| Background:The turmeric is Zingiberaceae dried root. Curcumin, the main chemical ingredient of turmeric, is polyphenolic compound. It is commonly used in clinical treatment of abdominal pain, gastritis. And also because of its color stability and extremely low toxicity, curcumin has been widely used in food additives and dyes. The pharmacological effect of curcumin is including anti-tumor, and reversing tumor multidrug resistance to chemotherapeutic drugs, anti-inflammatory, anti-oxidation, anti-atherosclerotic, anti-HIV.Colorectal cancer remains a significant cause of morbidity and mortality worldwide, ranking No.5in the common malignant tumors in China, the inferior gastric cancer, esophageal cancer, liver cancer, lung cancer. But the statistics in recent years show that, as the people’s living standards improving, diet changed, the incidence of colorectal cancer in China is increasing year by year, and colorectal cancer has become one of the fastest rising incidence of malignancy in most regions Therefore, how to prevent the occurrence of colorectal cancer and patients with colorectal cancer have been early diagnosis and treatment are hot topics.Chemotherapy is the important part of comprehensive treatment for patients with colorectal cancer. The FOLFIRI regimen is composed of irinotecan,5-FU and LV, which is the current standard chemotherapy of patients with advanced colorectal cancer. With irinotecan widely used in clinical application, it has found that, the efficiency of irinotecan for the tumor cells was49%mainly due to their multidrug resistance, irinotecan is semi-synthetic camptothecin derivative extracted from chinese unique plant Camptotheca acuminata, its antitumor mechanisms is that Irinotecan (predominantly in the form of SN38) binds to the Top I-DNA complex, stabilizing it and preventing re-ligation, Collision with advancing replication forks results in the formation of double stranded DNA breaks. These breaks activate cell cycle arrest in G2phase and, if unrepaired, can cause cell death, especially in the S phase of the cell toxicity. DNA topoisomerases (topos) are essential enzymes that regulate the topological state of DNA during cellular processes such as replication, transcription, recombination, and chromatin remodeling. Topoisomerase Ⅰ (Topo Ⅰ) is a ubiquitous nuclear enzyme which catalyzes the relaxation of superhelical DNA generating a transient single strand nick in the duplex, through cycles of cleavage and religation. Topoisomerase Ⅱ (Topo Ⅱ) mediates the ATP-dependent induction of coordinated nicks in both strands of the DNA duplex, followed by crossing of another double strand DNA through the transiently broken duplex. Although the biological functions of Topoisomerases are important for ensuing genomic integrity, the ability to interfere with enzymes or generate enzyme-mediated damage is an effective strategy for cancer therapy and, in this connection, DNA topos (Ⅰ and Ⅱ) proved to be the excellent targets of clinically significant classes of anticancer drugs. Actually, specific Topo Ⅰ and Topo Ⅱ inhibitors reversibly trap the enzyme-DNA complexes, thus converting Topos into physiological poisons, able to produce permanent DNA damage, which triggers cell death. Due to the high expression of topoisomerase in the tumor tissue, the topoisomerases are the targets for many antineoplastic agents. The expression of topoisomerase I was downregulated in the colorectal cancer cells treated by Irinotecan, which results in the loss of localization of the enzyme, also have been found to correlate with resistance to irinotecan. At the same time, it has reported that Patients with high Topo1gained very marked benefit from irinotecan, therefore, upregulated the expression of topoisomerase I is conducive to increase the target for irinotecan, and enhance its killing effect to tumor cells, which results in reversing the multidrug resistant to irinotecan in colorectal cancer cells.Curcumin combines with the chemotherapeutic drug is Top of the present study, previous studies have found that curcumin could enhance the efficacy of the chemotherapy drugs through different mechanisms, and also regulate reversal of multidrug resistance to chemotherapeutic drugs in colorectal cancer cells. It has found that curcumin via the regulation of epithelial growth factor receptor (EGFR) and insulin-like growth factor1receptor (IGF-1R) signaling pathway, inhibited the expression of the epidermal growth factor receptor (EGFR) and insulin-like growth factor1receptor (IGF-1R), resulted in enhancing killing effect of5-FU and oxaliplatin to colon cancer HCT-116and HT-29cell lines.This study using curcumin combined with irinotecan to treat colorectal cancer lovo cells, is to find out whether curcumin can enhance killing effect of irinotecan to lovo cells, whether the synergism exists between curcumin and irinotecan, and also to investigate whether curcumin can upregulate the expression of topoisomerase I in colorectal cancer lovo cells to increase acting points of irinotecan, which is aimed at providing the experimental basis for their clinical joint use.Objective:Investigating the influence of curcumin combined with irinotecan to the growth of colorectal cancer Lovo cells in vitro to define whether synergism exists between curcumin and irinotecan on colorectal cancer lovo cells, which is aimed at providing the experimental basis for their clinical joint use.Method:1.Using MTT assay to detect the proliferation activity of colorectal cancer lovo cells treated by crcumin and irinotecan respectively at different times:making up different concentrations of curcumin and irinotecan solution, which the concentration of curcumin solution is1.25,2.5,5,10,15,20,30,40μg/ml respectively, and the concentration of irinotecan solution is1.925,3.85,7.7,15.4,30.8,61.6,132.2μg/ml respectively, colorectal cancer lovo cells cultured in vitro were digested to be inoculated in96-well plates with6replicates by5*103/well, we set up the experimental group, negative control group and blank group. Pre-formulated curcumin and irinotecan solution were added to colorectal cancer lovo cell cultured in vitro for24,48,72,96,120h under dark condition. Detecting the proliferation activity of colorectal cancer lovo cells by MTT assay to draw the growth inhibition curve and screen out the Maximum concentrations of curcumin, which has no inhibitory effect on the proliferation of lovo cells. And using