| Objectives: 1. To investigate the effectsof 3-bromopyruvate(3-BrPA), a glycolytic inhibitor, on cell proliferation and cell death in colon cancer cells. 2. To investigate whether 3-BrPA induce different forms of cell deathin colon cancer cells. 3. To explorethe anti-tumor effects of 3-BrPAin vivo.Methods: 1. The growth inhibition of colon cancer cells induced by 3-BrPA was detected by MTT assay. 2. Cell colony formation assay was used to check the growth inhibition induced by 3-BrPA in colon cancer cells. 3. HexokinaseⅡ(HKⅡ) kit was used to detect intracellular hexokinaseⅡlevelin drug-treated human colon cancer cells SW480 and HT29. 4. The intracellular ATP levels in SW480 and HT29 cells treated with 3-BrPA was measured by the ATP Assay Kit. 5. Cell death was assessed by flow cytometry with PI/Annexin V- staining. 6. Changes of mitochondrial membrane potential in colon cancer cells SW480 and HT29 treated with 3-BrPA for 24 h were detected by the cationic dye JC-1 kit. 7. Changes of nucleus of colon cancer cells SW480 and HT29 were assessed by DAPI fluorescent staining. 8. Theultrastructural details of colon cancer cells SW480 and HT29 treated with3-BrPAwere respectivelyanalyzed by transmission electron microscopy(TEM). 9. The expression oftransporterproteins, HK Ⅱ, apoptosis-related proteins, necropotosis-related proteins and autophagy-related proteins in colon cancer cells SW480 and HT29 was analyzed by Western blot. 10. The anti-tumor effects of 3-BrPA in vivowas explored in nude mice xenograft models.Results: 1. 3-BrPA inhibited colon cancer cell viability.(1) SW480 and HT29 cells were treated with 3-BrPA(0, 40, 80, 160, 320 μmol/L). The proliferation of colon cancer cells SW480 and HT29 were decreasing with the increasing of 3-BrPA concentrations, and SW480 cells were more sensitive to 3-BrPA in the same concentration.(2) SW480 and HT29 cells were treated with 3-BrPA(0, 40, 80, 160, 320 μmol/L), the intercellular spaces increased in drug group compared with the control group, and the number of suspension cells increased under the microscope.(3) Cell colony formation was used to respectively verify the effect of 3-BrPA(0, 10,20, 30μmol/L) and(0, 30, 40, 50μmol/L) on SW480 and HT29 cells, which were treated with 3-BrPA for 5-7 days. Obvious anti-proliferation effect with low-concentration of 3-BrPA occurred in SW480 and HT29 cells. 2. 3-BrPA induced HKⅡand ATP reduction via MCTs transportation in colon cancer cells.(1) SW480 and HT29 cells were treated with different concentrations of 3-BrPA for 24 h, then both levels of HKⅡwere decreased, which were assessed by enzyme-linked immunosorbent assay kit.(2)The results of ATP experiment showed that the intracellular ATP levels were concentration-dependent in SW480 and HT29 cells, which were treated with 3-BrPA(0, 40, 80, 160, 320 μmol/L) for 5 h.(3) SW480 and HT29 cells were treated with various concentrations of 3-BrPA for 24 h. Western blotresults showed that the expression of HKⅡ, MCT1 and MCT2 were decreased with the increasing concentration of 3-BrPA, while the expression of MCT4 was not changed obviously. 3. 3-BrPA induced apoptosis in colon cancer cells. (1) SW480 and HT29 cells were treated with different concentrations of 3-BrPA(0, 40, 80, 160, 320 mmol/L) for 24 h, and the mitochondrial membrane potential was assessed with JC-1 staining by fluorescence microscopy. The results showed that red fluorescence transited to green fluorescence with the increasing concentration of 3-BrPA. The results illustrated that 3-BrPA induced the early-apoptosis on colon cancer cells.(2) SW480 and HT29 cells were treated with different concentrations of 3-BrPA(0, 40, 80, 160, 320 mmol/L) for 48 h, and nuclei were stained with DAPI and visualized by fluorescence microscopy. The results of DAPI showed that both the dense nuclear stain and nuclear fragmentation appeared in 3-BrPA group compared with the control group.