| Research source:Natural Science Foundation of Guangdong Province (9151051501000003)Purpose and significance:liver cancer is a complicated result of many factors,genes and stages, in which cell signal transduction plays a vital role for cancer occurrence and development. Recent studies have found that the incidence and development of liver cancer is closely related with Wnt/β-catenin (P-catenin) signaling pathway, changes in which may be the common pathway of tumorigenesis. In this study, we try to explore the anti-liver cancer molecular mechanism of Biejiajian Pills from an entry point of Wnt/β-catenin (β-catenin) signaling pathway, so as to provide a solid theoretical foundation for the clinical use of Biejiajian pills and the development of anti-liver cancer Chinese medicine.Methods:we cultured Human hepatoma cells (Hep3B, HepG2),and then measured expression ofβ-catenin as the key regulator of Wnt pathway in tumor cells by flow cytometry and FrpHEmRNA and DKK-1mRNA by RT-PCR after 24 hours culture with 200ul high-dose and medium-dose Biejiajian Pills Medicated serum. Research content and processes:1. drug serum PreparationSix male Wistar rats of 200±10g were randomly divided into normal group, Biejiajian pills high-dose and medium-dose group. Rats of the later groups got 3 days medication every 12 hours by intragastric administration of 12g/kg and 6g/kg Biejiajian pills(approximately 2ml each).Rats were anesthetized with chloral hydrate after 1 hour following last perfusion (fasting 12h without water limit before perfusion), blood sample were got from abdominal aorta and then placed for 5 min, followed by 10min centrifugation of 1000 rpm/min, and then lml supernatant of each were transfered to EP tubes for water bath inactivatation (56℃,30min), at last drug serum was gathered after filtration through micropore membrane。2.cell recovery and cultureHuman hepatoma cells (Hep3B, HepG2)culture bottles were put into 40℃water bath shaker of 60 rev/min for its dissolution,then transferred to 37℃water bath with slow manual shaking for 3 min, followed by 5 minutes centrifugation of 1000 rev /min。After centrifugation,supernatant were removed and replaced by DMEM mixed with 10% fetal bovine serum, then culture bottles were removed to incubator(37℃, 5% CO2), and good adherent condition was observed 10min later.Then two kinds of cells were both cultured with the mixture of 10% fetal calf serum and DMEM in Incubator(37℃,5% CO2)with a cell passage speed of 2 Generation/week.3.Measurement ofβ-catenin expression in liver cancer cells divered by Biejiajian Pills by flow cytometryCells Were trypsinized into single cell suspension 24h after drug serum addition, washed two times with PBS followed by 30min fixation with 2% paraformaldehyde, the supernatant was removed after centrifugation with the rest cells hanging in PBS +0.1 Triton-100+10% goat serum mixed staining Solution for 30 min.After a second centrifugation,the supernatant was replaced by rabbit anti-human P-catenin antibody for 60 min greenhouse Incubation, added goat anti-rabbit IgG-FITC for 30 min Incubation at room temperature after washed 2 times,and then washed another 2 times,at last filtrated through over 400 mesh nylon net for detection.4. Detection of FrpHEmRNA and DKK-1mRNA by RT-PCR.Total RNA of cells cultured with Biejiajian pills medicated serum were extracted according to TrizolReagent synopsis. GAPDH primer sequences:upstream primer 5’-CACAGTCCATGCCATCACTGC-3’, downstream primer 5’-GGTCT ACATGGCAACTGTG AG-3’(609 bp); FrpHE primer sequences:upstream primer 5-CCGTGCTGCGCTTCTTCTTCTGTG-3’, downstream primer 5’-GCGGG ACTTG AGTTCGAGGG ATGG-3’(461bp); DKK-1 primer sequences:upstream primer 5’-AGGCGTGCAAATCTGTCTCG-3’, downstream primer 5’-TGCATTTGGAT AGCTGGTTT AGT-3’(502 bp) (all primers were synthesized by The shanghai Boya Biotechnology corporation). According to the product documentation, reverse transcription reaction conditions were 50℃30min; PCR conditions were 94℃2min for hot start,94℃30sec for denaturation,55℃30sec for annealing,72℃1min for extension,40 cycles in total.Results:Compared with the control group,β-catenin levels in human hepatoma cells (Hep3B, HepG2) which were cultured with Biejiajian Pills medicated serum for 24h decreased significantly, while FrpHEmRNA and DKK-1mRNA expression Increased significantly.Conclusion:The classical Wnt/β-catenin signaling pathway is a hot spot in the field of tumor molecular biology research in recent years. Metastasis is not only one of the basic characteristics but also an important influence factor of liver cancer prognosis. Studies have found that liver cancer metastasis has a certain relationship with Wnt/β-catenin signaling pathway.β-catenin (β-catenin) expression,which is the key regulatory factor of Wnt/P-catenin signaling pathway,is positively correlated with lymph node and Intrahepatic metastasis in primary hepatocellular carcinoma (hepatocellular carcinoma, HCC) patients. P-catenin in this process has a dual role:on the one hand, P-catenin act as an important connecting bridge between E-cadherin and cytoskeleton to maintain E-cadherin adhesion; on the other hand, as a key effector of Wnt pathway,β-catenin can be translocated into nuclear to promote the tumor metastasis-related genes expression. Therefore, inhibition of the expression ofβ-catenin is expected to be a new target for anti-tumor metastasis therapy. sFRP (secreted Frizzled related protein) is a secreted glycoprotein, it maybe be able to compete with the receptor to bind wnt protein, or directly bind Wnt, thus block the Wnt signaling pathway. Studies have reported that in glioma cells, sFRP expression is associated with the MMP-2 activity, and can inhibit theβ-catenin phosphorylation,therefor inhibit glioma cell migration.The results showed thatβ-catenin expression of liver cancer cells (Hep3B, HepG2) which were cultured with Biejiajian Pills medicated serum was significantly lower than those in the control group, while the inhibitor FrpHEmRNA and DKK-1mRNA expression increased significantly compared with the control group. This prompts that Biejiajian pills can achieve the purpose of anti-tumor metastasis throughβ-catenin, FrpHE and DKK-1mRNA as well as other targets of Wnt signaling pathway inhibition. |
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