| Background: Tumor cells are more likely to use glycolysis to generate energy even in the presence of oxygen,known as “Warburg effect”.As pyruvate analogs,3-bromopyruvate(3-Br PA),can inhibit glycolysis in cancer cells and induce cell death.Monocarboxylate transporter 1(MCT1)is a membrane protein,which can transport lactate,pyruvate and other monocarboxylic acid.However,the role of MCT1 in the antitumor effect of 3-Br PA needs further study.Here we focus on determining the cytotoxicity of 3-Br PA in breast cancer cells and assessing the putative role of MCT1 on this effect.Objective: 1.To observe the inhibitory effect of 3-bromopyruvate(3-Br PA)on the proliferation of breast cancer cells.2.Explore the effect of 3-Br PA on breast cancer cell apoptosis.3.To explore the role of MCT1 in the apoptosis of breast cancer cells induced by 3-Br PA.Methods: 1.Immunohistochemistry and Western blotting were used to detect the expression of Hexokinase II(HKII)in human breast cancer tissues and multiple breast cancer cells.2.MTT assay was used to detect the inhibitory effect of 3-Br PA on the proliferation of multiple breast cancer cells.3.Western blotting was used to detect the expression of MCTs in multiple breast cancer cells.4.After blocking with AZD3965,a small molecule inhibitor of MCT1,the sensitivity of breast cancer cells to 3-Br PA was examined.5.Select MDA-MB-231,MDA-MB-453,MCF-7,and CAL-51 cells for follow-up study,using MTT assay to detect the inhibition of 3-Br PA on the proliferation of breast cancer cells.6.PI staining was used to detect the effect of 3-Br PA on the apoptosis rate of four breast cancer cells.7.The change of nuclei after 3-Br PA treatment on four breast cancer cells was detected by DAPI staining.8.The effect of 3-Br PA on the expression of HKII,lactate dehydrogenase(LDH),lactic acid and ATP levels in breast cancer cells was detected by ELISA.9.Through the small interference(si RNA)experiment,the expression of MCT1 protein in breast cancer MCF-7 and CAL-51 cells was down-regulated,and the sensitivity of two breast cancer cells to 3-Br PA was detected.10.Using c DNA to overexpress the MCT1 protein of breast cancer MDA-MB-231 and MDA-MB-453 cells,observe the changes of the sensitivity of breast cancer cells to 3-Br PA.11.The effect of 3-Br PA on the viability of four human primary breast cancer cells with different MCT1 expression levels was examined using the MTT assay.Results: 1.Upregulation of HKII expression in breast cancer tissues and cells(1)After immunohistochemical detection of human normal breast tissue and breast cancer tissue,the expression of HKII in breast cancer tissue was significantly higher than that in normal breast tissue.(2)Western blotting was used to detect the expression of HK II in normal breast epithelial cells MCF-10 A and eight breast cancer cells.The results showed that the expression of HKII in eight breast cancer cells was significantly higher than that in normal breast epithelial cells MCF-10 A.2.The sensitivity of breast cancer cells to 3-Br PA is related to the expression of MCT1(1)Eight breast cancer cells were treated with different concentrations of 3-Br PA(0,25,50,100,200 ?M).MTT results showed that: as the drug concentration increased,the survival rates of CAL-51?BT-474?HCC1395?HCC1954?T-47 D,MCF-7 cells were significantly reduced,but the cell viability of MDA-MB-231 and MDA-MB-453 did not change significantly.(2)Western blotting analysis of the expression of MCT1,MCT2,MCT3,and MCT4 in eight breast cancer cells showed that MCT1 was almost not expressed in MDA-MB-231 and MDA-MB-453 cells,but was expressed in other six cells.;MCT2 was not expressed only in T-47D;MCT3 was hardly expressed in the eight cells;MCT4 was expressed in all eight cells.(3)After treatment of different concentrations of AZD3965(0,0.01,0.1,1,10 ?M)for breast cancer CAL-51,BT-474,HCC1395,HCC1954,T-47 D,MCF-7 cells,MTT results showed that: 0.1 ?M AZD3965 has almost no proliferation inhibitory effect on the cells.Therefore,when the combination of 0.1?M AZD396 and different concentrations of 3-Br PA(0,25,50,100,200 ?M)was used,the proliferation inhibition of six breast cancer cells was significantly reduced.3.Effect of 3-Br PA on the survival and apoptosis of breast cancer cells(1)In order to further observe the sensitivity of breast cancer cells to 3-Br PA,four breast cancer cells including MDA-MB-231,MDA-MB-453,MCF-7,and CAL-51 were selected for further study.The results of MTT showed that four breast cancer cells were treated with different concentrations of 3-Br PA(0,25,50,100,200 ?M).Compared with the control group,with the increase of the drug concentration and the prolonged action time,the survival rate of MDA-MB-231,MDA-MB-453 cell did not significantly change,while the MCF-7,CAL-51 cell survival rate was significantly reduced.