| Background and Aims:The morbidity of chronic liver disease is very high in China, among which hepatocellular carcinoma is the most serious disease. With the development of the radical resection operation, liver transplantation and local radiofrequency ablation technology, the five-year survival rate of hepatocellular carcinoma(HCC) patient has been improved greatly. However, the diffusion, metastasis and postoperative recurrence of cancer are still important factors which restrict the prognosis. Therefore, systemic adjuvant therapy with drugs has been gradually highlighted.Matrine and oxymatrine are the major alkaloids of radix sophorae flavescentis, which are the main pharmacological active ingredients in the radix sophorae flavescentis. As a common kind of drugs which are derived from traditional Chinese medicine, they are widely used in the clinic treatment for colitis, gynecological disease. Apart from these, matrine and oxymatrine are employed for the treatment of liver diseases, such as chronic hepatitis and liver fibrosis as well. Researchers declared matrine and oxymatrine had exact curative effects in preventing and delaying the process of liver fibrosis, which improvedg the patients of chronic hepatitis. Plenty of in vitro and in vivo experiments has showed anti-tumor effects of matrine and oxymatrine for kinds of tumor diseases, such as non-small cell lung cancer, melanoma, leukemia, nasopharynx cancer, gastric cancer, pancreatic cancer, colon cancer, HCC, carcinoma of gallbladder,and so on. When the hepatocellular carcinoma cells are treated with matrine and oxymatrine, they could suppress the proliferation of cells, and induce apotosis, and reduce the occurrence of invasion as well. However, the anti-tumor effects of matrine and oxymatrine are not satisfactory for they have to been employed with high dose to reach obvious effects. In order to improve the pharmacological effects, researchers modified the structures of matrine/oxymatrine and they got a new chemical compound (13-methylamino-18-sulfo-matrine), which was called M19. Then, taking M19 as a precursor, a series of matrine derivatives were synthesized(called WM-1 series of matrine derivatives). Previous studies had proved that M19 could reduce the phosphorylation of AKT. It was proved that M19 had more obvious effects in inhibiting liver fibrosis when compared with matrine and oxymatrine. Meanwhile, people found that M19 may have some anti-tumor effects.The over expression and activation of EGFR are closely related with malignant proliferation and poor differentiation of tumor cells. Studies have found that various malignant tumors showed high expression of EGFR, such as gastric cancer, colon cancer, liver cancer, lung cancer, prostate cancer, and there is a high correlation between the expression level of EGFR and tumor metastasis. At the same time,people also found that EGFR can regulate the transcription and expression of matrix metalloproteinases(MMPs) by adjusting the downstream signaling pathways, mainly MAPK/ERK signal pathway.In many kinds of malignant tumors, matrix metalloproteinases(MMPs) play an important role in the metastasis and invasion behavior of tumor cells, especially the matrix metalloproteinases-2 and-9(MMP-2, MMP-9). Many kinds of solid malignant tumor, like lung cancer, breast cancer, gastric cancer, HCC, express high level of MMP-2 and MMP-9. Generally cells secret MMPs to extracellular in the form of zymogen, so body can regulate the function of MMPs by regulating MMPs plasminogen activator (such as plasmin), and inhibitors of matrix metalloproteinases(TIMPs).This study was planed to explore the influence in cell behavior, such as cell proliferation and invasive ability, when the matrine derivatives were employed for HCC cells. Then the change on signal pathways and protein expression level in HCC cells would be tested to explore the possible molecular mechanism for the anti-tumor effects of matrine derivatives.Methods1. Use the MTT method to pick out the one, which has more strong and stable effect for HCC cells, from WM-1 series of matrine derivatives, to conduct the subsequent experiments in sensitive HCC cell lines.2. Different concentrations of matrine derivatives WM-108 were employed in different HCC cell lines to detect their effects on proliferation inhibition, and the relationship between the proliferation inhibition and the concentration or time.3. Take AnnexinV/PI flow cytometric assay to test the apoptosis level of Hep3B cells, which have been treated with WM-108.4. Adopt wound healing and transwell experiment