Screening Of Cripto-1 Expression Inhibitor From Chinese Medicine Compounds And Analysis Of Its Antitumor Activity

Previous studies have shown that Cripto-1（CR-1）,as an oncogene,is low-expressed or not expressed in normal tissues of adults.However,it is abnormally high-expressed in various types of tumors such as breast cancer,pancreatic cancer and ovarian cancer,and plays an important role in the initiation and development of cancer.Therefore,Cripto-1 is a potential target for anti-tumor drug screening and treatment.Before this study,we had already screened out 31 potential small molecule compounds that can inhibit the expression of Cripto-1 protein from more than 700 traditional Chinese medicine monomers,by constructing a screening model targeting the promoter and m RNA 3’UTR of Cripto-1.Based on previous studies,in order to find out the best compounds for inhibiting the expression of Cripto-1,we further screened the 31 potential small molecule compounds for inhibition of Cripto-1 expression using several pairs of cell lines which contained one cancer cell line and one normal cell line and both have high Cripto-1 expression.The results showed that compounds TI150 was the best candidate.And we further studied its anti-tumor effect and molecular mechanism.1.Screening of candidate compounds and preliminary analysis of their anti-tumor activities（1）TI150 screeningFirstly,in order to screen out candidate compounds which were effectively toxic to tumor cells while non-toxic to normal human cell lines,we studied the impact of 31 compounds on the viability of 5 cancer cell lines including gastric cancer cell line SGC7901,cervical cancer cell line He La,and hepatoma carcinoma cell line Hep G2,SMMC7721,and Bel7402,which are all highly expressing endogenous Cripto-1,and 3 corresponding normal cell lines including GES-1（gastric mucosa cells）,H8（cervical epithelial cells）,L02（normal hepatic cell）by MTT assay.We calculated the IC₅₀and IC₁₀of 31 candidate compounds on five tumor cell lines and 3 normal cell lines,and found that the candidate compounds TI150 was the best one with high anti-tumor activity and low toxicity to normal cells.It can highly suppress the viability of He La and SGC7901,but has low toxicity to H8 and GES-1 normal cell lines.Therefore,we further studied the anti-tumor activity of TI150 and its mechanism.（2）The effect of TI150 on Cripto-1 expressionWe analyzed Cripto-1 expression by Western blotting in TI150 treated SGC7901,He La,Hep G2,SMMC7721,and Bel7402,and we found that TI150 was able to inhibit Cripto-1expression in all of the 5 cancer cell lines.（3）The anti-tumor activity of TI150 in vitroSince TI150 can inhibit Cripto-1,an oncogene,expression in cancer cells,we next evaluated the anti-tumor activity of TI150 by MTT assay using 5 cancer cell lines mentioned above,and 2normal cell lines A549（lung cancer）and SKOV3（ovarian carcinoma）with low endogenous Cripto-1 expression.Results showed TI150 was capable of suppressing the viability of all the 5cancer cell lines in a time and concentration dependent manner.Importantly,the effect was stronger in cancer cells with higher level of endogenous Cripto-1 than that with lower endogenous Cripto-1 expression.These results indicated that TI150 inhibits the activity of a variety of tumor cells and may exert its anti-tumor activity by suppressing the expression of Cripto-1.2.Study on the anti-tumor mechanism of TI150（1）Effects of TI150 on apoptosis of He La and SGC7901 cellsTo elucidate the specific mechanism of the inhibition of the compound TI150 on tumor cells viability,we first examined its effect on the apoptosis of tumor cells.The results of TUNEL assay showed that both He La cells and SGC7901 cells showed obvious apoptotic nucleic morphology such as fragmented nuclear and chromatin condensation after treated with TI150.Western blot results showed that TI150 significantly increased Cleaved caspase-9 and Cleaved caspase-3 levels after treatment.These results suggested that TI150 could induce apoptosis in He La cells and SGC7901 cells.（2）Effects of TI150 on the proliferation of He La and SGC7901 cellsNext,we tested the effect of TI150 on the proliferation of He La and SGC7901 cells.Clone formation assay and Brd U incorporation assay showed reduced number of clones formation and Brd U incorporation,which indicated down-regulated proliferation.Cell cycle analysis by flow cytometry showed that the ratio of S phase He La and SGC7901 cells increased significantly after TI150 treatment.Western blot showed that the expression of Cyclin E and CDK2 protein was down-regulated and the expression of P21 protein was up-regulated in SGC7901 cells;the expression of Cyclin E,Cyclin A and CDK2 protein in He La cells was down-regulated,and the expression of P21 protein was up-regulated,which was consistent with the results of flow analysis,indicating that the compound TI150 could cause S-phase arrest of the cells.These results indicated that TI150 could inhibit the proliferation of He La cells and SGC7901 cells.