Research On The Inhibitory Effect Of Matrine Derivative WM622 On Hepatoma Cells And Its Mechanism

Liver cancer is one of the most common malignant tumors.Among them,hepatocellular carcinoma（HCC）is the most common form.The occurrence of hepatocellular carcinoma is generally started with liver damage caused by chronic liver disease,which in turn leads to hepatic fibrosis,liver cirrhosis,and ultimately lead to hepatocellular carcinoma.Traditional treatments include surgery,intervention,radiotherapy,chemotherapy,and biological immunotherapy.Looking for effective chemotherapy drugs,and researching the pathogenesis of liver diseases and the mechanism of the drugs,is one of the most important research directions.Matrine is an alkaloid isolated from the dried roots of Sophora flavescens with a wide range of biological activities such as anti-bacterial,anti-inflammatory,analgesic,immunomodulatory,anti-arrhythmic,anti-hepatic fibrosis and anti-tumor activity.Matrine can be used in liver fibrosis,chronic hepatitis B and other diseases in clinical practice.The anti-tumor effect of matrine is extensive,mainly in the inhibition of tumor cell proliferation,promotion of apoptosis process,inhibition of tumor cell invasion and angiogenesis,and induction of tumor cell differentiation.It also has immunomodulatory effects and reduce the toxicity caused by chemotherapy and radiotherapy.M-19 is a new type of matrine derivative that has been researched in recent years.The pharmacological activity of M-19 is various,especially for a serious of tumor cells.The activity of M-19 is much stronger than that of matrine.The disadvantage of M-19 is as same as matrine.The water-solubility is good and the stability is even as bad as matrine and is easily decomposed.Based on previous studies,matrine and matrine derivatives M-19 were used as lead compounds,and sophocarpine as the starting material.Basic pharmacophores were retained and drug fragments were introduced into the structure.A series of new compounds were synthesized,and a new type of high activity and low toxicity matrine derivative numbered WM622 was selected.Extensive pharmacological studies had been conducted on WM622 and the results suggested that it had good activity on HCC-LM3 and Hep3B cells.In cell experiments,WM622 has a significant inhibitory effect on the proliferation of hepatoma cells,and can inhibit tumor cell migration,promote apoptosis,and block the cell cycle in the G0/G1phase.Computer-assisted drug design was used to simulate the interaction between WM622 and target proteins.It was speculated that WM622 could bind to EGFR and PTEN proteins,and it was confirmed by experiments that WM622 could inhibit the EGFR-AKT/PI3K pathway.In tumor-bearing mice,WM622 has a significant inhibitory effect on tumor growth.Therefore,the study provides a new research basis for the study of anti-hepatoma drugs.Methods:1.Synthesis of matrine derivatives and screening for high activity,low toxicity matrine derivative.Three classes of matrine derivatives were synthesized by using matrine and matrine derivative M-19 as the leading compounds.The MTT method was used to determine the inhibition rate of each compound against different hepatoma cell lines at a certain concentration.The IC₅₀ value was measured and the matrine derivative WM622was screened out as the compound with high activitiy and HCC-LM3 and Hep3B were screened out as sensitive cell lines.2.The effect of WM622 on the biological activity of liver cancer cells and liver cancer stem cells in vitro.In order to detect the inhibitory effect of WM622 on liver cancer cells and liver cancer stem cells,several tests were carried out:（1）MTT and colony formation experiment were carried out to detect the effect of WM622 on cell proliferation.（2）The would healing and transwell chamber experiment were taken to demonstrate the effect of WM622 on the migration ability of hepatocellular carcinoma cells.（3）The proportion of apoptotic cells in WM622-treated liver cancer cells was detected by Hoechst33258 staining and PI,AV-FITC double staining and then by flow cytometry to evaluate the effect of WM622 on the apoptosis of hepatocellular carcinoma cells.