| [Background and Objective]Human hepatocellular carcinoma (Hepatocellular, carcinoma, HCC) because of the limited treatment and high mortality rate, as a result the leading causes of cancer-related death worldwide. At present, the effective method of early treatment of liver cancer is still traditional, operation treatment, but the radiotherapy and chemotherapy treatment effection is not exact, the majority of patients can not tolerate the toxicity of chemotherapy and expensive medical bills, so its prognosis has not been fundamentally changed, clinical on the urgent need to find a new treatment method.Tumor differentiation therapy is an important discovery in clinical therapy of tumor. The so-called differentiation induced by some chemicals can cause malignant cells reversed immature, differentiation. The basic characteristics of induced differentiation of tumor is not to kill the tumor cells, but the induction of tumor cell differentiation is closed to normal cells, clinical treatment is the classic of retinoic acid and arsenic induced leukemia cells to normal mature cells, induced leukemia cell apoptosis, inhibit tumor growth. The present study confirms that the ginseng, tanshinone and other traditional Chinese medicine can make some malignant phenotype reversion of some tumor cells and differentiation and the differentiated treatment of malignant tumor are possible.The sophocarpine is the dried root of Sophora flavescens and plants from leguminous plants of the genus Sophora alopecuroides and ground part of effective monomer extracted, it is a rich source of drugs, drug toxicity and cheap, effective treatment of a variety of inflammation, but so far no use of sophocarpine preparation in treatment of antitumor drugs report. The purpose of this study is to observe the sophocarpine induce differentiation of liver cancer, and to explore its mechanism of action.[Methods]1. Effect of Sophocaprine on inducing differentiation of malignant cells including CSCs into mature hepatocytes(1) Effect of Sophocaprine on expression of liver-specific genes in hepatoma cell linesExpression of liver-specific genes in hepatoma cells was detected by real-time PCR on the first day of dealed with sophocarpine.(2) Effect of Sophocaprine on expression of CSCs in hepatoma cell linesThe expression level of CD133and CD90was detected by real-time PCR on the first day of dealed with sophocaprine. Furthermore some genes involved in the establishment or maintenance of pluripotency were detected by real-time PCR, inclding P-catenin, Oct3/4, NANOG, c-Myc.2. Effect of Sophocarpine on the biological characteristics of hepatoma cells(1) Effect of Sophocarpine on the ability of proliferation and colony formation of hepatoma cellsTo detect the proliferation ability, the hepatoma cells were plated into a96-well plate and then treated with different dose of sophocarpine. The number of viable cells was determined at different time pionts. To evaluate colony formation ability,3×103hepatoma cells were seeded onto6cm culture dishes24hours after treatment and grown for2-3weeks. Colonies were fixed and visualized via crystal violet staining. HCC cells treated with different sophocarpine concentrations (0,1,10,100,1000μmol/L) for48h were resuspended in medium containing0.5%low melting point agarose and seeded into plates containing medium with1%solidified agarose in triplicate. Colonies were photographed and counted after2to3weeks.(2) Effect of Sophocaprine on the Cell invasive assay of hepatoma cells and cell migration assayThe invasive activity of HCC cells was assessed using24-well transwell inserts with8um pores coated with Matrigel. The cells (2×104) with serum-free medium were seeded into the upper chamber. Medium supplemented with10%fetal bovine serum and50μg/ml Fibronectin was placed in the lower chamber as chemoattractants. After48h of incubation, the cells on the upper surface of the filter were removed with a cotton swab, and cells which invaded through the Matrigel and were adherent to the lower surface of the filter were fixed and stained with0.5%crystal violet and counted under a light microscope. Cell migration assay was performed