Antitumor Effects Of 3-bromopyruvate And 5-fluorouracil Against Human Colorectal Cancer Cells Via Cell Cycle Arrest And Induction Of Apoptosis

Objectives: 1.To explore whether 3-bromopyruvate(3-BP)can enhance the sensitivity of colon cancer cells to low-dose 5-fluorouracil(5-FU).2.To investigate the mechanism of 3-BP enhances the sensitivity of 5-FU in colon cancer cells.3.To explore whether 3-BP can enhance antitumor effect of 5-FU in tumor-bearing nude mice.Methods: 1.The effect of drugs alone and in combination on the survival rate of colon cancer cells SW480 and HT-29 were detected by MTT assay.2.Colony-forming assay was used to detect the growth inhibition induced by 3-BP and 5-FU combination or alone.3.The changes of cell viability of SW480 and HT-29 cells were detected by PI single-flow cytometry after 24 h.4.The changes of mitochondrial membrane potential in SW480 and HT-29 cells were detected by JC-1 kit after 24 h of therapy alone and in combination.5.The changes of nuclear morphology of SW480 and HT-29 cells in human colon cancer cells were detected by DAPI fluorescence staining for 24 h.6.To detect the effect of drugs on the levels of ATP in human colon cancer SW480 and HT-29 cells after 6 h with ATP test kit.7.Effects of ROS levels in SW480 and HT-29 cells of human Colon Cancer Cells after 6 h of therapy alone and in combination.8.The effect of drugs alone and in combination after 24 h on the cell cycle of human colon cancer SW480 and HT-29 was detected by PI flow cytometry.9.The effects of drugs alone and combination on the expression of apoptosis-related proteins and cell cycle-associated proteins in human colon cancer cells SW480 and HT-29 were detected by Western blot.10.The anti-tumor effect of 3-BP combined with 5-FU was detected by tumor-bearing nude mice in vivo.Results: 1.Effects of 3-BP and/or 5-FU on the viabilities of SW480 and HT-29 cells.(1)We found that treatment of the cells for 24 h with 5-FU at concentrations ? 64 ?M had little effect on cell viability.A similar observation was made for 3-BP at concentrations ? 50?M.Because of these results,we pretreated the SW480 and HT-29 cells with 50 ?M 3-BP for 2 h and exposed them to 10 ?M 5-FU in subsequent experiments.The combined treatment inhibited the growth of the SW480 cells more than it inhibited that of the HT-29 cells.(2)The 3-BP plus 5-FU treatment also markedly reduced colony formation in the SW480 cells but had a lesser effect on the HT-29 cells.The results showed that 3-BP combined with 5-FU was more cytotoxic to the SW480 cells than it was to the HT-29 cells.2.Combined 3-BP and 5-FU treatment induces apoptosis in SW480 and HT-29 cells.(1)To detect whether 3-BP has a synergetic effect with 5-FU in inducing apoptosis in SW480 and HT-29 cells,we treated the cells with both 3-BP and 5-FU.The results showed a similar pattern of colony formation.Cell morphology was examined by light microscopy using differential interference contrast optics and PI staining.It can be observed that the apoptotic rate of the SW480 cells was higher with the combined treatment than with a single agent.Nevertheless,the combined treatment did not increase the apoptotic rate of the HT-29 cells.(2)We also used JC-1 as a fluorescent marker to detect changes in mitochondrial membrane potential.The results shows that the combined 3-BP and 5-FU treatment altered mitochondrial membrane potential in the SW480 cells but not in the HT-29 cells(3)DAPI staining was used in the detection of cell death,and observed with a fluorescence microscope,following treatment with 50 ?M 3-BP and 10?M 5-FU or both for 24 h.we found that 3-BP combined with 5-FU in the SW480 cell group,the nucleus into the thick,nuclear fragmentation and other cells late apoptosis phenomenon;but in HT-29 cells did not observe significant nuclear fragmentation.3.Combined 3-BP and 5-FU treatment induces ROS generation and activates mitochondria-dependent apoptosis in SW480 cells.(1)We investigated the effect of ROS on cell viability by assessing ROS generation in the SW480 and HT-29 cells.We found that,the combined 3-BP and 5-FU treatment increased ROS levels more than either of the agents did.Furthermore,pretreatment with NAC downregulated ROS levels.(2)We assessed the effects of 3-BP and/or 5-FU on cell viability in the presence of NAC,which is a ROS scavenger and can therefore reduce oxidative stress.The results indicated that the presence of NAC decreased the combined effect of the two agents on the SW480 cells.(3)The combined 3-BP and 5-FU treatment increased Bax expression and reduced Bcl-2 expression more than either agent did.Bax and Bcl-2 are apoptosis-related proteins that are important in the mitochondrial apoptotic pathway.4.Combined 3-BP and 5-FU treatment decreases ATP production in SW480 and HT-29 cells.(1)3-BP is an energy-depleting drug that can suppress ATP production.The results showed that compared to treatment with a single agent,the combined treatment caused a marked decrease in ATP production in the SW480 cells and a slight decrease in ATP production in the HT-29 cells.5.Effect of 3-BP and/or 5-FU on cell cycle in SW480 and HT-29 cells.(1)We analyzed the cell cycles of the SW480 and HT-29 cells since cell proliferation is largely related to the regulation of cell cycle progression.The results demonstrated that,5-FU increased the percentages of the SW480 and HT-29 cells in their respective G0/G1 phases.On the other hand,3-BP induced cell cycle arrests at S phase in the SW480 cells and at G2/M phase in the HT-29 cells.Furthermore,in the SW480 cells,pretreatment with 3-BP for 2 h followed by the 5-FU treatment induced a higher G0/G1 phase arrest than the 5-FU treatment alone did.However,we did not observe a similar phenomenon in the HT-29 cells.(2)To confirm the results obtained,we measured the expression levels of cell cycle-related proteins,which play a key role in the cell cycle.We found that the combined treatment induced upregulations of P21 and P53 and downregulations of CDK2 and CDK4 in the SW480 cells.However,the effects of the combined treatment on the expression levels of the aforementioned proteins in the HT-29 cells were minimal.6.3-BP enhanced the antitumor effect of 5-FU in vivo.(1)To further investigated the synergistic antitumor effect of 3-BP and 5-FU,SW480 cells were xenografted into nude mice and the tumor-bearing animals were treated with 3-BP or 5-FU.At day 24,the mice were sacrificed.Treatment with low-dose 3-BP(3 mg/kg)or low-dose 5-FU(30 mg/kg)alone slightly reduced SW480 cell growth compared with NS.However,the sizes and weights of the SW480 tumors were significantly reduced by combined 3-BP and 5-FU treatment.(2)The results of the H&E staining experiment showed the presence of loose cancer cells,nucleus deformation,and large necrotic areas in the mice administered the combined treatment.However,these features were either absent or less marked when the mice were treated with either 3-BP or 5-FU.(3)The results obtained from the H&E staining of the kidneys and livers of the mice indicate that the hepatotoxicity and nephrotoxicity caused by the combined treatment are minimal.Conclusion: 1.3-BP combined with 5-FU has a synergistic effect on human colon cancer SW480 cells.2.3-BP combined with 5-FU produces synergistic antitumor effect by inducing the activation of mitochondrial apoptosis and cell cycle arrested of human colon cancer SW480 cells.3.3-BP combined with 5-FU has good anti-tumor effect in tumor-bearing nude mice,the hepatotoxicity and nephrotoxicity caused by the combined treatment are minimal.