The Influnce And Function Of MCL-1 And FBW7 Proteins In The Formation Of Polyploid Tumor

Objective:At present, the malignant tumor is still the greatest killers and a threat to human health. Found in clinical work, tumor drug resistance and relapse is closely related to the polyploid formation.The spindle poisons, such as taxol and vincristine etc, are the first-line tumor chemotherapy drugs, the most important mechanism of killing tumor is to induce cell apoptosis.In our previous study we found spindle poisons can induce tumor cells apoptosis in some cell line, but will form polyploidy tumor instead in the others. the polyploid tumor cells are more resistant to commonly used chemotherapy drugs, and its formation is associated with high expression of BCL- 2. The formation of polyploid tumor is associated with a variety of regulating abnormal molecular mechanisms, the high expression of BCL- 2 induced tumor cells apoptosis escape,P21 degradation caused spindle assembly checkpoint out of control,which lead to mitotic slippage and endoreduplication may be the important reasons for the formation of polyploid tumor, but has not been determined.Foreign scholars research also found that about 90% of solid tumor is aneuploid, chromosome amplification and the drug resistant phenotype increased lead to polyploid tumor really resistance with chemotherapy and radiotherapy, polyploid subcloning tumor cells with worse prognosis.Wertz IE et al. Study in Nature magazine showed that MCL-1(myeloid cell leukemia-1) is an important member of BCL- 2 family, meanwhile the pro-survival protein MCL-1 is a crucial regulator of apoptosis triggered by antitubulin chemotherapeutics. During mitotic arrest, The polyubiquitylation and degradation markedly of MCL-1 directed its interaction with the important ubiquitin ligase complex E3 FBW7/Cdc4(SKP1-cullin-1-F-box complex,SCFFBW7), MCL-1 protein levels declined markedly, potentiating cells death.But in ovarian cancer, non-small cell lung cancer and other tumor cells often found that The degradation of MCL-1 was blocked in tumour cells and the expression of MCL-1 protein declined markedly that lacked FBW7 or had loss-of-function mutations in FBW7, which increasing mitotic slippage, endoreduplication,conferring resistance to antitubulin therapeutics and promoting chemotherapeutic-inducedpolyploidy. Other scholars found that loss of FBW7, over-expression of MCL-1 is a common phenomenon frequently found in various types of human cancer, including breast cancer, colon cancer and T-cell acute lymphoblastic leukaemia(T-ALL).T-ALL cell lines with defective FBW7 are particularly sensitive to the multi-kinase inhibitor Sorafenib, Sorafenib play a role in anticancer by inhibiting MCL-1 protein,but these defective FBW7 cells are resistant to the BCL2 antagonist ABT-737. If FBW7 reconstitution on the genetic level or MCL-1 depletion, these cells can restore sensitivity to ABT-737, establishing MCL-1 as a T-ALL therapeutically relevant bypass survival mechanism that enables FBW7-deficient cells to evade apoptosis.This experiment by applying the spindle poisons Nocodazole, paclitaxel(Taxol)as inducer, breast cancer cells MDA- MB- 231 as the research object, harvest cells after incubation cells different time together with drugs, to observe the cell morphology and chromosome ploidy change,to explore the role of MCL- 1 and FBW7 proteins level in the polyploid formation of breast cancer cells. And expectations of inhibitting MCL- 1 protein expression by Sorafenib, to achieve the purpose of reducing polyploid formation, which can be used as a new target for curing drug resistance based on the polyploid formation.Methods:1.with 100ng/ml Nocodazole spindle poison treated breast cancer cell MDA-MB-231,harvested cells in 0h, 6h, 12 h, 24 h, 48 h and 72 h, cell morphological change was observed under microscope, cell cycle and DNA-ploidy change were examined by flow cytometry, the expression of FBW7 and MCL-1 proteins was detected by Western blot.2.Sorafenib(a multikinase inhibitor) respectively with Nocodazole or Taxol treated MDA-MB-231,with Nocodazole or Taxol or Sorafenib alone as control. Subsequently,MCL-1 protein expression of each group was detected by Western blot after 48 h, cell cycle and DNA-ploidy change were examined by flow cytometry after 48 h, cell proliferation was observed by Methyl thiazolyl tetrazolium(MTT) assay after 48 and72 h.Results:1.After Nocodazole treated cells, polyploid characteristics of large cell size and nucleus appeared over time; Octaploid percentage increased significantly over time,which was 0h(0.8±0.2)%, 6h(8.5±2.3)%, 12h(7.8±2.0)%, 24h(9.9±0.9)%,48h(28.2±0.8)% and 72h(35.1±4.9)%, respectively(F=91.417, P<0.001); Expression of FBW7 protein was decreased significantly but the expression of MCL-1 protein was increased significantly, especially after 48 h.2.Harvested cells in 48 h, the octaploid percentage(8.6±0.1)% of Nocodazole +Sorafenib group was significantly lower than(28.2±0.8)% of Nocodazole group(t=12.557, P=0.006), expression of MCL-1 protein was decreased, and cell proliferation was decreased(t=17.872, P<0.001). Expression of MCL-1protein, the octaploid percentage(0.8±0.7)% of Taxol+Sorafenib group was significantly lower than(10.5±1.3)% of Taxol group as well(t=10.92, P<0.001), expression of MCL-1protein was decreased as well, and cell proliferation was decreased as well(t=9.175,P<0.001).Conclusion:1.Low expression of FBW7 protein and overexpression of MCL-1 protein were correlated with the formation of breast cancer polyploid.2.Sorafenib could reduce polyploid tumor cells by inhibiting MCL-1 protein expression,which may provide new ideas for clinical treatment of tumor chemoresistance.