according to the protocol of invasion assay, except that the cells were seeded onto the uncoated filter.3. Sduty on the mechanism of differentiation therapy by Sophocarpine(1) sophocarpine reverses liver stem cell malignant phenotype through down-regulating the activity of a novel AKT/GSK-3β/β-catenin axisThe expression of the important passways of tumor cells, including Ras/MAPK, PIK3CA/AKT and Wnt/β-catenin signaling pathways, and maintenance and proliferation of stem/progenitor cells are tightly regulated by comprehensive signaling network involving JAK/STAT3, NOTCH, PTEN, Akt/FOXO3a, etc. Dysregulation of these pathways may lead to aberrant proliferation or neoplastic transition of stem/progenitor cells. AKT phosphorylation is usually enhanced as a consequence of PI3K activation or PTEN suppression. With this report, we unveiled that upregulated and suppressed AKT phosphorylation respectively in HCC. As GSK-3β was downstream of AKT, and AKT phosphorylates and deactivates GSK3-beta. Many case reports that GSK-3β (Ser9) phosphorylation was elevated, the GSK-3β activity was inhibited. When GSK3β activity has been inhibited, β-catenin translocates to the nucleus and acts as a coactivator of transcription factors involved in cell proliferation, survival and angiogenesis. To futher study the relationships between GSK-3β and β-catenin, we regulated p-AKT activity with50ng/mL IGF and decreased10μmol/L LY294002to observe the effort on HCC.(2) Sophocarpine inhibits epithelial to mesenchymal transition (EMT) incuced by TGF-βTGF-β and sophocarpine were used to deal with MHCC-97H and LM3, then the EMT markers were detected by RT-PCR.4. The anti-tumor effect of Sophocarpine in vivo(1) Effect of Sophocaprine on the subcutaneous tumor through intratumoral injection2×106of LM3cells were injected into the armpits of mice to establish a subcutaneous tumor model. At the sixth days, mice were randomly assigned to treatment groups. Injection of normal saline and sophocarpine was administered intratumorally every other day for four weeks. Tumor size was measured with calipers. At the time of euthanasia, tumors were removed for further analysis.(2) Effect of sophocarpine on the transplanted orthophoric tumor of liverBalb/c mice were anesthetized, and2×106of LM3cells in100μl of serum-free DMEM were injected into the liver. Either sophocarpine or normal saline was injected via the intraperitoneal injection five a week for4w, after10d of tumor cell inoculation. Mice were killed and livers were removed for analysis.5. Statistical analysisStatistical analyses were performed with SPSS software (16.0version), with a P value <0.05considered significant[Results]1. Sophocarpine reverses liver stem cell malignant phenotype(1) Sophocarpine could upregulate some liver function mRNA gene expression in liver cancer cellsLiver tumor cells were dealed with sophocarpine by24h, significant the liver function gene mRNA levels of ALB, G-6-P, ALDOB, BR, CYP1a3were increased in both MHCC-97H and LM3, compared with control groups.(2) Sophocarpine could down-regulate some cancer stem/progenitor cells mRNA gene expression in liver cancer cellsLiver tumor cells were dealed with sophocarpine by24h, cancer stem/progenitor cells gene mRNA levels of CD133、CD90、Epcam decreased, We also detected that the mRNA levels of Oct3/4, β-catenin, NANOG, c-Myc and AFP, which are richly expressed in human embryonic stem cells and involved in the establishment or maintenance of pluripotency were declined.2. Sophocarpine suppresses the ability of proliferation and migration of hepatoma cells(1) Sophocarpine inhibited proliferation and colony formation of hepatoma cells in vitroThe proliferation assay showed that sophocarpine significantly suppressed the proliferation of LM3and MHCC-97H cells and on the thirdth the suppression rate was up to more than50%with a dose-dependent manner. Meanwhile, sophocarpine also reduced the ability of colon formation. Furthermore, sophocarpine could also significantly suppressed the proliferation ability of primary HCC cells from human. (2) Sophocarpine reduced the migration and invasion ability of HCC cells in vitroAfter treated