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@#[Abstract] Objective: To investigate the effect of double oxidase 2 (DUOX2) on the sensitivity of colorectal cancer (CRC) cells to 5-fluorouracil (5-FU). Methods: CRC cell lines DLD-1, SW480, HCT116, SW620 and normal intestinal epithelial cell line NCM460 were selected, and the expression of DUOX2 in these cell lines were detected by qPCR. DUOX2 expression in HT-29 and HCT116 cells was stably knocked down by lentivirus infection technique. The knockdown efficiency was detected by qPCR and WB. Cells in sh-Control and sh-DUOX2 groups were treated with 5-FU at different concentrations (0, 5, 10, 20, 40, 80, 120 μg/ml). The effects of 5-FU on cell proliferation, apoptosis and cell cycle were detected by CCK-8 method and flow cytometry. HT29 cell transplanted xenograft model in nude mice was constructed to observe the effect of DUOX2 gene on the treatment efficacy of 5-FU. Results: the expression level of DUOX2 mRNA in CRC cells was significantly higher than that in NCM460 cells (P<0.05 or P<0.01). Compared with sh-Control group, the mRNA and protein expressions of DUOX2 in sh-DUOX2 group were significantly decreased (all P<0.01); the sensitivity of cells to 5-FU was enhanced, the apoptosis rate and the ratio of cells at G0/G1 phase were significantly increased (all P<0.01), and the ratio of cells at G2 and S phase was significantly decreased (all P<0.01). There was no significant difference in tumor volume and mass between sh-Control group and sh-DUOX2 group without 5-FU treatment (all P>0.05), but the volume and mass of transplanted tumor in sh-DUOX2+5-FU group after 5-FU treatment was significantly lower than that in sh-Control+5-FU group (all P<0.01). Conclusion: The sensitivity of CRC cells to 5-FU can be significantly enhanced by knocking down DUOX2 gene.
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Objective To evaluate the effect and mechanism of down-regulating the expression of REV7 on the radiosensitivity of human colon cancer cell HCT116.Methods HCT116 cells were cultured and the expression of REV7 was down-regulated by RNA interference technique.HCT116 cells were divided into the blank group,negative control transfected with negative RNA oligo group and REV7 expression down-regulation transfected with REV7 RNA oligo group,respectively.The cell proliferation was determined by colony formation assay.The expression levels of the proteins of relevant genes were detected by Western blot.The level of cell apoptosis and non-homologous end joining was evaluated.Results The colony formation rate was significantly reduced in THE REV7 siRNA group after 6Gy irradiation (P＜0.05).The down-regulating efficiency rate of REV7 gene was ＞ 60％ in the REV7 siRNA group.The expression levels of γ H2AX and Caspase9 were significantly up-regulated,whereas those of KU80 and XRCC4 were remarkably down-regulated in the REV7 siRNA group (all P＜0.05).Conclusions The radiosensitivity of human colon cancer cell HCT116 can be increased by down-regulating the expression of REV7.The underlying mechanism may be related to the lower incidence rate of non-homologous end joining.
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Objective@#To evaluate the effect and mechanism of down-regulating the expression of REV7 on the radiosensitivity of human colon cancer cell HCT116.@*Methods@#HCT116 cells were cultured and the expression of REV7 was down-regulated by RNA interference technique. HCT116 cells were divided into the blank group, negative control transfected with negative RNA oligo group and REV7 expression down-regulation transfected with REV7 RNA oligo group, respectively. The cell proliferation was determined by colony formation assay. The expression levels of the proteins of relevant genes were detected by Western blot. The level of cell apoptosis and non-homologous end joining was evaluated.@*Results@#The colony formation rate was significantly reduced in THE REV7 siRNA group after 6Gy irradiation (P<0.05). The down-regulating efficiency rate of REV7 gene was>60% in the REV7 siRNA group. The expression levels of γH2AX and Caspase9 were significantly up-regulated, whereas those of KU80 and XRCC4 were remarkably down-regulated in the REV7 siRNA group (all P<0.05).@*Conclusions@#The radiosensitivity of human colon cancer cell HCT116 can be increased by down-regulating the expression of REV7. The underlying mechanism may be related to the lower incidence rate of non-homologous end joining.
