Effects Of Biejiajian Pills On Wnt/β-catenin Signaling Pathway Molecules And Target Genes In Hepatocellular Carcinoma Cells

Hepatocellular carcinoma (HCC) is one of the most common malignant tumour in China, ranking third in the global cancer death.About 250,000-1,000,000 people die of it every year. The main treatments of HCC are surgery, radiation, chemotherapy and biological treatment.However, all kinds of treatment is unable to inhibit tumor growth and metastasis fully. According to the signs and symptoms of HCC,it can be called Jiju or Zhengjia in Traditional Chinese medicine(TCM). HCC is the result of a variety of pathogenic factors synergy and closely related to the deficiency of qi.,The prevention and principle of TCM is mainly involved to strengthen body resistance, relieve stasis, eliminate pathogenic factors, promote circulation, as well as resolve and soften hard masses. Treatment of TCM in HCC maining refer to reduce the patients’ clinical symptoms, improve the quality of survival, prolong survival period curative effect, reduce side effects, etc.Hepatocellular carcinoma lack of typical symptoms, early onset hidden, often ignored by people, so that missed diagnosis misdiagnosis, brings irreparable loss to patients. Clinical symptoms, the disease often have entered the middle and late, bring certain difficulty to cure. Among them, the invasion and metastasis are the basic characteristics of liver cancer, is also the key factors influencing the effect of the treatment of liver cancer, made for the treatment of tumor recurrence and metastasis after becoming the key factor of prolong patient survival. Invasion and metastasis of HCC is a multi-step, complex process. Every link involves a series of gene regulation. The Wnt/beta - catenin signaling pathway gene mutations and/or abnormal expression lead to cell proliferation, metastasis and invasiveness.Wnt signaling pathway is a very conservative signaling pathways in biological evolution. In normal cells, it is non-activation.Its abnormal expression is closely related to human diseases. According to molecular mechanism, Wnt signaling pathways can be classed to classic Wnt pathway and the non-classic Wnt pathway, and classic Wnt pathway, called Wnt/β-catenin pathway, is composed of Wnt, transmembrane receptor Frizzled (Fz) family receptor, cytoplasmic protein and downstream target gene. Classic Wnt signaling pathways play a temporary role in organ development, differentiation of embryonic axis and cell regeneration, while its exceptionally sustainable activities involved in the development of a variety of malignant tumors.When Wnt/β-catenin is non-actived, Wnt, acting as extracellular signaling molecules, is combined to specific protein receptor Frizzled on cell membrane, P-catenin is bound to Axin, APC and GSK3P, called the destruction complex, then β-Catenin is phosphorylated by GSK-3β and degraded, ultimately β-catenin remain at a low level in the cytoplasm. When Wnt/β-catenin is actived, Wnt is combined to specific protein receptor Frizzled on cell membrane, under the synergy of LRP5/6, Dsh is recruited to the Frizzled receptor, which leads to phosphorylation of GSK-3β and cannot Phosphorylate β-catenin, resulting accumulation of P-catenin in cytoplasmic. β-catenin,acting as important signaling molecules in Wnt/β-cateni signaling pathways, plays an important role in cell adhesion, regulating cell growth, differentiation and apoptosis, etc.p-catenin translocates to the nucleus and combines with T-cell factor/lymphoid enhancer factor (LEF/TCF). Activation of CD44v6, VEGF,MMP-2,cyclinDl, c-myc and a series of proto-oncogenes can result in cell proliferation, differentiation and maturation. GSK-3β, play an important role in the process of beta-catenin phosphorylation, can promoteβ-catenin phosphorylation and accelerate the degradation. When GSK-3β deactivatd by phosphorylation, P-catenin can avoid being phosphorylation and gradually accumulated in the cytoplasm.Biejiajian pills consists of 23 herbs and is one of the most important prescriptions described in the "Synopsis of Golden Chamber," the classic book of TCM. Biejiajian pills has the abilities to promote circulation, relieve stasis, strengthen body resistance,eliminate pathogenic factors, as well as resolve and soften hard masses; therefore, it is suitable for patients with liver fibrosis and cirrhosis,liver and spleen enlargement, uterine fibroids.In the clinical treatment of liver cancer, Biejiajian pill have curative effect, low side effect, etc. ObjectiveBy studying the influence of Biejiajian pills on signal molecule activity and target gene expression of Wnt/β-catenin signaling pathway on HepG2, to explore the key molecule of Wnt/β-catenin signaling pathway in metastasis and invasiveness of HCC. Illuminating the relationship between HCC, exploring mechanisms of how Biejiajian pills inhibit the metastasis and invasiveness of HCC Methods and Contents1.Serum preparation:Wistar rats were kept at 21-25℃, relative humidity of 50-60%, and half day and night surroundings. Animals were provided water and food uncontrolled. Thirty-two rats were randomly divided into high-dose group(Group H), low-dose (Group L),negative