A Novel Camptothecin Analogue Inhibits Colon Cancer Development And Downregulates The Expression Of MiR-155 In Vivo And In Vitro

Objective This study was to investigate the anti-colon cancer activity of FL118 systematically and trying to elucidate the action mechanism accounting for FL118's better anti-colon cancer activity prior to other camptothecin analogues with respect to epigenetics(micro RNA).Methods Human colorectal carcinoma cell HCT-116 was employed as experimental subject to test the anti-colon cancer activity properties of FL118.All statistical analyses were performed using student's t test(T test).MTT assay was performed to detect the cell viability affected by FL118 of HCT-116 cells.FL118 was added to each well at a series of concentration: 0.01/ 0.1/ 1/10/100/1000 n M.Cells treated with SN38(100 n M,the active metabolite of irinotecan)served as positive group.The OD value of each well was measured by microplate reader at 490 nm.Scratch wound assay was performed to detect the cell migration affected by FL118 of HCT-116 cells.FL118 was at 10 n M.The migration of cells was observed 24 h and 48 h after incubated with FL118.Photos of the wound were collected.Brd U cell proliferation assay was performed to detect the cell proliferation affected by FL118 of HCT-116 cells.Cells were treated with FL118 at 10 n M for 24 hours.1x Brd U label was added into proliferating cells two and a half hours prior to the end of FL118 treatment.The OD value of each well was measured by microplate reader at 450 nm.Flow cytometry was applied to detect apoptosis and cell cycle under the treatment of FL118(10 n M).PI was used to detect the cell cycle distribution.Cells were starved for 12 hours for synchronization before FL118 administration.Cells were collected after being treated with FL118 at 10 n M for 24 hours.Subsequently,cells were immobilized and stained by PI.At last,cells were detected one by one after filtered by nylon net(400 mesh)in flow cytometry at 488 nm.The portion of each cell cycle stage(G0,G1,S,G2/M)was analyzed by Motif analysis software.Annexin V-FITC/PI was used to detect the apoptosis of cells.After being treated by FL118 at 10 n M for 36 h,cells were collected and incubated with Annexin V-FITC and PI.Apoptosis detection was measured by flow cytometry after filtered by nylon net(400 mesh).Apoptotic and necrotic cells were analyzed by quadrant statistics.Xenograft models were established to observe the effect of FL118 on tumor growth.Female BALB/c nude mice(age 49 to 56 days,weight 15 to 22g)were obtained to establish xenograft models.HCT-116 cells were injected subcutaneously.Mice were divided into three groups: control group(injected vehicle only),lower dose group(treatedwith FL118 at 0.75mg/kg),and higher dose group(treated with FL118 at 1.5mg/kg).The treatment schedule is one treatment per week for four weeks.During the treatment,the tumor volume and body weight were measured every other day.The tumor volume was calculated as V=0.5×(length × width2)and measured by caliper.And the weight of mice were measured by electronic balance.When the tumor volume is more than 1500 mm3 or the mouse is moribund,mice were sacrificed to collect the tumors that were then stored in liquid nitrogen.Also,q RT-PCR was performed to detect the level of mi R-155 in colon cancer cells and tumor samples after FL118 administration.The doses of FL118 on HCT-116 cells for 24/48 h were 1/10/100 n M.The results of q RT-PCR were analyzed by CFX Manager TMSoftware.Results FL118 effectively inhibited cell viability,mobility and proliferation,induced cell cycle arrest at S phase and accelerated cell apoptosis of colon cancer.Moreover,FL118 obviously limited tumor growth in tumor-bearing mice.Intriguingly,FL118 significantly decreased the expression of mi R-155(p<0.05)both in vitro and in vivo.Conclusion A novel camptothecin analogue FL118 effectively inhibited colon cancer development and significantly downregulated the expression of mi R-155 in vivo and in vitro.On the basis that mi R-155 is closely associated with the pathogenesis and progression of colon cancer,its downregulation resulting from FL118 should be noted,and urges further study.