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Objective:To explore the molecular mechanism of cantharidin(CTD) in inducing morphological changes and dissociation in human colon cancer HCT116 cells, in order to study the correlation with tumor metastasis and provide a new basis for clinical use of cantharidin. Method:Different concentrations of CTD (0, 2.5, 5, 10, 15, 20 μmol·L-1) were added to each hole to culture for 7 to 10 days, so as to determine the inhibitory effect of CTD on HCT116 cells; and changes of F-actin cytoskeleton and integrin in HCT116 cells were detected by immunofluorescence staining. Western blot analysis of protein expressions of RhoA,RhoB,RhoC,Cdc42 and Rac1/2/3 of Rho family were performed before and after cantharidin treatment. overexpression of RhoA was constructed by plasmid transient transfection, and effect of 10 μmol·L-1 cantharidin on the morphology and adhesion of overexpressing cells was also observed. Result:Cantharidin induced cytoskeletal remodeling and decreased integrin content in HCT116 colon cancer cells. RhoA protein was a member of Rho enzyme family with the greatest variation after cantharidin action. The proportion of transfected RhoA cells with green fluorescence was about 60%, the expression of RhoA protein in constructed RhoA overexpression cells was significantly increased, compared with wild-type HCT116 cells (PPConclusion:Cantharidin can inhibit the expression of RhoA protein, induce the morphological changes of HCT116 cells and weaken the adhesion to the basement, thereby inhibiting cell migration.
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Objective@#To investigate the effect and mechanism of LncRNA ANRIL on the radiosensitivity of HCT116 cells line and nude mouse transplant tumors.@*Methods@#The expression of LncRNA ANRIL in colorectal cancer cells was detected by qPCR. The negative control siRNA, ANRIL siRNA, miR-NC mimic, miR-195 mimic, miR-NC inhibitor and miR-195 inhibitor were transfected into HCT116 cells, and marked as negative control group, silencing ANRIL group, overexpressing miR-NC group, overexpressing miR-195 group, inhibiting miR-NC group and inhibiting miR-195 group, and the HCT116 cells without any treatment were marked as the blank control group. The clone formation assay was used to detect radiosensitivity of colorectal cancer cells, flow cytometry was used to detect apoptosis. The web site, StarBase, was used to predict the downstream miRNAs of ANRIL and dual luciferase reporter gene assay was used to further verify. Subcutaneous tumor transplantation assay was used to detect the effect of ANRIL on the growth of colorectal cancer cells after irradiation.@*Results@#After irradiation with 2, 4, 6 and 8 Gy, the cell survival fraction of silencing ANRIL group was significantly decreased when compared with that of negative control group (P<0.05), and the radiosensitivity ratio was 1.52. The apoptosis rate of the silencing ANRIL+ 4 Gy group was significantly higher than that of the negative control+ 4 Gy group ((27.86±2.78)% vs. (12.06±1.46)%, P<0.05). The results of the experiment on nude mouse transplant tumors showed that the tumor volume in the negative control group was lower than that of the silent ANRIL group on days 13, 16, 19, 22 and 25 ((234±66) mm3, (273±63) mm3, (296±72) mm3, (321±85) mm3 and (403±94) mm3 vs. (357±79) mm3, (485±124) mm3, (617±143) mm3, (764±174) mm3 and (985±221) mm3P<0.05). MiR-195 is a target gene of ANRIL, and inhibition of miR-195 can reverse the inhibitory effect of silencing ANRIL on radiosensitivity, apoptosis and xenografts of HCT116 cells.@*Conclusions@#LncRNA ANRIL regulates the radiosensitivity of colorectal cancer cells by miR-195, which may provide a new sensitizing target for clinical colorectal cancer radiotherapy.