control (Group NC) and positive control(Group P). Group H、 Group L、Group N was fed with gavage of Biejiajian pill at 20-fold and 10-fold clinical doses and normal saline. Group P was fed with 50 mg/kg sorafenib in distilled water. Each group gaind gavage daily for 3 days. Animals were anesthetized by intraperitoneal injection of 3% sodium pentobarbital at the 4th day,Venous blood was collected from the abdominal aorta, stewing 1h, then serum was separated(4000rpm,30 min),administered with a high dose 56℃ for 30 min,0.22 μm filter eliminatd bacteria and stored at-20℃2.Cell culture:HepG2 were cultured in 10% FBS DMEM at 37℃, humidified CO2 (5%) incubator.When reaching the logarithmic growth phase, the cells were subcultured, seeded into 6 well plat at 2.5×106/ml, Randomly divided into 5 groups: Drug serum group(group H/L/NC), sorafenib positive control group(group P) and group in which cells were fed with 10% FBS DMEM used as blank control (Group BC). After cells being adherent supernatant was absorbed,5% Drug-containing serum was added into the cell culture,48h later cells were determined by preliminary tests.3. The expression of β-catenin and GSK-3βand p-GSK-3β and AKT and p-AKT were measured by Western blotting and Immunofluorescence.4. The expression of GSK-3β and p-GSK-3p and AKT and p-AKT were measured by Western blotting5. Luciferase assay were used to confirm the effect of Biejiajian pill on β-catenin/TCF4 complex activities.6. The expressions of CD44v6 and VEGF were detected using, immunohistochemistry.7. The expressions of MMP-2 were determined by Western blotting.8. The expressions of DKK-1 and FrpHE mRNA were determined by RT-PCR.Data were presented as mean ± standard deviation (SD). Statistical analysis was performed with one-way analysis of variance (ANOVA).Results were considered statistically significant when P< 0.05.Results1.The expression of β-catenin measured by Western blotting:Compared with group BC and N, the expression of β-catenin protein in the nuclei of group H and group P can be reduced markedly(P<0.05).There are no difference among group L、 BC and NC (P> 0.05). Compared with group BC and NC, the expression of β-catenin protein in the cytoplasm of group H and group P can be reduced markedly(P<0.05).There are no difference among group L、BC and NC(P>0.05).It shows that Biejiajian can significantly reduce the expression of β-catenin protein both in the cytoplasm and the nuclei on HepG2 in a dose-dependent manner. Immunofluorescence further show that the results.2.The expression of GSK-3β、p-GSK-3β measured by Western blotting: Compared with group BC and N, the expression of p-GSK-3β of group H and group P can be reduced markedly(P< 0.05).There are no difference among group L、BC and NC (P> 0.05).The expression of GSK-3β between groups had no significant difference(P>0.05).It shows that Biejiajian pill had no effect on expression of GSK-3 beta, but it can significantly reduce the expression of phosphorylated GSK-3 beta on HepG2.3.The expression of AKT、p-AKT measured by Western blotting:Compared with group BC and N, the expression of p-AKT of group H and group P can be reduced markedly(P<0.05).There are no difference among group L-. BC and NC (P >0.05).The expression of AKT between groups had no significant difference (P> 0.05).It shows that Biejiajian pill had no effect on expression of AKT, but it can significantly reduce the expression of p-AKT on HepG2.4. Luciferase assay was used to determine the effect of P-catenin/TCF complex activities. Result shows that Biejiajian Pills suppressed the activated T-cell factors in a dose-dependent manner as compared with the control groups. However, it was weaker than sorafenib on β-catenin/TCF complex activities.5.The expressions of CD44v6 and VEGF were detected by immunohistochemistry:Compared with group BC and NC, the expression of CD44v6 and VEGF of group H can be reduced markedly(P< 0.05).There are no difference among group L、 BC and NC (P>0.05). It means that Biejiajian pill can decreased expression of CD44v6 and VEGF in a dose-dependent manner.6.The expression of MMP-2 measured by Western blotting:Compared with group BC and NC, the expression of MMP-2 of group H and group P can be reduced markedly(P< 0.05).It means that Biejiajian pill can decreased expression of MMP-2.7.The expressions of DKK-1、FrpHE mRNA were determined by RT-PCR: Biejiajian Pills showed obvious up-regulation of DKK-1 mRNA expression in a dose-dependent manner (P<0.05). The expression of FrpHe mRNA showed no significant differences (P> 0.05)Conclusions1.Biejiajian Pills can significantly reduce the expression of β-catenin by decreasing the phosphorylation of GSK-3β and AKT, suppressing the activated T-cell factors.2.Biejiajian Pills can cause down-regulation of the target genes protein CD44v6, VEGF and MMP-2.3.Biejiajian Pills can significantly reduce the expression of DKK-1, The anticancer effect of Biejiajian pill may be related to inactivation of the Wnt signaling pathway.Biejiajian Pills can regulate and control the Wnt signaling pathway, thus inhibiting liver cancer cell proliferation, invasion, metastasis and the formation of tumor angiogenesis, further inhibiting tumor metastasis and invasion.