with sophocarpine, the invasion assay also showed that sophocarpine suppressed the ability of migration and invasion in HCC cells.3. Sophocarpine reverses liver stem cell malignant phenotype through down-regulating the activity of a novel AKT/GSK-3p/p-catenin axis and EMT induced by TGF-β(1) Sophocaprine reverses liver stem cell malignant phenotype through down-regulating the activity of a novel AKT/GSK-3β/β-catenin axisThe expression of the important passways of tumor cells, including Ras/MAPK, PIK3CA/AKT and Wnt/β-catenin signaling pathways, and maintenance and proliferation of stem/progenitor cells are tightly regulated by comprehensive signaling network involving JAK/STAT3, NOTCH, PTEN, Akt/FOXO3a, etc. Dysregulation of these pathways may lead to aberrant proliferation or neoplastic transition of stem/progenitor cells. AKT phosphorylation is usually enhanced as a consequence of PI3K activation or PTEN suppression. With this report, we unveiled that upregulated and suppressed AKT phosphorylation respectively in HCC. Our report results revealed that sophocaprine could inhibit phosphorylation AKT activation significantly. As GSK-3β was downstream of AKT, and AKT phosphorylates and deactivates Gsk3-beta. Many case reports that GSK-3β (Ser9) phosphorylation was elevated, the GSK-3β activity was inhibited. When GSK3β activity has been inhibited, β-catenin translocates to the nucleus and acts as a coactivator of transcription factors involved in cell proliferation, survival and angiogenesis. In consistent with this study, we detected the p-GSK-3β was declined treated with sophocarpine. We also found that sophocaprine could inhibit Wnt/β-catenin reporter gene activity (data no show). To futher study the relationships between GSK-3β and β-catenin, we regulated p-AKT activity with50ng/mL IGF, we found that sophocarpine could significantly inhibite the activities of p-AKT, p-GSK3β and p-β-catenin in HCC. And we next downregulated the activity of p-AKT with10μmol/L LY294002, and we dectected that sophocaprine and the inhibitor had synergistic effectiveness in HCC. Our data suggest that sophocaprine could through decreasing the activity of a novel AKT/GSK-3β/β-catenin axis to prevent hepatocytes from aberrant proliferation.(2) Sophocarpine inhibits epithelial to mesenchymal transition (EMT) incuced by TGF-P TGF-β was capable of epigenetically modulating CD133expression via inhibition of DNA methyltransferases in Huh7cells, implying a novel role of TGF-β in the regulation of liver cancer stem cells. Accumulating evidence has demonstrated that TGF-β modulates the expression of numerous genes relevant to tumor development. Moreover, high TGF-β levels have been correlated with advanced clinical stage of HCC.Our results confirmed that sophocaprine could inhibite EMT induced by TGF-β, which may provide a possible explanation for its effects in terms of tumor control and reduced cancer metastasis.4. Sophocarpine exhibites substantial anti-tumor effect in vivo(1) Intratumoral injection of sophocarpine regressed established tumor growthIn the control animals, the tumors grew progressively. On the contrary, tumors in sophocarpine treated mice were significantly smaller than that in control mice, including the final weight measurement. Real time PCR revealed that stem marker genes were decreased treatment of sophocaprine. In the examination of immunohistochemistry, treatment with sophocarpine in tumor decreased expression of Ki67.(2) Intraperitoneal injection sophocarpine suppressed the transplanted orthophoric tumor of liverTumors in sophocarpine treated mice were significantly smaller than that in control mice,[Conclusions]1. Sophocarpine reverses liver stem cell malignant phenotype2. Sophocarpine suppresses the ability of proliferation and invasion of hepatoma cells.3. Sophocarpine integrantly prevents hepatocytes from aberrant proliferation through down-regulating the activity of a novel AKT/GSK-3β/β-catenin axis and EMT induced by TGFβ.4. Sophocarpine exhibites substantial anti-tumor effect in vivo, which may present as an effective therapeutic agent for HCC. |
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