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@#[Abstract] Objective: To investigate the impact of FBXW7 gene mutation on cell proliferation, apoptosis, migration, and invasion processes of colorectal cancer HCT-116 cell line. Methods: Recombinant plasmids carrying wild-type and mutant-type FBXW7 SNP were constructed and transfected into HCT-116 cell line; the FBXW7 protein expression level in HCT-116 strains after transfection was detected by Western blotting. Subsequently, cell proliferation capacity was tested by CCK-8 assay; tumor cell colony formation ability was tested by HTCA; cell apoptosis function was tested by FCM; cell migration and invasion were tested by scratch assay and Transwell assay, respectively. Results: Higher HBXW7 protein expression level was detected in HCT-116 strain transfected with wild-type HBXW7 in comparison to the control group (strains transfected with mutant-type HBXW7), negative-control (strains transfected with empty plasmids), and blank-control (strains untransfected) (all P<0.05). Compared with the other groups, strains transfected with wildtype HBXW7 exhibited significantly reduced proliferation, colony formation, migration and invasion ability (all P<0.05), but obviously increased apoptosis rate (P<0.05). Conclusion： ：FBXW7 gene mutation can down-regulate its protein expression, and further promote the proliferation, migration and invasion as well as inhibit the apoptosis of HCT-116 cells.
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Objective To investigate the effect of evodiamine (Evo) on the autophagy and proliferation of colon cancer HCT-116 cells and the underlying mechanism. Methods The effect of Evo on proliferation of HCT-116 cells was detected by CCK-8 method. After being processed with Evo (3 and 6 μmol/L) for 48 h, the number of autophagic vesicles were detected by MDC method. The amount of ROS in HCT-116 cells was measured by DHE assay, and the protein related with autophagy and AMPK/mTOR pathway was detected by Western blotting. After the treatment of Evo (6 μmol/L) combined with autophagy inhibitor 3-MA (3-methyladenine) or apoptosis inhibitor Z-DEVD-FMK respectively for 48 h, Western blotting was used to detect the expression of autophagy and apoptosis-related protein in HCT-116 cells. Results Compared with the control group, Evo inhibited the proliferation of HCT-116 cells in a dose-dependent manner; After treated with Evo (3 and 6 μmol/L) for 48 h, the amount of intracellular ROS and autophagic vesicles were increased, the protein expression levels of LC3, p-AMPK, and mTOR were increased while the expression of p62 was increased. After being treated with Evo and autophagy inhibitor, the protein expression of LC3 was decreased while activated Caspase-3 was increased; Combination of Evo and apoptosis inhibitor increased the expression of LC3 and inhibited the expression of activated Caspase-3. Conclusion Evo can activate autophagy of HCT-116 cells through AMPK/mTOR pathway and inhibit the proliferation, and the effect of autophagy and apoptosis on cells are complementary.
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Objective To discuss the inhibition mechanism of chlorogenic acid for colorectal cancer cell line HCT116. Methods Colorectal cancer cell line HCT116 were treated with chlorogenic acid, and the untreated group was blank control. Then, the gene expression chip was used to screen the differential expression gene before and after processing, the Real-time PCR technique was used to identify IGFBP3, MALAT1, SOX4, and NDRG1, and the Western blotting detected the level of protein expression of NDRG1, which belongs to up-regulated gene. Results The gene expression spectra chip test showed that there were 161 up-regulated expression gene of colorectal cancer (the fold change was larger than 2), and 64 down-regulated expression genes (the fold change was less than 0.5) after chlorogenic acid treatment. Also, these genes were mainly related to the function of cell signal transduction, biological process and cellular component. The results of Real time PCR showed that the mRNA expression of IGFBP3 and NDRG1 were up-regulated significantly (P < 0.05) after chlorogenic acid treatment. Western Blotting results showed that the NDRG1 gene protein expression was up-regulated significantly (P < 0.05) after chlorogenic acid treatment. Conclusion The gene expression spectrum chip combined the Real-time PCR technology, which were used to screen the differential expression gene before and after the chlorogenic acid treatment, in order to reveal the inhibition mechanism of chlorogenic acid for colorectal cancer cells.