the statistical software SPSS13.0to calculated the half maximal inhibitory concentration(IC5o) of irinotecan on lovo cells at different times;2. Using the flow cytometry to detect the apoptosis rates of lovo cells treated by curcumin and irinotecan at different time:3*105ml lovo cells were seeded in60mm2Petri dish, treated by co-administration method (the ratio preparation of curcumin and irinotecan was1:1, including the experimental concentrations of curcumin were5,10μg/ml respectively, the experimental concentration of irinotecan was56.201μg/ml, control group and experimental groups were set up with6replicates) and Sequential administration method (first treating lovo cells by5μg/ml,10μg/ml curcumin for24h,48hours respectively and then transforming into irinotecan for24h, the concentrations of curcumin and irinotecan as above) of the two drugs. At the time of each termination point detecting the apoptosis rate of lovo cells in each group by flow cytometry;3. Using RT-PCR, immunofluorescence, western blot to detect the expression of topoisomerase I mRNA and protein of lovo cells treated by different concentrations of curcumin at different times:3*105/ml lovo cells were seeded in60mm2Petri dish, and control group and experimental groups were set up with3replicates, then treating lovo cells by5,10μg/ml of curcumin for24,48h, At the time of each termination point detecting he expression of topoisomerase Ⅰ mRNA and protein of lovo cells by RT-PCR, immunofluorescence, western blot. Gel-Pro Analyzer software was employed to quantitively analysed Photodensity after scaning western films. The ratio of interest Protein and β-actin(IOD) was used to reflect relative expression of protein.Results:1. The effect of curcumin on the proliferation of lovo cells: Used different concentrations of curcumin in colorectal cancer Lovo cells, with the concentration of curcumin increased and the time prolonged, the proliferation levels of lovo cell were significantly decreased, curcumin inhibited the proliferation of colon cancer lovo cells in time and dose-dependent way, in which the5μg/ml curcumin lost the inhibition effect. Compared to the negative control group, the absorbance value of lovo cells measured by570nm wavelength showed no significant difference (P24h=0.518, P48h=0.433, P72h=0.222, P96h=0.648, P120h=0.117)(Figure1below).2. The effect of irinotecan on the proliferation of lovo cells: At different time periods, the half inhibitory concentration of irinotecan to lovo cells is different in different concentrations(IC5024h=56.201μg/ml, IC5048h=21.183μg/ml, IC5072h=20.211μg/ml, IC5096h17.246μg/ml, IC50120h=7.891μg/ml), and the inhibition of proliferation of lovo cells was also in time-and dose-dependent manner (Figure2).3. The apoptosis rate of Lovo cells induced by different administration modes of Curcumin combined with irinotecan in different time:Ⅰ.Mixing administration:Contrast with the single-agent irinotecan group at the same time, treating lovo cells by combining the5μg/ml curcumin with irinotecan for24h,48hours, of which the mixture concentration ratio was1:1, the early apoptotic rate of lovo cells between the two groups was significant differences (P=0.029), while the late and total apoptotic rate were no significant differences (P=0.314,0.229) for24h; the early, late and total apoptotic rates had a significant difference (P=0.008,0.001,0.000) between the two groups for48h.Ⅱ.Sequential administration:first treating lovo cells by5μg/ml,10μg/ml curcumin for24h,48hours respectively and then transforming into irinotecan for24h, compared with the control group, the apoptotic rate of irinotecan to lovo cells increased significantly in curcumin treatment group, with the concentration of curcumin increased and the time prolonged, the apoptotic rate increased distinctly. In which statistical differences also existed.Ⅲ. Comparison of the different administrations:Compared with Mixed administered treatment group, the early, late and total apoptotic rates of lovo cells in the sequential administration treated by5μg/ml curcumin for24h were significantly higher (P<0.05); The early, late and total apoptotic rates were also significantly higher for48h (P<0.05).4. The expression of topoisomerase Ⅰ protein induced by different concentrations of curcumin in lovo cells at different time: Compared with the control group, the expression of topoisomerase I protein in experimental group could be upregulated in lovo cells for24,48h, and with the concentration of curcumin increased and the time prolonged, the expression of topoisomerase I protein was increasing.5. The expression of topoisomerase I mRNA induced by different concentrations of curcumin in lovo cells at different time: Compared with the negative control group, the expression of topoisomerase I mRNA in the5μg/ml treatment group has no significant difference (P=0.952), but significantly increased in the10μg/ml treated group (P=0.049) for24hours; Compared with the negative control group, the expression of topoisomerase I mRNA in the5μg/ml treatment group has no significant difference (P=0.952), but significantly increased in the10μg/ml treated group(P=0.031) for48hours.6. The expression of topoisomerase I protein induced by different concentrations of curcumin in lovo cells at different time: Compared with the negative control group, the expression of topoisomerase I protein was significantly increased in the5μg/ml treatment group (P=0.011), but no statistical differences in the10μg/ml treated group (P=0.384) for24hours; Compared with the negative control group, the expression of topoisomerase I protein in the5μg/ml treatment group was significantly decreased (P=0.006), but significantly increased in the10μg/ml treated group (P=0.001) for48hours.CONCLUSION:1. Both curcumin and irinotecan can effectively inhibit the proliferation of colorectal cancer lovo cells in time-dose dependent way.2. Curcumin can enhance the killing effect of irinotecan to lovo cells. They cooperate with each other to exert inhibitory effect on colorectal cancer lovo cells. Compared to the mixed administration, curcumin is more effective in enhancing the killing effect of irinotecan to lovo cells by using the Sequential administration.3. Curcumin can upregulate the expression of topoisomerase I mRNA and protein of lovo cells, which may enhance the killing effect of irinotecan to lovo cells by increasing acting points of irinotecan. |
PDF Full Text Request