Dynamin-related Protein 1 Is Involved In MCL-Induced Breast Cancer Cell Apoptosis

Objective: To investigate the role and related mechanisms of Dynamin-related protein 1(Drp1) involved in micheliolide(MCL)-induced human breast cancer cells apoptosis.Methods: Both MCF-7 and MDA-MB-231 cells were infected with EGFP(control group), Drp1-K38A(a dominant negative mutant of Drp1 group) and Drp1-WT(Drp1 wild type group) expression vectors via lentivirus and treated with MCL. The protein expression level of Drp1 was detected by Western blot. The morphological structure and mitochondrial membrane potential was observed by laser scanning confocal microscope in breast cancer cells. After transduction, cell viability was assessed by MTT. Cell apoptosis and intracellular ROS were measured using flow cytometry. Western Blot detected the cytochrome C protein release and PARP cleavage protein. MTT assay measured the inhibitory ratio of MCL again, when provided the NAC on the breast cancer cells.Results: 1. MCL could inhibit breast cancer cell proliferation in vitro. Mitochondrial fission protein Drp1 were upregulated in breast cancer cells after addition of MCL, 2. EGFP, Drp1-K38 A and Drp1-WT overexpression of MCF-7 and MDA-MB-231 breast cancer cell lines was successfully constructed, and laser confocal microscope showed that the mitochondrial fragmentation rate of MCF-7 and MDA-MB-231 cells with overexpression Drp1-WT were higher than EGFP and Drp1-K38 A counterparts was observed by(P<0.05). 3. EGFP, Drp1-K38 A and Drp1-WT breast cancer cells incubated with various concentrations of MCL for 24 h. Results showed that after treatment more Drp1-WT breast cancer cells shown more necrosis and apoptosis shapes. Then we used hoechst 33342 colored the nuclei of Drp1-WT cells shown the same phenomenon. 4. After used with various concentrations of MCL for 48 hours, The most obvious sensitivity effect was observed at the concentration of 10 μM for both cell lines which suggested that breast cancer cells with Drp1 overexpression were more sensitive to MCL than EGFP and Drp1-K38 A counterparts.(P<0.05).5. Flow cytometry assay showed that the apoptosis rate of MCF-7 and MDA-MB-231 cells were increased after MCL treatment. Similarly, the apoptosis rate of MCF-7 and MDA-MB-231 cells with Drp1-WT overexpression were enhanced which indicated that MCF-7 and MDA-MB-231 cells with high Drp1-WT levels were more sensitive to MCL induced cell death.(P<0.05). 6. After treatment with MCL, cytochrome C release and PARP cleavage were both enhanced after overexpression of Drp1-WT. 7. When treated with MCL, generation of reactive oxygen species(ROS) in Drp1-WT cells was higher compared with EGFP and Drp1-K38 A cells. 8. Fluorescence microscope showed that overexpression of Drp1-WT, but not Drp1-K38 A led to ?Ψm depolarization in breast cells after treatment with MCL. 9. When pretreated cells with N-acetyl-L-cysteine(NAC), the ROS scavenger, before MCL administration, the inhibitory of MCL on different breast cancer groups was not obvious difference.Conclusions: We indicated that increased Drp1 is involved in MCL induced breast caner cell apoptosis via the ROS pathway in this process. Taken together, our findings identify a feed-forward mechanism whereby early Drp1 up-regulation under MCL treatment, resulting in mitochondrial fission and increased ROS generation, loss of mitochondrial membrane potential, augmented cytochrome c release that induces PARP cleavage and amplifies the apoptotic signal in breast cancer cells. These findings pave the way to new potential therapeutic treatments of breast cancer cells.