（4）The distribution and proportion of the cell cycle affected by WM622 were detected by PI staining and flow cytometry.（5）CD133⁺cells were obtained by magnetic cell separation（MACS）,and the stemness was confirmed by the tumorsphere formation assays.The effect of WM622 on the number of spheres were examined.The effect of WM622 on the proliferation of CD133⁺and CD133^-cells were detected.3.The molecular mechanism of WM622.Possible target proteins and signaling pathways of WM622 were speculated by the method of CADD.The mechanism of WM622 was comfirmed by Western Blot.4.The anti-liver cancer effect of WM622 in vivo.HCC-LM3 cell suspension was subcutaneously injected into the back of 4-week-old BALB/c nude mice to establish tumor-bearing mouse model.The high-dose group（34 mg/kg）,low-dose group（17 mg/kg）,5-Fu group（20 mg/kg）,and blank control group（PBS）were administered intraperitoneally every other day.Tumor tissue was harvested and measured 10 times after administration.The tumor growth curve and mouse body weight curve were plotted.HE staining was used to observe the tumor organization structure.The expression levels of related proteins in tumor tissues were detected by HE staining,immunohistochemistry and Western Blot.Results:1.Synthesis of matrine derivatives and screening for high activity,low toxicity matrine derivative.Three classes of matrine derivatives were synthesized.The inhibition rates at 0.1 mg/mL were determined by MTT assay.The IC50 values were measured and WM622 was screened out as high activity,low toxicity matrine derivative.HCC-LM3 and Hep3B were screened out as sensitive cell lines.2.The effect of WM622 on the biological activity of liver cancer cells and liver cancer stem cells in vitro.The following conclusions were got from this part（1）The proliferation of HCC-LM3 and Hep3B was inhibited by WM622 dose-and-time-dependently,and the toxicity to normal hepatic cell lines was lower than that to hepatocarcinoma cells.The plate colony formation rate of HCC-LM3 and Hep3B decreased significantly with the increase of WM622 concentration.（2）The wound healing area of HCC-LM3 and Hep3B decreased significantly,and the number of transwell cells decreased significantly with the increase of WM622 concentration.（3）The apoptotic cells were observed as scattered granular or rod-like nuclei under fluorescence microscope after stained with Hoechst 33258.The number of apoptotic cells gradually increased with the increase of WM622 concentration.（4）After PI and AV-FITC staining,the proportion of apoptotic cells detected by flow cytometry increased.The proportion of cells in G1 phase increased,suggesting that WM622 can block the cell cycle in G0/G1 phase.（5）The liver cancer cell lines were divided into CD133⁺cell population and CD133^-cell population by MACS.The sphere formation rate of CD133⁺cells was significantly higher than that of CD133^-cells.The proliferation of CD133⁺cells was inhibited by WM622 dose dependently.3.The molecular mechanism of WM622.The EGFR and PTEN were found to be better for the binding of WM622 by the CADD method.The Western Blot was performed to further validates that the EGFR-PI3K/AKT pathway was inhibited by WM622 in tumor cells.4.The anti-liver cancer effect of WM622 in vivo.With the drug administration of WM622,the tumor volume growing slower.The general morphological characteristics of tumor tissue such as large nuclear and cytoplasm,deep stained nuclei,disorganized tissue structure can be seen under microscope by HE stained sections.Immunohistochemical and Western Blot experiment demonstrated that the EGFR-PI3K/AKT pathway of tumor cells was inhibited by WM622.Conclusion:In vitro,WM622 can inhibit the proliferation and migration of hepatocellular carcinoma cell HCC-LM3 and Hep3B dose-and time-dependently,promote apoptosis,and block the tumor cell cycle in G0/G1 phase.And it can inhibit the proliferation of liver cancer stem cells.In vivo,WM622 inhibits tumor growth dose-dependently.The mechanism of WM622 is inhibiting the EGFR-PI3K/AKT pathway in tumor cells.