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OBJECTIVE:To study the effects of Lactobacillus fermentum(Lb-f)on inhibition of cytoxan(CTX) on HCT116 cells of human colorectal cancer in vitro and in vivo. METHODS:HCT116 cells were cultured in vitro. MTT assay was used to in-vestigate the effects of 4,2,1μg/ml CTX and combined with 10μg/ml Lb-f on the survival rate of HCT116 cells. HCT116 cell xe-nograft tumor nude mice model was induced to investigate the effects of 100,50,25 mg/kg CTX and combined with 180 mg/kg Lb-f on tumor volume,tumor weight,relative tumor inhibition rate,the sensitization effect of chemotherapy (by q value),the number of peripheral blood leucocyte and platelet. RESULTS:Compared with CTX alone,CTX combined with Lb-f had no signifi-cant effect on survival rate of HCT116,relative inhibition rate of tumor volume in nude mice, relative inhibition rate of tumor weight and q value (P>0.05),but increased the number of peripheral blood leucocyte and platelet (P<0.05). CONCLUSIONS:No synergistic effects of Lb-f is found on the inhibition of CTX on the growth of HCT116 in vitro and in vivo;Lb-f can inhibit the decrease of peripheral blood leucocyte and platelet of HCT116 cell xenograft tumor nude mice induced by CTX.
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Aim To investigate the effect of triptolide ( TP) on human colorectal cancer HCT116 cell prolif-eration and explore the potential mechanism of TP in treating colon cancer. Methods MTT assay was used for estimating the survival rates of HCT116 cells ex-posed to different concentrations and different duration time of TP. Western blot ( WB ) was used for testing the expression of LC3-Ⅱ. MDC staining was employed to detect cell autophagy. Flow cytometry ( FCM) was applied to test the effects of TP on cell apoptosis. Re-sults TP could inhibit cell proliferation in HCT116 cells. The expression of LC3-Ⅱwas enhanced with the increase of the concentration of TP after exposed to dif-ferent concentrations of TP for 48 h and the duration time of TP after exposed to 40 nmol·L-1 TP,and the number of acid autophagic vacuoles in HCT116 cells had increased, which was observed by fluorescence mi-croscope. The HCT116 cells apoptotic rates in TP group had decreased compared to control group and de-creased significantly compared to TP +3-MA group. The HCT116 cells apoptotic rates in TP group had in-creased compared to control group and increased signif-icantly compared to TP+RAPA group. Conclusion TP could inhibit the proliferation of human colon canc-er HCT116 cells and induce apoptosis in HCT116 cells, which indicates that synergy may exist between autophagy and apoptosis.
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Objective To explore the role of sphingosine kinase 2 (SphK2) in 5-fluorouradl (5-FU)-induced apoptosis in human colon cancer HCT116 cells. Methods Hoechst33342 staining was used to examine the apoptosis morphological changes of HCT116 cells 48 h after treatment with 5-FU; flow cytometry was used to detect the apoptotic ratio of HCT116 cells and the effects of SphK2 (down-regulation and over-expression) on 5-FU-induced apoptosis were analyzed. Western blotting analysis was used to examine the changes in the activated forms of SphK2 and apoptotic marker protein in HCT116 cells after exposure to 5-FU. Results 5-FU induced significant apoptosis in HCT116 cels as reflected by apoptosis characteristics-condensation and fragmentation of chromatin. Interference of SphK2 significantly increased 5-FU-induced apoptosis in HCT116 cells compared with non-interfered group (P < 0.01), accompanied by increased apoptotic marker protein. On the contrary, the apoptosis ratio of the cells with SphK2 over-expression was significantly decreased compared with vehicle plasmid-transfected group (P< 0.01), accompanied by decreased apoptotic marker protein. Moreover, 5-FU greatly increased activated SphK2. Conclusion SphK2 negatively regulates 5-FU-induced apoptosis in HCT116 cells.
