| Hepatocellular carcinoma(HCC) is the leading cause of cancer in the world.It is the fifth most common malignancy and its incidence is rising in the world. About 560000 new patients with liver cancer was diagnosised each year,more than half of those people are in China.It is also one of the common types of malignant tumors with poor prognosis, metastasis and high mortality. Although great progress have been made in clinical treatment,the overall prognosis of HCC population remains poor, the fatality rate second only to lung cancer, gastric cancer, and is third leading cause of death in malignant tumor.The 5-year survival rate for HCC patients is less than 5%.Adhesion and invasion is the key factor affect the therapeutic activity of HCC. It is important to explore the mechanism of adhesion and invasion of HCC and discover new drugs to treat HCC.Traditional Chinese Medicine(TCM) has been describing and treating cancer with varying success for millennia. Biejiajian pill consists of 23 herbs and is an important prescription described in the "Synopsis of Golden Chamber," the classic book of TCM. The herbs are turtle glue, salt peter, ground beeltle, scarabaeidae, pillworm, peony, hives, peachseed, ginseng, donkey-hidegelatin, Radix Bupleuri, Radix Paeoniae alba, Radix Scutellariae, Pinellia Tuber, Cortex Magnoliae officinalis, dried ginger, cinnamomum twig, Herba Dianthi, Semen Lepidii, blackberry lily.According to TCM, Biejiajian pill has the abilities to promote circulation, relieve stasis, strengthen resistance of body, eliminate pathogenic factors, as well as resolve and soften hard masses. Therefore, it is suitable for patients with liver fibrosis and cirrhosis. Biejiajian pill has been shown to can inhibit tumor proliferation and metastasis. Some of the ingredients, such as rhubarb, radixbupleuri, and scutellariae radixhave beenshown to inhibit tumor proliferation and metastasis via disturbing cellular signalingpathways such as PI3K/Akt/GSK3β, MAPK/ERK, and Cdk7/Cdc2/p34. As the cingredients of Chinese drugs are complex, and a number of compositions are not effective before bio-transformation in the body, serumpharmacology is very useful to evaluate the pharmacological effects of TCM herbalmixtures in vitro. In recent years, several Chinese herbal prescriptions, such as Baoganning, Xiao-xu-ming decoction, and Dahuang Zhechong pill, have been studied by this method.The Wnt/β-catenin signaling pathway is a closely related to tumors,and it is a hot topic in field of tumor molecular biology.The Wnt/β-catenin pathway is also required for cell proliferation, invasion and metastasis of malignant neoplasm.The Wnt/β-catenin pathway is composed of Wnt, the extracellular cysteine-rich domain of a Frizzled (Fz) family receptor,β-catenin, destruction complex and transcription factors that belong to the T cell family (TCF)/lymphoid enhancer family(LEF). Binding of Wnt proteins initiates a signaling cascade, which results in activation of β-catenin. Cytoplasmic P-catenin is bound in a complex with Axin, APC and GSK3β, which collectively comprise the destruction complex. β-catenin is phosphorylated by GSK-3β at specific serine/threonine residues located at the N-terminal region of the protein, which is facilitated by the Axin and APC.Binding of Wnt proteins to Frizzled (Fz) receptor,a seven-pass transmembrane protein,on the surface of cells induces association with the LRP5/6. This coreceptor complex triggers activation of the canonical Wnt pathway. Dishevelled (Dvl) is recruited to the Frizzled receptor, and the Fz/Dvl complex in turn relocates Axin to LRP5/6. Axin-bound GSK-3β and CK1 then phosphorylate LRP5/6 and which leads to inactivation of GSK-3β.The absence of P-catenin phosphorylation releases it from the Axin/APC/GSK3 complex, resulting in accumulation of cytoplasmic P-catenin. It then translocates to the nucleus through an unknown mechanism which may involve interaction with either components of the nuclear pore complex LEF/TCF. Once in the nucleus,β-catenin binds to LEF/TCF, initiating transcription of target genes,such as c-myc,cyclinDl,COX-2,CD44,MMP-2 and MMP-9.Wnt/β-catenin pathway participate in the carcinogenesis,metastasis and invasiveness of HCC. Approximately 20-40% of HCCs bear abnormal cytoplasmic and nuclear accumulation of P-catenin, and the level of β-catenin is positively correlated with both lymph node and intrahepatic metastasis as well as highly related to the prognosis of patients. E-cadherin, the classical cadherin, forms cell-cell contacts through homotypic interactions, which can result in the formation of stable junctions. The recruitment of P-catenin is required for this cytoskeletal linkage, which is essential for the stabilization and formation of E-cadherin-mediated cell-cell junctions, referred as adherens junctions,when P-catenin binds to the carboxyl terminus of E-cadherin and promotes transport of it through the secretory pathway. COX-2 is one target gene of Wnt/p-catenin signaling pathway, overexpression of COX-2 affects many mechanisms involved in HCC, such as angiogenesis, stimulation of cell growth, inhibition of cell apoptosis,and promotion the invasiveness and metastatic capacityof tumor cells.Cyclin Dl,a key inducer of transition from the G1 to S phase of cell cycle is regulated by P-catenin-TCF complex.Thus β-catenin activation is absolutely