(3) SW480 and HT29 cells were treated with 3-BrPA(0, 40, 80, 160, 320 mmol/L) for 24 h, then cells were detected by flow cytometry with PI/Annexin V-FITC staining. The results showed that percentages of cell death in 3-BrPA group were higher compared with the control group.(4) To investigatethe micro-change of SW480 and HT29 cells, both cell lines were treated with different concentration of 3-BrPA, then cells were analyzed by TEM.The cytoplasm and cell membrane were intact in control group, while the cytoplasm were shrinked, chromation margination in groups which were treated with 160 mmol/L or 320 mmol/L 3-BrPA.(5) The combination of 3-BrPA withthe pan-caspase inhibitor z-VAD was used to investigate the effect of 3-BrPA on cell lines, the results showed that the cell viability in group treated with z-VAD and 3-BrPA increased compared with the 3-BrPA group.(6)The results of Western blot showed that the expressions of anti-apoptosis proteins were decreased with the increasing concentration of 3-BrPA(0, 40, 80, 160, 320 mmol/L), while the expressions of pro-apoptosis proteins were increased in 3-BrPA treated cells. 4. 3-BrPA induced necroptosis in colon cancer cells.(1) The ultrastructures of cells were detected by TEM, the results showed that cytoplasm and cell membrane were intact in control group, while the organelle was swelling, membrane integrity disappeared and other characters of necrosis appeared in groups with 160 mmol/L or 320 mmol/L 3-BrPA.(2) The combination of Necrostin-1(10 mmol/L), a necroptosis inhibitor, and 3-BrPA were used on colon cancer cells to investigate the effect of 3-BrPA on necroptosis, the results showed that the cell viability in combination group were higher than 3-BrPA group, which means the cells in combination group were protected.(3) SW480 and HT29 cells were treated with 3-BrPA(0, 40, 80, 160, or 320 mmol/L) for 24 h. The expression of apoptosis-related proteins RIP1 was decreased by Western blot analysis. 5.3-BrPA induced autophagy in colon cancer cells.(1) We found that autophagy appeared in HT-29 cells in addition to apoptosis and necroptosis. The autophagy body could be found in 3-BrPA group compared with the control group, which were analyzed by TEM.(2) The autophagy inhibitor 3-MA(5 mmol/L) and 3-BrPA were both used on HT29 cells, the results showed that the cell viability of HT29 cells in combination group were protected compared with control group.(3) HT29 cells were treated with 3-BrPA(0, 80, 160, 320 mmol/L) for 24 h. The results showed that the expression of autophagy-related proteins LC-(40) was decreased,while the expression of LC- Ⅱ was increased with the increasing concentration of 3-BrPA,which analyzed by Western blot. 6. In vivo efficacy of 3-BrPA in nude mice model.(1) From the results of tumor growth curve and tumor volumes, we found that the average volumes in PBS-treated groups were 1750 ± 66 mm3, while the 3-BrPA-treated groups were 890±40 mm3, the daunorubicin(DNR) treated groups were 790±50 mm3, which means 3-BrPA had a good anti-tumor effect compared with control group. The results of H&E staining of tumors showed that there were rich blood vessels and large atypia nucleus in the tissues in control group, while chaotic distribution of blood vessel, karyopyknosis, and inflammatory cell infiltration in drug group, which indicated 3-BrPA induced extensive necrosis and inhibited tumor growth.Conclusion: 1. 3-BrPA inhibits the cell viability of colon cancer cells. 2. 3-BrPA may induce multiple cell death in colon cancer cells. 3. 3-BrPA possesses anti-tumor ability in nude mice. |
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