Role Of Mcl-1 In Deciding Sensitivity Of 3-bromopyruvate Induced Apoptosis In Breast Cancer Cells

Objectives:1. To investigate the effect of 3-Br PA(3-bromopyruvate) on the proliferation and apoptosis of breast cancer cells.2. To investigate whether 3-Br PA induced apoptosis depends on caspase.3. To explore the effect of ROS in 3-Br PA induced apoptosis in breast cancer cell.4. To explore whether 3-Br PA induces apoptosis through PI3K/Akt signaling pathway.5. To explore the function of Mcl-1 in deciding the sensitivity of 3-Br PA induced apoptosis.Methods:1. MTT assay were used to detect the proliferation inhibition induced by 3-Br PA in MDA-MB-231 and MDA-MB-435 breast cancer cells.2. Cell apoptosis was assessed by flow cytometry with propidium iodide(PI) staining or PI/Annexin V staining.3. The ROS level was measured by the DHE Assay Kit after MDA-MB-231 cells treated with 3-Br PA(160 mmol·L-1) for 1 h, 3 h or 6 h.4. The intracellular ATP level was measured by the ATP Assay Kit after MDA-MB-231 cells and MDA-MB-435 cells treated with 3-Br PA( 40, 80, 160 mmol·L-1).5. Western blot was used to explore the expressions of apoptosis key protein Mcl-1 and effect of PI3K/Akt signaling pathway.6. Small interfering RNA silenced Mcl-1 gene expression, and the expression of related protein were analyzed by Western blot.7. Upgulating the expression of Mcl-1 and observe the changes of 3-Br PA inducedapoptosis in MDA-MB-231 cell line.Results:1. Different concentrations of 3-Br PA regulates cell proliferation and apoptosis in MDA-MB-231 and MDA-MB-435 cells1.1 In order to examine the effect of 3-Br PA-induced proliferation inhibition in breast cancer cells MDA-MB-231 and MDA-MB-435, cells were treated with different concentrations of 3-Br PA(10, 20, 40, 80, mmol·L-1). The results demonstrated that the rate of cell proliferation had an inverse relationship with both the time of 3-Br PA exposure and concentration. However, 3-Br PA did not induce cytotoxicity inthe MDA-MB-435 cell line.1.2 PI staining and PI/Annexin V staining were used to determine the type of cell death induced by 3-Br PA in MDA-MB-231 cells. After 24 h of 3-Br PA treatment, the percentage of the apoptotic cells was increased significantly from 5.5 to 22.2%, and the percentage of necrotic cells increased from 0.9 to 10.7%(Fig. 1d). Taken together, these data suggest that 3-Br PA induces apoptosis in MDA-MB-231 cells and that MDA-MB-435 cells are not sensitive to 3-Br PA.2. 3-BrPA-induced apoptosis is independent of caspase activity2.1 To verify whether 3-Br PA-induced cell death observed in MDA-MB-231 cells was a result of apoptosis, we performed immunoblotting to detect caspase activation. The cleaved products of caspase-8 and caspase-3 were detected in the MDA-MB-231 cells, indicating that 3-Br PA-induced cell death was apoptotic.2.2 Treat MDA-MB-231 cells with 160 mmol·L-1 3-Br PA for 1 h, 3 h or 6 h. In this study, 3-Br PA treatment increased the generation of ROS. These results indicate that 3-Br PA-induced apoptosis might occur as a result of increased ROS production.2.3 In addition, it is well documented that cancer cell death induced by 3-BrPA depends on the depletion of the cellular ATP pool. Our studies in two breast cancer cell lines confirm this mechanism. When MDA-MB-231 and MDA-MB-435 cellswere treated with 3-Br PA(40,80,160 mmol·L-1) for 6 h, ATP levels rapidly decreased in both cell lines in a dose- dependent manner.2.4 To determine whether caspases play a role in cell death induced by 3-Br PA, we used the pan-caspase inhibitor z-VAD-FMK to treat the 3-Br PA sensitive MDA-MB-231 cells. z-VAD treatment did not inhibit 3-Br PA-induced cell death, indicating a caspase-independent killing mechanism.3. 3-Br PA decreases the expression of Mcl-1 in MDA-MB-231 cells through the PI3 K pathway3.1 To confirm whether 3-Br PA downregulated the expression of Mcl-1 through the PI3K/Akt pathway, we test the expressions of Mcl-1 and p-Akt. Our data revealed that 3-Br PA downregulated the expressions of Mcl-1 and p-Akt in MDA-MB-231 cells.3.2 Treated MDA-MB-231 cell with PI3K/Akt pathway inhibitor LY294002, the expression of Mcl-1 in MDA-MB-231 cell was decresased. On the basis of these results, we hypothesize that 3-Br PA decreases the expression of Mcl-1 through the PI3K/Akt pathway.4. Downregulation of Mcl-1 induces apoptosis in MDA-MB-435 cells4.1 Western blot analysis confirmed that Mcl-1 expression was reduced substantially by the Mcl-1-specific si RNA, downregulation of Mcl-1 expression in the MDA-MB-435 cells resulted in increased sensitivity to 3-Br PA-induced antiproliferation.4.2 Treated MDA-MB-435 cells with 160 ?mol·L-1 3-Br PA for 24 h after silence of Mcl-1 expression. Compared with the control group, western blot analysis showed that treatment group had certain activation of Caspase-3.5. Upregulation of Mcl-1 protects MDA-MB-231 cells from apoptosis5.1 To further confirm the role of Mcl-1 in 3-Br PA-induced apoptosis, we transfected complementary DNA encoding Mcl-1 into MDA-MB-231 cells. Western blot analysis confirmed that Mcl-1 expression was increased substantially by the Mcl-1 c DNA. The overexpression of Mcl-1 significantly increased cell viability after the administration of 3-Br PA, indicating that the cells were protected from apoptosis.5.2 The cleaved products of caspase-3 were nearly undetectable in the Mcl-1-overexpressing MDA-MB-231 cells(Fig. 5c). Taken together, these results further confirm that Mcl-1 plays an important role in 3-Br PA-induced apoptosis.Conclusion:1. 3-Br PA induced apoptosis in MDA-MB-231 cells.2. The process of 3-Br PA induced apoptosis in MDA-MB-231 breast cancer cells accompanies the increased reactive oxygen species3. Regulation of Mcl-1 expression may be one of the mechanisms of 3-Br PA induced apoptosis in breast cancer cells.