of H460 cell line treated with apigenin.We found that the increasing concentrations and the longer time of apigenin culturing on H460 cell line,the apoptotic rate increased compared with negative control.The result of cell cycle assay showed that most H460 cells existed in G0/G1 phase.Compared with negative control,the proportion of cells in G2/M phase increased with the increasing concentrations and time of apigenin interferring H460 cell line.Therefore,apigenin mainly blocked H460 cell line in G2/M phase,inhibiting cell mitosis,and thus achieve the purpose of inhibiting cell proliferation.Last but not the least,we mainly inquiried the potential mechanism of apigenin on the effects of toxicity in response to cisplatin in NSCLC cell lines.Western blot and qRT-PCR methods were applied to detect the protein and mRNA levels of Nrf2,Nrf2-mediated genes(NQO1,HO-1 and GSTA1)expression changes in H460 cell line treated with different concentrations of apigenin for 24 h compared to the skeleton protein ?-actin,gene GAPDH).In addition,we added another two groups(t-BHQ 50 ?M,apigenin 40 ?M + tBHQ 50 ?M).Tert Butylhydroquinone(t-BHQ)has known as a agonist of Nrf2.The results showed that with increasing concentrations of apigenin treating on H460 cell line,the down-regulatin rate of Nrf2,NQO1,HO-1 and GSTA1 in protein and mRNA levels was more obvious.Compared with negative control group,the protein and mRNA expression of Nrf2 in t-BHQ 50 ?M group appeared up-regulated with statistical significance(p<0.05).The over-expression effects of other genes were not obvious in protein levels with no significant difference,but the expression of mRNA increase obviously.Compared with the effect of t-BHQ 50 ?M group,the protein and mRNA expression of Nrf2 in the group(apigenin 40 ?M + t-BHQ 50 ?M)was obviously lowered.Compared with the effect of apigenin 40 ?M group,celery,the group(apigenin 40 ?M + t-BHQ 50 ?M),the protein and mRNA expression of Nrf2 behaved slightly higger.Although t-BHQ can reverse the suppressing effect of apigenin in Nrf2,the results of Nrf2 downstream molecules were not consistent with Nrf2.Nrf2 mediated molecules may regulated by some other nuclear factors.The above results showed that the effects of apigenin inhibition expression of Nrf2-ARE signaling pathway might be in coordination with the potential mechanism of apigenin on the effects of toxicity in response to cisplatin in NSCLC cell lines.To sum up,apigenin had obvious inhibition effect of NSCLC cell proliferation,blocked cell cycle and induced cell apoptosis.Small doses of apigenin could play a positive role in anti-tumor effects of cisplatin in NSCLC cell lines,the mechanism of which might be associated with the inhibition role of apigenin in Nrf2-ARE signaling pathway.This thesis mainly aimes to improve the effects of chemotherapy drug therapy,and provides a new approach for collaborative treatment of lung cancer.