（3）Effects of TI150 on glucose metabolism in He La cells and SGC7901 cellsThen we considered whether TI150 affected the glucose metabolism process of He La and SGC7901 cells while affecting their vitality.We detected the expression of glucose metabolism related proteins in SGC7901 and He La cells after TI150 treatment by Western blot,and results showed that TI150 partially inhibited HK2,GLUT1,and GLUT4 expression in SGC7901 and He La cells,indicating that the compound TI150 impacted the aerobic glycolysis of He La cells and SGC7901 cells to a certain extent.3.Study on the correlation between the anti-tumor activity of TI150 and its inhibition of Cripto-1 expressionBased on the above results,we confirmed that the compound TI150 was able to inhibit the viability of a variety of tumor cells,and it could significantly suppress Cripto-1 expression in tumor cells.Therefore,we then investigated whether the anti-tumor effect of the compound TI150was correlated with its inhibition of Cripto-1 expression in tumor cells.We overexpressed Cripto-1 in He La cells and SGC7901 cells and then treated with TI150,and further verified whether the anti-tumor effect of the compound TI150 was affected by Cripto-1 overexpression.（1）Effects of overexpression of Cripto-1 on viability of tumor cells by TI150We first examined the effects of the TI150 on the viability of He La cells and SGC7901 cells with overexpression of Cripto-1.The results showed that the inhibitory effect of TI150 on the viability of He La cells and SGC7901 cells with overexpression of Cripto-1 was significantly impaired.（2）Effects of Cripto-1 overexpression on apoptosis and proliferation of tumor cells induced by TI150Subsequently,we verified the effect of overexpression of Cripto-1 on the anti-tumor effect of TI150 from two aspects inducing apoptosis and proliferation.We tested the effect of TI150 on apoptosis of He La and SGC7901 cells which overexpressed with Cripto-1 by Western blot.Results showed that after Cripto-1 overexpression,the expression of apoptosis marker Cleaved-caspase-3 was significantly down-regulated.At the same time,Brd U incorporation assay showed that Brd U incorporation in He La and SGC7901 cells was increased after Cripto-1 overexpression.These results suggested that after overexpression of Cripto-1,the effects of TI150 on inducing apoptosis and inhibiting proliferation were significantly reduced.（3）Effects of Cripto-1 overexpression on inhibition of glucose metabolism in tumor cells by TI150We have previously shown that TI150 can inhibit the expression of glucose metabolism-related protein HK2 GLUT1 GLUT4 in SGC7901 and He La cells to a certain extent.Subsequently,we detected the expression of glycemic metabolism-related protein HK2 GLUT1GLUT4 in He La cells and SGC7901 cells after overexpression of Cripto-1.The results showed that the expression level of GLUT1,GLUT4,HK2 in He La cells and SGC7901 cells overexpressing Cripto-1 was still up-regulated to a certain extent after being treated with the compound TI150.These results suggested that the ability of TI150 to inhibit glucose metabolism of tumor cells was significantly weakened after overexpression of Cripto-1.Taken together,we concluded that the compound TI150 plays an anti-tumor role at least to a certain extent by inhibiting the expression of Cripto-1 protein.4.Analysis of signal pathway related to anti-tumor effect of the target compound TI150（1）The effect of TI150 on MAPK/ERK signaling pathway in tumor cellsWe screened a large number of key proteins involved in a variety of signaling pathways by Western blot,and found that the MAPK/ERK signaling pathway played a certain role in the anti-tumor effect of the compound TI150.After the treatment of TI150,the phosphorylation level of ERK protein in He La cells and SGC7901 cells decreased significantly,and with the increase of the concentration of compound TI150,the reduction of ERK protein phosphorylation level became stronger,suggesting that compound TI150 may play an anti-tumor role by inhibiting the activation of ERK.（2）Study on the necessity of MAPK/ERK signaling pathway in the anti-tumor effect of TI150Furthermore,we verified whether the MAPK/ERK signaling pathway played a role in the anti-tumor effect of TI150.Through Western blot,MTT,and Brd U incorporation assays,we found that the inhibitory effect of the compound TI150 on cell viability,the activation of Cleaved caspase-3,the apoptotic marker protein,and the inhibitory effect on cell proliferation were inhibited after the overexpression of ERK protein in SGC7901 cells and He La cells.These results suggested that the compound TI150 could exert its anti-tumor effect by inhibiting MAPK/ERK activity.（3）Study on the correlation between MAPK/ERK signaling pathway and Cripto-1 expressionThe compound TI150 can exert its anti-tumor effect by inhibiting the MAPK/ERK signaling pathway.Meanwhile,the antitumor effect of compound TI150 is closely related to its inhibition of Cripto-1 protein expression.Therefore,we then studied the correlation between MAPK/ERK signaling pathway and Cripto-1 protein expression.After the overexpression of Cripto-1 in SGC7901 cells and He La cells,the levels of ERK protein and phosphorylated ERK protein were increased to a certain extent.Meanwhile,the expression of Cripto-1 protein was significantly inhibited after ERK protein overexpression in SGC7901 cells and He La cells.The above results suggested that the compound TI150 exhibited its anti-tumor effects by inhibiting the expression of Cripto-1 protein and further inhibiting the MAPK/ERK signaling pathway.In conclusion,the compound TI150 has an anti-tumor effects which can induce apoptosis and inhibit proliferation of tumor cells,and influence glucose metabolism of tumor cells to a certain extent,which is closely related to its ability to target Cripto-1.