critical in the normal regeneration process of liver.The antitumor relationship between Biejiajian pill and the Wnt/β-catenin signaling pathway has not been reported.The aim of this study was to investigate the effect of Biejiajian pill on tumor cell proliferation and invasiveness, and the mechanism involved in the Wnt/β-cateninsignaling pathway.ObjectiveTo explore the mechanism of Biejiajian Pill for fighting against proliferation, adhesion and invasion of HepG2 by investigating the effect of Biejiajian Pill on proliferation, the cell cycle, invasion and the expression of β-catenin, phospho-β-catenin(Ser675), cyclinDl and COX-2.Methods1.Serum preparationWistar rats were kept at room temperature (21-25℃), a relative humidity of 50-60%, and a 12 h light/12 h dark cycle. Animals were provided water and food adlibitum. The research was conducted in accordance with the Guidelines for the Careand Use of Laboratory Animals of SMU. Biejiajian pill was dissolved with saline to make a suspension.Rats, male andfemale, were randomly falled into groups, One group servedas the negative control (Group N) and given a gavage of saline of the same volume as the drug suspension. Another group was set as the positive control(Group P) with 50 mg/kg sorafenib in saline. The two remaining groups were intragastrically (Group H,20-fold time) or a low dose (Group M,10-fold time) of the Biejiajian suspension, respectively. Each group received gavage daily for 3 days. Rats were fed the last time 2h before the blood collection, and the animals were anesthetized byintraperitoneal injection of 30 mg/kg sodium pentobarbital 1 h after the last gavage.Venous blood was collected from the abdominal aorta, then serum was separated(4000x,30 min),administered with a high dose 56℃ for 30 min, and stored at -20℃ until the experiment.2.Cell cultureHepG2 were cultured in DMEM medium supplemented with 10% FBS at 37℃ in a humidified CO2 (5%) incubator. The cells were subcultured until they reached the logarithmic growth phase and then were treated with rat serum from rats that weretreated with or without Biejiajian pill or sorafenib. Cells that were not treated with rat serum were used as a blank control (Group BC). Drug-containing serum was added into the cell culture up to 10% of the culture medium, as determined by preliminary tests.3.The experiental methodBy the Cell Counting Kit-8 (CCK8) assay to observe the effect of Biejiajian pill oncell viabilit on HepG2.By using PI staining method and flow cytometry to detect distribution of the cell cell cycle. Using cell adhesion assays to explore adhesion in different basement membrane components (Matrigel, FN, LN) in the influence of the Biejiajian pill on HepG2,transwell assays was used to study the invasion of Biejiajian pill on HepG2.The expression of β-catenin, cyclinDl, phospho-β-catenin(Ser675) and COX-2 were measured by Western Blot.Results1.the cell viability decreased significantly in the presence of Biejiajian pill, and this effect occurred in a time-dependent manner. Obserced the cell count of different groups under a light microscope at 100×. We found that H<M<N and there was no difference between N and BC.Compared with N/BC,the OD values of H/M were lower (p<0.05).2.The percentages of HepG2 cells in the Gl, S, and G2/M phases of growth were comparable between Group N and Group BC (P>0.05).Compared with the controls, Biejiajian pill moderately reduced the percentage of cells in the S phase and blocked the G1 phase. These effects were similar to the cells treated with sorafenib.3.Cell adhesion experiment results:the effects of experiments showed that different basement membrane components (Matrigel, FN, LN) arrived 40μg/well when the basement membrane components can achieve maximum adhesion rate. And aging experiment showed that incubate time≥ h or more when the basement membrane components can achieve maximum adhesion rate. H and M group adhesion rate is less than N group and the control group significantly (P<0.01), Biejiajian pill preparation of medicated serum of liver cancer cell adhesion has significant inhibitory effect, and the inhibition enhanced with drug concentration.4. According to time-response relationship,the time of maximum invasiveness of HepG2 cells is around 48 h.migrated cells were stained with crystal violet;The cell counts of different groups under a light microscope at 200×magnification of nine random views.Both sorafenib and Biejiajian pill decreased the invasiveness,compared with N/BC,H/M showed lower invasiveness(p<0.05), there was no difference between N and BC(p>0.05).5. Biejiajian-treated sera significantly decreased the expression of P-catenin in a dose-dependent manner.there were difference between P/H/M and N/BC P<0.05),there was no difference between P and H(P>0.05),there was no difference between N and BC(P>0.05).6.Biejiajian-treated sera significantly decreased the expression of cyclin Dl, phospho-β-catenin (Ser675) and COX-2 in a dose-dependent manner.there were difference between P/H/M and N/BC P<0.05),there were difference among P,H and M(P<0.05),there was no difference N and BC(P>0.05).ConclusionsOur results suggested that Biejiajian pill suppressed tumor cell proliferation and invasion, and this antitumor effect might be through inhibition of the Wnt/β-catenin pathway. |
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