(2)Different concentrations of 3-Br PA(0,25,50,100,200 ?M)acted on four cells for 24 h,and were observed under an inverted microscope: compared with the control group,The morphology and density of MDA-MB-231,MDA-MB-453 cells did not change significantly,but the morphology and density of MCF-7 and CAL-51 cells changed significantly after drug treatment.(3)Different concentrations of 3-Br PA(0,25,50,100,200 ?M)acted on four cells for 24 h,Flow cytometry PI single staining experiment results showed that compared with the control group,the apoptosis rates of MDA-MB-231 and MDA-MB-453 cells did not change significantly,but the apoptosis rate of MCF-7 and CAL-51 cells was significant.increase.(4)Different concentrations of 3-Br PA(0,25,50,100,200 ?M)acted on four cells for 24 h,the effect of 3-Br PA on the nucleus of MDA-MB-231,MDA-MB-453,MCF-7 and CAL-51 cells was detected by DAPI fluorescent staining.The results showed that compared with the control group,the nucleus of MDA-MB-231 and MDA-MB-453 cells after drug treatment did not change significantly,but the nucleus of MCF-7 and CAL-51 cells changed significantly after drug treatment,nuclear dense dyeing and nuclear fragmentation increased significantly.4.Effect of 3-Br PA on energy metabolism of breast cancer cells(1)After 24 h of treatment with 100 ?M 3-Br PA on four breast cancer cells,the HKII kit showed that compared with the control group,HKII activity of MDA-MB-231 and MDA-MB-453 cells did not significantly changed,while HKII activity of MCF-7,CAL-51 cells was significantly reduced.(2)After 100 ?M 3-Br PA was applied to four breast cancer cells for 24 h,the LDH kit showed that the LDH activity of MDA-MB-231 and MDA-MB-453 cells was not significantly different from that of the control group,while the LDH activity of MCF-7,CAL-51 cells was significantly reduced.(3)After 100 ?M 3-Br PA was applied to four breast cancer cells for 24 h,the lactic acid ELISA kit test results showed that there was no significant difference in lactic acid content between MDA-MB-231 and MDA-MB-453 cells compared to the control group,while the MCF-7,CAL-51 cell lactate content decreased significantly.(4)The ATP kit showed that the ATP levels did not change significantly after treatment with 100 ?M 3-Br PA for 6 h in MDA-MB-231 and MDA-MB-453 cells.However,the production of ATP in MCF-7 and CAL-51 cells was significantly reduced.5.Si RNA down-regulates MCT1 expression in breast cancer cells and reduces the sensitivity of cells to 3-Br PA(1)si RNA interference experiments down-regulated the expression of MCT1 in MCF-7 and CAL-51 cells.Western blotting showed that MCT1 si RNA1752 could significantly reduce MCT1 expression in two breast cancer cells.(2)Cells transfected with MCT1 si RNA1752 were treated with different concentrations of 3-Br PA(0,25,50,100,200 ?M)for 24 h,MTT assay and PI staining experiment were used to detect the survival rate and apoptosis rate of the two breast cancer cells.The results showed that the sensitivity of MCF-7 and CAL-51 cells to 3-Br PA was significantly reduced after transfection.7.Overexpression of MCT1 increases the sensitivity of breast cancer cells to 3-Br PA(1)The MCT1 c DNA was transfected into MDA-MB-231 and MDA-MB-453 cells.The results of Western blotting confirmed that the expression of MCT1 in both breast cancer cells increased significantly.(2)MDA-MB-231,MDA-MB-453 cells transfected with MCT1 c DNA were treated with different concentrations of 3-Br PA(0,25,50,100,200 ?M)for 24 h,MTT and PI staining were used to detect the survival rate and apoptosis rate of the cells.The experimental results showed that the sensitivity of the two cells to 3-Br PA was significantly increased.7.Inhibitory effect of 3-Br PA on proliferation of human primary breast cancer cells with high expression of MCT1 protein (1)Western blotting assay detected the expression of MCT1 in four human primary breast cells.The results showed that two primary breast cancer cells(PBCC #1,PBCC #2)hardly expressed MCT1,and two other primary cells(PBCC #3,PBCC #4)MCT1 was significantly higher.(2)In order to observe whether the inhibition of 3-Br PA on primary breast cancer cells is consistent with the expression of MCT1,MTT assay was used to detect different concentrations of 3-Br PA(0,25,50,100,200 ?M).The survival rate of the primary breast cancer cells after 24 hours showed that 3-Br PA had almost no effect on the survival rate of the two primary cells(PBCC #1,PBCC #2)that did not express MCT1,but the cell survival rate of primary cells that expressed MCT1(PBCC #3,PBCC #4)was significantly reduce.Conclusion: There is a significant difference in the sensitivity of breast cancer cells to 3-Br PA,and this sensitivity difference is determined by the expression of MCT1. |
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