to detect the effect on invasion and metastasis ability for HCC cells after the intervention of WM-108.5. Use Western blot and RT-PCR to test the change of transcription and expression level of some protein in tumor cells when different concentrations of matrine derivatives WM-108 were used to intervene Hep3B.6. Use gelatin zymography method to detect the activity change of matrix metalloproteinase which was secreted by Hep3B cells to the culture medium after intervention.7. We established subcutaneous and liver orthotopic HCC models in nude mice with Hep3B and then gave them drug intervention. The effect of WM-108 on tumor growth and the general situation of mice were detected, and the tumors were gotten for pathological HE staining after intervention.Result1. Matrine derivatives can significantly inhibit the proliferation of HCC cellsa. WM-1 series of matrine derivatives had effects of proliferation inhibition on several human HCC cell lines, like SMMC-7721, HepG2, Hep3B, HCCC-9810,and the effects of WM-108 were more obvious and stable.b. WM-108 had stronger effect of proliferation inhibition on Hep3B when it was compared with matrine and this effect would be stronger with the increase of time and concentration.The half inhibition concentration(IC50) for 24 hours was 127.3±18.49mg/L.c. WM-108 could play obvious proliferation inhibition effect for liver cell line just under high level(>150mg/L).2. The Effects of WM-108 on cell apoptosis, invasion and migration for HCC cells.a. WM-108 could significantly promote Hep3B cell apoptosis under certain concentration and concentration of 200mg/L can lead to above 30% cells necrosis.b. WM-108 could reduce the migration capacity for Hep3B and LM3 under low concentration 10mg/L,20mg/L,30mg/L).c. Transwell experiments confirmed that the capacity of penetrating matrigel was declined for Hep3B and LM3 cells after the intervention of WM-108.3. Matrine derivatives WM-108 could regulate some cell signal transduction pathways and the expression of some proteins to reduce the proliferation, invasion and migration of Hep3B.a. After the intervention of WM-108, the phosphorylation level of EGFR in Hep3B cells was decreased, and the phosphorylation levels of AKT and ERK in signaling pathways downstream dropped too.b. It was confirmed that the transcription and expression of MMP-2 and MMP-9 in Hep3B cells were reduced after the intervention of WM-108 by the examination of RT-PCR and Western blot. However, the transcription level of TIMP-1 and TIMP-2 were not changed obviously.c. When the gelatin zymography method was used, we found the activity of matrix metalloproteinase in the culture medium were reduced.4. The experiments in nude mice model proved that matrine derivatives WM-108 could show some anti-tumor effects in vivo.a. WM-108 can delay the growth speed of tumors at the dose of 50mg/kg when compared with control group. But the effect was lower than that of 5-Fu at the dose of 30mg/kg.b. The harmful effect of WM-108 on general condition of nude mice was less than that of 5-Fu.c. The tumors from WM-108 groups shared the similar performances of HE staining with tumors from 5-Fu groups. They had larger necrosis areas in the center of tumor tissues and there were some small necrosis areas around the tumor center.ConclusionWe conducted this study to detect the effect of matrine derivative WM-108 on HCC. Then the effects on signal pathways and expression of proteins were explored to investigate the possible mechanism of these effects. From the experiments, we found WM-108 had stronger effects of proliferation inhibition on HCC cells when it was compared with matrine. The drug could promote apoptosis at appropriate concentration, however, when the concentration was too high, it showed obvious effect leading tumor cells to necrosis. The intervention of WM-108 could drop the phosphorylation level of EGFR, and then induce the decline of phosphorylation levels of AKT and ERK in signaling pathways downstream, which could inhibit the proliferation of cells and induce the apoptosis. WM-108 could reduce the migration and invasion capacity of HCC cells, and the possible mechanism was the drug’s down regulation of PI3K/AKT, MAPK/ERK signal pathways, which would lead to the decline of the expression level of MMP-2 and MMP-9. When giving some WM-108 to HCC model for a period(50mg/kg,5 times,1 time per 2 days), the tumor growth in nude mice were inhibited, and the harmful effect of general condition on nude mice was much less than that of 5-Fu. |
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