The Research Of Cisplatin-induced Neuroblastoma Cell Senescence And Its Experimental Mechanism

Background and objective: Neuroblastoma(NB)is a common extracranial solid tumor in childhood.Despite aggressive multiple therapy,more than 50% of children with high-risk neuroblastoma relapse,and survive rate after relapse is rare.High doses of chemotherapeutic drugs can cause severe side effects on children,while increasing the intensity of chemotherapy does not significantly improve the long-term survival of high-risk children.Reducing the dose of chemotherapeutic drugs may not directly induce apoptosis,but may induce senescence in tumor cells.And this way may also temporarily inhibit tumor cell proliferation and reduce treatment-related side effects.This study will investigate whether low-dose cisplatin can induce neuroblastoma cell senescence and its molecular mechanismMethods: The NMYC-amplified neuroblastoma cell line(IMR32)was used as the study object,and the effects of different concentrations of cisplatin on cell growth and cell cycle were examined by thiazolyl blue colorimetry(MTT)and flow cytometry,to compare the corresponding changes in IMR32 cells under the action of cisplatin.After treatment with cisplatin for 48 h,senescence associated-β-galactosidase(SA-β-gal)staining was used to detect cell senescence.Western Blot was used to detect the expression of NMYC and p-NMYC proteins in senescent cells.The senescence-associated sectroy phenotype(SASP)phenomenon was studied by RT-PCR and Western Blot techniques.Results: 1.IMR32 was treated with cisplatin at different concentrations(0.5 μmol/L,1 μmol/L,2 μmol/L and 5 μmol/L).The results of MTT indicated that there was no difference in cell proliferation rate in each group after the treatment of cisplatin for 24 h,and the proliferation of cells in the 5 μmol/L group was significantly inhibited at 48 h,and it was more obvious at 72 h.After 48 hours of drug treatment,the proliferation rate of 2 μmol/L group gradually slowed down,especially 72 hours,which was significantly different from the other two groups(0.5 μmol/L and 1 μmol/L).The 0.5 μmol/L group had almost no significant effect on cell proliferation.2.The results of flow cytometry showed that G2/M phase cells respectively accounted for 30.95%,57.58%,70.14%,and 38.69% with the treatment of cisplatin at different concentrations(0.5 μmol/L,1 μmol/L,2 μmol/L,5 μmol/L)for 48 h,which was significantly different from the control group(G2/M phase cells accounted for 10.76%).Most of the cells treated by cisplatin were arrested in the G2/M phase,and the blocking rate of cisplatin(2 μmol/L)could reach 70.14%,and flow apoptosis assay showed that apoptosis was not obvious.The blocking rate of cisplatin at a concentration of 5 μmol/L was 38.69%,and apoptosis assay showed an increase in apoptotic cells.3.The morphological changes of cells treated with low-dose cisplatin(2 μmol/L)for 24 h were not obvious.But the cell morphology changed significantly with low-dose cisplatin(2 μmol/L)on cells for 48 h.Compared with the control group,the number of cells obviously decreased,and volume increased significantly,and cell death was not obvious.Cells were stained by SA-β-gal,which was treated by cisplatin(2 μmol/L)for 48 h.It could be seen that the number of blue-stained cells was significantly higher than control in the administration group,indicating that cisplatin could induce senescence of neuroblastoma cells.4.Cells were analyzed for the expression of NMYC and p-NMYC proteins by Western Blot.The results showed that there was no significant change in NMYC and p-NMYC proteins in the cells of the medicated group,compared with the control group at 6h and 24 h.As the drug worked for a longer period of time,the expression of NMYC and p-NMYC proteins were down-regulated in cells after 48 hours of action of cisplatin.5.The most prominent cytokine associated with SASP is interleukin 6(IL-6).The SASP phenomenon after tumor cell senescence was studied.The results of PCR showed that the expression level of interleukin 6(IL-6)m RNA was up-regulated in senescent cells,compared with the control group.Conclusions:1.Different concentrations of cisplatin could inhibit cell proliferation,in which low-dose cisplatin mainly blocked the cell cycle in the G2/M phase and high-dose cisplatin induced apoptosis.2.Low-dose cisplatin could induce senescence in neuroblastoma cells to inhibit tumor growth,and NMYC protein was down-regulated in senescent cells with an increase in IL-6 m RNA expression.