Antitumor Effects Of Curcumin On The Proliferation,Apoptosis And Metastasis Of Human Colorectal Carcinoma Cells

Research BackgroundColorectal cancer(CRC)is one of the most common malignant tumors in humans.The therapeutic means that colon cancer are operation excise,radiation oncology,chemotherapy,immune therapy,and molecular target therapy.Early surgical excision is mainly suitable for a limited transfer of cancer patients,such as stage Ⅰ colon cancer.Ⅱ stage and Ⅲ colon cancer are treated by surgery combined with chemotherapy.The treatment of advanced colon cancer is mainly chemotherapy.During the treatment of various stages of colon cancer,chemotherapy has become the primary treatment.Fluorouracil(5-FU)is commonly chemotherapeutic agents in the treatment of colon cancer.Fluorouracil is an essential drug for colorectal cancer.Fluorouracil is the most widely used anti-tumor drug in clinical practice and has a broad spectrum of anticancer properties.However,long-term administration of 5-FU will cause strong toxic and side effects in patients,such as severe gastrointestinal reactions,bone marrow suppression,neurotoxicity,and drug resistance.So,it is necessary to develop new anticancer drugs.In recent years,researchers have made positive progress in isolating and screening natural anti-tumor active ingredients with no toxicity or low toxicity in Chinese herbal medicine.Curcumin is a type of polyphenol extracted from turmeric,and it is the main component of turmeric.Many studies demonstrated that curcumin might possess anti-infection,anti-inflammatory,antioxidant,and tumor growth inhibitory properties.It has significant inhibitory effect on all kinds of common tumors.However,the underlying molecular mechanism of curcumin’s inhibition of colon cancer cells remains unclear.ObjectiveThe aim of the present study was to investigate the effects of curcumin on the viability,migration and apoptosis of human colorectal carcinoma cells.We verify whether the anti-tumor effect of curcumin can reach the level of fluorouracil.Besides,We explored the possible underlying molecular mechanisms to determine whether curcumin may be considered a potential drug for treating patients with colorectal cancer.MethodsDifferent doses of curcumin and fluorouracil treated colorectal cancer cells in vitro.The following experimental methods were used to detect the anti-tumor effects of curcumin.1.The proliferation ability of HCT-116 cells and SW620 cells that were treated with different doses of curcumin and 5-FU was analyzed using a Cell Counting Kit-8(CCK-8)assay.To explore the IC50 of curcumin on colon cancer cells,and formulate the following experimental groups’ concentration.2.The proliferation ability of HCT-116 and SW620 cells that were treated with different doses of curcumin and 5-FU for 24,36,and 48 h was analyzed using a CCK-8 assay.3.Cell viability was detected using a colony formation assay.4.The cell cycle was detected using flow cytometry-based on propidium iodide(PI).5.The morphological change of apoptosis was detected using fluorescence microscope.6.The apoptosis of HCT-116 cells and SW620 cells were detected using flow cytometry assay.7.The relative expression level of Fas、FADD、caspase-8、caspase-3 mRNAs were detected using real-time RT-PCR(RT-qPCR)analysis.8.The relative expression level of NF-κB、Fas、FADD、caspase-8、caspase-3proteins were detected using Western blot(WB)assay.9.The migration ability of HCT-116 cells were detected using a wound healing assay.10.The invasion ability of HCT-116 cells was detected using a Transwell assay.11.The colon cancer HCT-116 cells metastasis in mouse lung was detected by manual metastasis model assay.12.MMP-9 expression in HCT-116 cells was analyzed using a zymography assay.13.The effects of curcumin on the expression levels of E-cadherin and Claudin-3 were analyzed using western blot assay.Results1.The CCK-8 assay results indicated that the IC50 of HCT-116 cells was 22μM.The IC50 of colon cancer SW620 cells treated with curcumin for 24 h was 49 μM.2.The CCK-8 assay results indicated that the proliferation of HCT-116 cells and SW620 cells in the drug treatment groups differed compared with that in the control group.The proliferation inhibitory effect