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Objective To study the effects of Irinotecan (CPT-11) on human colorectal cancer HCT-116 and HT-29 cells and investigate the potential mechanisms.Methods The effect of Irinotecan on the proliferation of HCT-116 and HT-29 cells was determined by MTT assays.The invasive capacity was measured by transwell assays,and the apoptosis of the tumor cells was detected by flow cytometry after stained with Annexin-V and PI.The difference between the current of ATP-sensitive potassium ion of HCT-116 and HT-29 was examined by patch clamp.Results It was found that 1.0-64.0 μg/ml CPT-11 could inhibit the proliferation and the invasive capacity of HCT-116 and HT-29 cells at both dose-and time-dependent manner.The IC_(50) of HCT-116 and HT-29 were 39.3 and 19.5 μg/ml respectively.Cytometry showed that the apoptotic rates were increased from 14.8% and 9.3% to 36.9% and 27.9% after the treatment of 32.0 μg/ml and 16.0 μg/ ml CPT-11,which were close to their IC_(50).The proportion of G_0/G_1 and S of HCT-116 and HT-29 was enhanced from 27.4% and 17.4% to 95.9% and 98.2%.Transwell assay indicated that the invasiveness of HCT-116 and HT-29 was reduced by 40.8% and 47.5%.The patch clamp showed that CPT-11 reduced the I_(KATP) of cell membrane at a negative dose-dependent manner.Conclusion CPT-11 could have a significant impact on the proliferation,invasiveness,cell cycle,and the apoptosis of human colorectal cancer cell HCT-116 and HT-29.HT-29 was more sensitive to CPT-11 than HCT-116.The inhibitory effect of CPT-11 on cell proliferation might be linked to its inhibition of ATPsensitive potassium channel.
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BACKGROUND: Genistein and daidzein are two major soybean isoflavones. They have received increasing attention because of their possible roles for cancer prevention. However, their mechanisms of action and molecular targets on the human colon cancer cells are not fully understood. METHODS: Human colon cancer HCT-116 cells were treated with genistein and daidzein to investigate their effects on the cell growth and this was analyzed with MTT assay. TUNEL assay and Hoechst33342 stain were carried out to identify apotosis. RESULTS: Daidzein was able to inhibit cell proliferation and induce apoptosis of the HCT-116 cells, but genistein didn't affect the cell growth. The ER antagonist ICI182780 didn't attenuate the antiproliferative and proapoptotic effects of daidzein: this means the effect of daidzein on the HCT-116 cells may not be dependent on the ER pathway. The other soybean isoflavone, genistein, attenuated the effects of daidzein on the HCT-116 cells and its mechanism should be elucidated. CONCLUSIONS: These data suggest that daidzein may act as a preventive agent on human colon cancer, and its mechanism of action doesn't involve the ER-dependent pathway.
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Humans , Apoptosis , Cell Proliferation , Colon , Colonic Neoplasms , Genistein , HCT116 Cells , In Situ Nick-End Labeling , Isoflavones , Glycine maxABSTRACT
Objective It has been known that NSAIDs can inhibit the growth of tumors through cyclooxygenase, but it is unknown that whether there are other mechanisms involved. It is essential to detect the effect of indomethacin for proliferation and apoptosis in the HCT116 and explore its anti tumor mechanism. Methods Human colon adenocarcinoma cell line HCT116 was treated with different concentration of indomethacin for 24 h and CDK 2,CDK 4, Bcl 2, Bax and p21 WAF1/CIP1 protein were detected by Western blot. Results Indomethacin down regulated the expression of CDK 2, CDK 4, Bcl 2 and up regulated the expression of p21 WAF1/CIP1 , however, the expression of Bax remained unchanged. Conclusions Inhibiting proliferation and inducing apoptosis contribute to the anti tumor activity of indomethacin by down regulating the expression of CDK 2, CDK 4, Bcl 2 and up regulating the expression of p21 WAF1/CIP1 .