Effects Of Heavy Ions (¹²C⁶⁺) On Radiation Sensitivity And Invasion And Metastasis Of Malignant Melanoma Cells B16F10

At present,the process of global industrialization is gradually accelerating,and the destruction of human living environment is increasingly aggravating.The ozone hole is piecemeal expanding;as a result,ultraviolet rays could not effectively shielded by atmosphere.A large number of ultraviolet ray reach the surface of the earth,and long-term exposure of human skin to ultraviolet ray causes abnormal metabolism of the human body and mutation of melanocytes to form melanoma.Melanoma is a kind of skin malignant tumor with the fastest growth rate,strong invasion,migration ability and high mortality rate,which mostly occurs in the skin and body surface,organs and organs of the mucous membrane and so on.Surgery treatment is mainly for early malignant melanoma.The cure rate is up to 95%after surgical resection,but most of patients are found late,which is in the middle and advanced stage.The chemotherapy mainly consists of dacarbazine（DITC）-based multi-drug combination.The objective effective rate is 7%¹3%.Temozolomide（TMZ）and formoterol（FTM）are used as chemotherapy drugs instead of DITC,but the effect is not significantly better than DITC.The best clinical effect is immunotherapy drug anti-PD-1 monoclonal antibody,the overall effective rate is 42%,the complete remission rate is 17%,but because of the high price of the drug,the use is limited.Clinical studies have shown that malignant melanoma is insensitive to conventional radiation（X-ray）radiotherapy,easy to recur and has a high rate of distant metastasis,and most of the patients die of lung metastasis.The effect of heavy ion(¹²C⁶⁺)radiotherapy is better than that of conventional radiotherapy.After heavy ion radiotherapy,the local control rate of patients is significantly improved.Radiotherapy is considered to be a standard model for the clinical treatment of malignant melanoma.The mechanism of the significant difference between conventional radiation（X-ray）and heavy ion(¹²C⁶⁺)radiotherapy in the treatment of malignant melanoma is still unclear.There are few literature reports.The NF-κB signaling pathway is closely related to tumor invasion and migration.Therefore,B16F10 was cultured in vitro to detect the effects of X-ray and heavy ion(¹²C⁶⁺)irradiation on cell proliferation,apoptosis,cycle,migration,EMT and NF-κB signaling pathway.The results of the study are as follows:1.Melanoma B16F10 cells cultured in vitro were irradiated with different doses of heavy ions(¹²C⁶⁺).The morphology of melanoma B16F10 cells was observed under inverted microscope.The morphological changes of apoptotic cells were observed under fluorescence microscope by Hoechst 33342/PI double staining.It was found that heavy ions(¹²C⁶⁺)irradiation could induce apoptosis of melanoma B16F10cells;2.The proliferation activity of malignant melanoma cell line B16F10 irradiated by heavy ion(¹²C⁶⁺)and conventional radiation（X-ray）was detected by CCK-8.The results showed that heavy ion(¹²C⁶⁺)irradiation could inhibit the proliferation activity of malignant melanoma cell line B16F10.The inhibition effect of X-ray is more obvious than that of X-ray;3.Western Blotting was used to detect the expression level of apoptosis-related in malignant melanoma cells B16F10 after X-ray and heavy ion(¹²C⁶⁺)irradiation.The result shows that heavy ion(¹²C⁶⁺)significantly inhibited the expression of B16F10 anti-apoptotic protein Bcl-2 and C-myc,and induced the expression of apoptotic protein Bax to promote the apoptosis of melanoma cells B16F10;4.The phase distribution of B16F10 cells irradiated by heavy ions(¹²C⁶⁺)was detected by flow cytometry.The results showed that G2/M phase arrest occurred after heavy ion(¹²C⁶⁺)irradiation.Malignant melanoma cell line B16F10 is highly sensitive to heavy ion(¹²C⁶⁺)radiation;5.Cell scratches were used to detect the cell migration ability of malignant melanoma cell line B16F10 irradiated by heavy ions(¹²C⁶⁺)and conventional rays（X-rays）.The results showed that compared with X-rays,Heavy ion(¹²C⁶⁺)irradiation significantly inhibited the migration of B16F10 cells;6.Western Blotting method was used to detect the expression of invasion and migration related proteins in B16F10 melanoma cells irradiated by heavy ions(¹²C⁶⁺)and conventional radiation（X-ray）.The result shows that heavy ions(¹²C⁶⁺)inhibited extracellular matrix lysis by restraining the expression of MMP-2 and MMP-9,and induced the expression of E-cadherin to enhanced cell adhesion and further inhibited cell invasion,migration and EMT;7.Western Blotting method was used to detect the expression of NF-κB signaling pathway protein after X-ray and heavy ion(¹²C⁶⁺)irradiation.The result shows that heavy ions(¹²C⁶⁺)could activate NF-κB signaling pathway more effectively than X-ray.The downstream proteins TNF-αcould participate in apoptosis,and the highest expression at 2.5GyE,while heavy ions(¹²C⁶⁺)irradiation has the greatest influence on cell proliferation and apoptosis in 2.5GyE,which presumed that TNF-αis involved in the regulation of apoptosis after heavy ions(¹²C⁶⁺)irradiation.