Effect Of Melatonin On Oncogenesis Of Retinoblastoma HXO-RB₄₄ Cells

Objective(1) To investigate inhibited effects of melatonin on proliferative activity of HXO-RB₄₄ cell line of Retinoblastoma and its related mechanism.(2) To investigate Effects of Melatonin on expression of VEGF in ARPE-19 cells induced by supernatant fluid of HXO-RB₄₄ cells and its related mechanism.(3) To investigate effects of melatonin on abnormal cell cycle and apoptosis in HXO-RB₄₄ cell line of Retinoblastoma and its related mechanism.(4) To study the inhibitory effect of melatonin on human RB transplanted subcutaneously in nude and its related mechanism.Method(1) The cells counting and tetrazolium dye-reduction assay (MTT) was used to determine the effect of melatonin (MLT) on HXO-RB₄₄ cell line of Retinoblastoma cell survival and proliferation for 24h, 48h and 72h at a series of concentration. The arrest rates of HXO-RB₄₄ cells proliferation by cells counting and MLT was calculated. The fluorescent intensity of ROS of HXO-RB₄₄ cells treated by MLT of 10^-5M at different times (12h, 24h, 36h, 48h) or treated by different concentration of MLT (10^-6, 10^-5, 10^-4, 10^-3M) for 24 hours were assayed by flow cytometry (FCM).(2) ARPE-19 cells in vitro culture were incubated with supernatant fluids of HXO-RB₄₄ cells, then melatonin at a series of concentration (10^-9, 10^-7, 10^-5 M) were added, incubated for 24h, the mRNA and protein expression of VEGF in ARPE-19 cells used supernatant fluids of HXO-RB₄₄ cells alone or combinated with MLT was monitored by RT-PCR, immunocytochemistry and Western-blot respectively.(3) HXO-RB₄₄ cells in vitro culture were incubated with melatonin at a series of concentration (10^-10, 10^-9, 10^-8, 10^-7M) and treated by MLT of 10^-7M at different times (4h, 8h, 12h, 24h),. The status of cell cycle distribution and apoptosis was analyzed by flow cytometry. The morphodifferentiation of apoptotic nuclei was analyzed by Hoechst-PI fluorescence staining. mRNA and Protein expression of P21, P27 gene was monitored by RT-PCR, immunocytochemistry and Western-blot respectively.(4) Human RB model transplanted subcutaneously in fifty nude mice were established and randomly divided into 5 groups, 10 mice were assigned into each group. 20, 40, 60 mg/kg MLT treated respectively in low, moderate and high doses groups for one week. The change of tumor volume and weight was detected, tumor morphology was detected by histopathology, the level of VEGF protein in tumor tissue was determined by immunohistochemistry and Western-blot respectively.Result(1) MLT at the concentration of 10^-7M or below didn't inhibit the proliferation of HXO-RB₄₄ cells in vitro. MLT at the concentration of 10^-6 M or exceed could inhibit the proliferation of HXO-RB₄₄ cells. The fluorescent intensity of ROS of HXO-RB₄₄ cells increased obviously after treatment with MLT of 10^-5 M in different times. With the augmentation of concentration in MLT from 10^-6M to 10^-4M, the degree of the fluorescent intensity of ROS in HXO-RB₄₄ cells were aggravated than ever.(2) The mRNA and protein expressions of VEGF were significantly increased in ARPE-19 cells in vitro culture incubated with supernatant fluid of HXO-RB₄₄ cells after 4h, 8h, 12h, 24h of incubation (P<0.01),. Compared with the control group, the mRNA and protein expressions of VEGF were significantly decreased in ARPE-19 cells in vitro culture incubated with melatonin at a series of concentration (10^-9, 10^-7, 10^-5M) for 24h(P< 0.01).(3) Hoechst-PI fluorescence staining showed karyopyknosis and nucleus cataclasm increased, with the augmentation of concentration in melatonin; the cells in G₀/G₁ phase were increased, the cells in S phase were decreased; Compared with the control group, the mRNA and protein expression of P21 and P27 were significantly increased in HXO-RB₄₄ cells in vitro culture incubated with melatonin.(4) The growth of tumor were obviously repressed treated by MLT of 40mg/kg, 60mg/kg (P<0.01), The inhibition ratio were 12.5, 27.89, 40.72, 44.52 (per cent) in MLT and VP-16 treated group respectively, the degree of haemorrhage and necrosis were aggravated in MLT and VP-16 treated groups. The expression of VEGF and ICAM-1 protein in MLT and VP-16 treated group was significantly lower than that in control group detected by immunohistochemistry and Western-blot respectively. The negative correlation between the concentration of MLT and the expression of VEGF and ICAM-1 protein was evident.Conclusion(1) MLT at the concentration of 10^-7M or below didn't inhibit the proliferation of HXO-RB₄₄ cells in vitro. MLT at the concentration of 10^-6M or exceed could inhibit the proliferation of HXO-RB₄₄ cells. The effects of antiproliferation of MLT may relate to increased ROS expression induced in HXO-RB₄₄ cells.(2) Supernatant fluid of HXO-RB₄₄ cells can enhance the expression of VEGF in ARPE-19 cells. Melatonin can inhibit the high VEGF expression in ARPE-19 cells induced by supernatant fluid of HXO-RB₄₄ cells.(3) Melatonin can block cell cycle in G₀/G₁ phase, intensificate the expression of P21, P27 mRNA and protein in HXO-RB₄₄ cell line of Retinoblastoma and induce cell apoptosis.(4) MLT of high concentrations can inhibit the growth of RB transplanted subcutaneously in nude mice. The expression of VEGF and ICAM-1 protein can be inhibited by MLT. There was negative correlation between the concentration of MLT and the expression of VEGF and ICAM-1 protein in transplantation tumor transplanted subcutaneously in nude...