increased gradually with increasing drug concentration and the treatment time.The inhibition rate in each group was significantly inhibited compared with the control group(*P<0.05).Besides,the high curcumin dose group’s inhibition rate was not significantly different from those of the 5-FU group(P>0.05).3.The results of colony formation assay indicated that the cell viability in each group was significantly inhibited,compared with the control group(*P<0.05).The cell’s vitality inhibitory effect was in a dose-dependent manner.Besides,the high curcumin dose group’s inhibition rate was not significantly different from those of the 5-FU group(P>0.05).4.The results of flow cytometry assay indicated that curcumin could significantly induce the arrest of HCT-116 cells in the S phase,compared with the control group(*P<0.05).5.The results of the morphological observation indicated that curcumin could significantly induce morphological changes in HCT?116 cells(*P<0.05).The effect was in a dose-dependent manner.Besides,the high curcumin dose group’s apoptosis rate was not significantly different from those of the5-FU group(P>0.05).6.The flow cytometry assay results indicated that the proportion of apoptotic cells significantly increased,after the HCT-116 cells and SW620 cells treated by curcumin for 24 h,compared with the control group(*P<0.05).Besides,the high curcumin dose group’s apoptosis rate was not significantly different from those of the 5-FU group(P>0.05).7.The results of wound healing assay indicated that the migration ability of HCT-116 cells in the drug treatment groups differed compared with that in the control group.The cell migration inhibitory effect increased gradually with increasing drug concentration.The healing rate in each group was significantly inhibited compared with the control group(*P<0.05).Besides,the high curcumin dose group’s migration healing rate was not significantly different from those of the 5-FU group(P>0.05).8.The results of the Transwell assay indicated that the invasion ability of HCT-116 cells were significantly inhibited by curcumin(*P<0.05),andthe inhibitory effect of cell invasion was dose-dependent manner.Besides,the invasion inhibition rate of the high curcumin dose group was not significantly different from those of the 5-FU group(P>0.05).9.The manual metastasis model assay results indicated that curcumin could significantly inhibit the arrest of HCT-116 cells in the lungs of mice compared with the control group(*P<0.05),after the HCT-116 cells treated by curcumin.10.The zymography assay results indicated that the expression level of MMP-9 was significantly reduced(*P<0.05).And its expression level decreased with increasing concentration.11.The results of the reverse transcription-quantitative PCR(RT-qPCR)analysis indicated the relative expression levels of Fas,FADD,caspase-8,and caspase-3 mRNA in HCT-116 cells were significantly increased compared with the control group(*P<0.05).12.The Western blot results indicated that the relative expression levels of Fas,FADD,caspase-8,caspase-3,and E-cadherin protein in HCT-116 cells were significantly increased,and the relative expression levels of NF-κB and Claudin-3 protein were significantly inhibited(*P<0.05).Conclusion1.Curcumin significantly inhibited the growth and proliferation of colon cancer HCT-116 and SW620 cells in a dose-and time-dependent manner,and its effect was comparable to that of 5-FU.2.Curcumin significantly induced HCT-116 cell cycle arrest in the S phase,thereby inhibiting their growth.3.Curcumin significantly induced apoptosis in HCT-116 and SW620 cells in a dose-and time-dependent manner,and its effect was comparable to that of5-FU.4.Curcumin significantly inhibited the migration and metastasis of HCT-116 cells in vitro and in vivo.The effect was in a dose-dependent manner,and its effect was comparable to that of 5-FU.5.The potential molecular regulatory mechanism of curcumin inducing apoptosis in HCT-116 cells may be related to the activation of the Fas death receptor signaling pathway.The potential molecular mechanism of curcumin inhibiting the metastasis of HCT-116 cells may be related to the inhibition of EMT.The common upstream target may be associated with NF-κB.