Experimental Study Of The Proliferation Inhibition Mechanism Of Oxymatrine On Human Colon Cancer LOVO

BackgroundThe chemical composition of single Chinese herbal medicine, especially effective monomers'research, is the foundation for the study of effective ingredients in compound. Oxymatrine (OM) is the effective active ingredients of Chinese herbal medicine, such as Sophora flavescens Ait, S. Alopecuroides L., S. Subprostrata Chun. Oxymatrine has strong anti-tumor properties, and anti-proliferation, anti-adhesion and anti-mobility functions for various cancer cells. Previous studies showed that Oxymatrine can directly inhibit the proliferation of colon cancer cells (SW116, SW480 cell), induce its apoptosis and influence its DNA cycle distribution. Induction of leukemia cell line differentiation was also reported. However, reports on the function of Oxymatrine for colon cancer cell line LOVO is few.ObjectivesThe research object of this study is LOVO cells, this paper discusses whether OM can inhibit the LOVO cells proliferation of function, and its mechanism is discussed, to investigate OM anti colorectal tumours role, as well as Guide clinical application and provide laboratory basisMethods1.Take LOVO cells in their logarithmic growth and cultivate in 96-well plates, use MTT colorimetric method for detecting the inhibition of Oxymatrine (OM) to human liver cancer cell line HepG2 and rat intestinal fossae stem cell line IEC-6 cell line proliferation.2. Use MTT colorimetric method detecting the survival rate of cells by different concentrations of Oxymatrine(OM) after 6h,24h,48h,72h,96h and 120h, compared with cells in normal culture, calculate inhibition rate of cell proliferation.3. Use MTT colorimetric method for detecting the influence of LOVO cell anti-proliferation after LPS stimulation by Oxymatrine(OM).4. Observe the morphological change of cells by microscope after using Oxymatrine(OM) with different concentrations.5. Detect the apoptosis rate and cycle distribution of LOVO cell after using Oxymatrine(OM) with different concentrations by flow cytometer.6. Detect the related proteins of apoptosis and adhesion as well as the related gene expressions of skeleton proteins by using RT-PCR technology.7. Immunohistochemically detect the LOVO apoptosis and adhesion related protein expression by the influence of Oxymatrine(OM).8. Quantitative ELISA detect the influence of Oxymatrine (OM) inflammatory cytokine secretion on tumors.Results1.2mg/mlOM and lmg/mlOM has significant effect on inhibiting the proliferation and killing effect to human liver cancer cell line HepG2 and rat intestinal fossae stem cell line IEC-6 cell.2. OM with different concentrations has different levels of proliferation inhibition and killing effect to LOVO cells, showing a dose-response relationship.2mg/ml group time-dependence is obvious. With the extension of time, the cells'floating, breakage, death and etc, increase apparently; the inhibition rates of 1,0.5,0.25 mg/ml group increase before 72h, and decline after 72h.3. OM has obvious effect on LPS-induced LOVO cells'over-proliferation.4. In 6h group, compared with controls, each dosing group all appears block trend of G1, S period; compared with controls, the cells go into the G2 period relatively increase in 1mg/ml,2mg/mlOM group of 24h; compared with controls, the cells go into the G2 period increase respectively in 2,1,0.5 mg/ml dosing group of 48h.5. The apoptosis ratio of cells appears time-effect and dosage-effect positive correlation after 6h,24h,48h drug effect. Compared with the control group, the ratio of cells apoptosis has statistical significance (P< 0.05) in 24h,48h of 2mg/mlOM group. And the ratio of cells apoptosis in 1,0.2, 0.1 mg/mlOM group has a trend of increase, but without statistical significance.6. Compared with normal cultured cells, P53 are cut after 24h,48h in 2mg/mlOM group; And P53 rises in the positive control group of 48hl00ng/mlTNF-a; OCLN also rises, but bcl-2, TUBAIA appear cut after 24h drug effect. There is statistical significance (P< 0.05) compared with normal cultured cells. c-myc express cut in 0.25mg/ml OM group after 48h, there was statistically significant (P< 0.05); c-myc,CDK4 express cut in 0.5mg/ml OM group after 48h, there was statistically significant (P< 0.01); c-myc,PSMD9 (proteasome 26S subunit, non-ATPase,9),CDK4 express cut in 0.5mg/ml OM group after 96h, there was statistically significant (c-myc express P< 0.05, PSMD9, CDK4 express P <0.01)7. In immunohistochemical detecting, after 24h OM-effect on cells, compared with normal cultured cells, the number of BCL-2 protein with normal expression found to be down obviously in lmg/mlOM group and 2mg/mlOM group; Ocludin protein expression increases.8. After 48h medication, compared with control group, the IL-6,IL-4 have a tendency to rise in lmg/mlOM group; the IL-Iβ, IL-6, IL-4, IL-12, TNF-a have a tendency to rise in 100ug/mlTNF-a group, but only IL-4 has statistical significance; both H-CRP groups have cut trend, but without statistical insignificance (P>0.05); IL-1β, IL-6, IL-4, H-CRP group in OM and TNF group have statistical significance; sTNFa-R in this experiment hasn't been detected.ConclusionOxymatrine(OM) can inhibit the proliferation on human colon cancer cell line LOVO. By influencing the gene expressions of bcl-2, tubulin, occludin etc, promote LOVO cell apoptosis, make the cell cycle stagnation in the G2 /M period, influence LOVO cells secrete cell factors related such as IL-1β, IL-6, IL-4, IL-12, TNF-a, H-CRP and may through increasing cell adhesion effect in order to reduce the LOVO cells invasion, transfer to exert its anti-tumor effect.