Curcumol Mediated LOVO Cell Line Of Coloretal Cancer Proliferation And Apotosis Via IGF-1R/VEGF Pathway

Objective:This research aimed to study different concentrations of curcumol in inhibiting the proliferation in the LOVO cell line of coloretal cancer and the impact to the expression of VEGF and IGF-1R. It was used to explore possible mechanism of the curcumol inhibiting the proliferation of colorectal cancer cells LOVO cells and to verify the exact targets of curcumol and provid the basis of the scientific and effectiveness of curcumol in clinical use.Methods : Curcumol was dissolved to ethyl alcohol as the mother liquid of the solution was 4 μ mol/m L.Curcumol was used to make up the culture medium in proportion as the concentration to 0.05、0.1、0.2、0.4μmol/m L.Culture medium which contained 1% anhydrous ethanol was applied to the negative control group,while the 5-FU was the positive control group.Proliferating inhibition of coloretal cancer cell line LOVO in different times(24 h, 48 h, 72 h, 96 h) was determined by MTT; The morphological change of cell apoptosis in the LOVO cell after 48 h being cultured in the medium containing curcumol was measured by fluorescent staining of Hoechest 33258;Apoptosis rate and cell cycle arrest of the LOVO cell after 48 h being cultured in the medium containing curcumol was analyzed through Flow cytometry;RT-PCR was done to detect the expression of m RNAs of VEGF and IGF-1R;And The expression of VEGF and IGF-1R in the LOVO cell after 48 h being cultured in the medium containing curcumol was performed by Western Blot.Results: It was showed that the LOVO cell was inhibited with the different concentration of curcumol, and its inhibition of cell proliferation was gradually enhanced with concentration increasing and time prolonging. There were significant differences between the negative control group and the groups with the concentration beyond 0.2 μ mol/m L after 24 h and 48h(P<0.01). With the inhibition of the drug, there were also significant differences between the negative control group and the groups with the concentration beyond 0.1μmol/m L after 72 h and 96 h(P<0.05, P<0.01), and the difference was significant in the the negative control group compared to the treatment groups after 120 h(P<0.05). It can be observed cell shrinkage,cell falls off, chromatin gathered, nuclear pyknosis, karyorrhexis, etc by ordinary optical microscope morphology with the concentration 0.4μmol/m L after 48 h through the Hoechest 33258 stained, while apoptotic cells were characterized by presenting dense thick strong blue fluorescence and visible apoptotic body with fluorescence microscopy, and the control group was the normal cells with the nucleus of the uniform dispersion of light blue fluorescence. There were significant differences between the negative control group and the positive group with different concentrations(0.05, 0.1, 0.2, 0.4μ mol/m L) in the LOVO cell after 48 h being cultured in the medium containing curcumol(P<0.01) by FCM. There were also significant differences in cell apoptosis rate between the negative control group and the groups with the concentration beyond 0.2 μ mol/m L(P<0.05), and with the increase of the concentration of curcumol, the proportion of living cells were shrinking gradually, increasing apoptosis, and showing obvious concentration dependence. According to curcumol influence on cell cycle depending on the increase of drug dose, it can be showed that the G1 phase of LOVO cell proportion increased, change of G2 phase was not obvious, and S phase of cells decreased by the cell cycle detecting. RT-PCR showed that curcumol can reduce the expression of VEGF and IGF-1R m RNA in LOVO cells with the concentration from 0.05 to 0.4 μ mol/m L, what’s more, the expression of VEGF and IGF-1R decreased as the curcumol concentration increasing, and the differences were significant(P<0.05).The Western Blot showed that curcumol can reduce the expression of VEGF and IGF-1R protein in LOVO cells with the concentration from 0.05 to 0.4 μ mol/m L, after 48 h being cultured in the medium containing curcumol. And the quantity of the expression was gradually decreased with concentration increasing.The differences were significant between the negative control group and treatment groups(P<0.01).Conclusions: Curcumol can inhibit the proliferation of LOVO cells in coloretal cancer in different time points, and the inhibition was concentration-time depending. It can induce LOVO cell apoptosis after 48 h cultured in the medium containing curcumol in vitro, making the proliferation stagnate at the G1 phase. And the mechanism may be made through pulling down the expression of genes and protein of VEGF and IGF-1R,playing the role of resisting the tumor.