| Background and Purpose:Maimendong and Qianjinweijing formula is an effective anti-lung cancer prescription that has been tested by long-term clinical practice under the guidance of Professor Zhou Zhongying,who is a Chinese medicine master.The previous study of our research group showed that this formula has certain effects on inhibiting tumor growth and enhancing immunity.A group of anti-tumor active components were obtained from this formula by drug separation and screening,among which isoliquiritigenin(ISL)and luteolin(LTL)have significant anti-lung cancer effect.Multiple signaling pathways are involved in the development of lung cancer.The aim of this study is to elucidate the regulation mechanism of ISL and LTL on targeting the relactive pathway.In order to provide experimental evidence for its treatment in lung cancer,and to find new directions for innovative cancer drug development.Methods:In the first part,the human non-small cell lung cancer(NSCLC)cell lines A549,H460 and H1299 were chosen as experimental cells to investigate the effect and molecular mechanism of ISL on the inhibition of tumor cell proliferation.And normal lung cell lines BEAS-2B as control cell lines.(1)The human lung cancer cell lines H460,A549 and H1299 were cultured in vitro.ISL was diluted with DMSO respectively into 5,10,15,20,30,40,60,80,and 100 ?mol/L.The cell viability was detected by CCK-8 assay.(2)The cell cycle and apoptosis were measured by flow cytometry analysis.(3)The mRNA expression of lung cancer related genes in H460 cells were determined by PCR array.(4)The protein expression levels of p-p65,IKB a,IKB p,Bax and Bcl-2 were detected by Western blot analysis.The activation of Caspase-3 and Caepase-9 were detected by Spectrophotometric.(5)The effect of ISL on nuclear translocation of NF-kB p65 was observed by immunofluorescence assay.The second part,(1)the human NSCLC cell lines A549,H460 and H1 299 were chosen as experimental cells in vitro,The inhibitory effect of LTL on its proliferation was detected by CCK-8.(2)The effect of LTL on cell apoptosis was detected by flow cytometry.(3)subcutaneous injection was used to establish a H460 xenotransplantation mouse model(BALB/c).(4)HE staining method for the tumor Histopathological evaluation.(5)TUNEL assay was used to detect cell apoptosis.(6)Ki-67 assay was used to detect cell proliferation.(7)Microarray analysis and qPCR were used to detect the expression of miR-34a-5p.Results:(1)CCK-8 detection analysis showed that ISL inhibited the proliferation of lung cancer cells in a concentration-and time-dependent manner.Among them,the inhibition of proliferation of H460 cells was most obvious,but not toxic to normal cells.Therefore,we performed subsequent experiments on H460 cells.(2)Flow cytometry analysis showed that after ISL treatment on H460 cells,the number of cells in S and G0/G1 phases was less than of the control group,while the number of cells in the G2/M phase was significantly more than that,indicating that ISL can cause G2/M phase arrest in H460 cells.(3)Annexin V assay showed that the apoptosis rate of H460 cells after treated with different concentration of ISL(15,30,60?mol/L)for 24 h increased from 7.7%which in blank group to 11.1%,14.2%and 20.3%respectively,indicating that the rate of apoptosis increased with the increase of ISL concentration,the difference was statistically significant.(4)PCR microarray analysis showed that there were 12 genes up-regulated by 2 times or down-regulated by 0.5 times or more in the ISL high-dose group compared with the control group,of which 7 genes were up-regulated,such as the up-regulation of NF-kB IE gene expression 4.9416-fold,and 5 down-regulated genes,such as the Bcl-2 gene,were down-regulated by 3-fold.(5)Western blot analysis showed that compared with the control group,the expression levels of p-p65,lkB a,IkB ? and Bcl-2 protein gradually decreased in the ISL-treated group,while the expression level of Bax protein gradually increased which in a dose-dependent manner.And compared with the control group,the activity of Caspase-3 and Caspase-9 gradually increased in the ISL-treated group.(6)Immunofluorescence assay showed that compared with TNF-a group,H460 cells were treated with TNF-a after under ISL treatment,the nuclear movement of NF-kB(p65)was significantly inhibited.It showed that ISL can significantly inhibit TNF-a-induced nuclear import of p65.The second part,(1)CCK-8 results showed that LTL could inhibit the proliferation of A549 and H460 cells in a dose-dependent manner.(2)Flow cytometry showed that LTL could promote early and late apoptosis of two cells.(3)Modeling results showed that LTL could inhibit tumor growth in the H460 xenograft mice model.(4)Tumor group.The pathological evaluation showed that LTL could promote the death of tumor cells.(5)TUNEL fluorescence results show that LTL could induce the apoptosis of tumor cells in the model mice.(6)Ki-67 fluorescence results show that LTL could inhibit the proliferation of tumor cells in the model mice.(7)Microarray analysis and qPCR results showed that LTL could increase the expression of miR-34a-5p in the tumor cells.Conclusion:The first part,ISL can effectively inhibit the proliferation of NSCLC cells,by inducing cell cycle arrest and promoting apoptosis.For NCI-460 cells,the mechanism may be related with the inhibition of the NF-kB signaling pathway,by blocking the translocation of NF-kB p65 into the nucleus,resulting in the inhibiton of its transcriptional activity and ultimately affecting the expression of downstream apoptotic proteins such as Bax and Bcl-2,as well as the activation of Caspase-3 and Caspase-9.The second part,LTL can inhibit the proliferation of NSCLC cell lines A549 and H460 and induce apoptosis.In the H460 xenograft tumor model,LTL can significantly inhibit tumor growth and induce apoptosis,and its mechanism of action is likely to be achieved through up-regulation of miR-34a-5p.These findings provide novel insights into the anticancer function of ISL and LTL,suggesting their potential as therapeutic agents for human NSCLC. |
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