Synthesis Of Emodin Derivatives And Study On The Mechanism Of Radiosensitization Effect On Nasopharyngeal Carcinoma Via Mitochondrial Pathway

Objective Emodin from guangxi Polygonum multiflorum is modificated to obtain emodin derivatives. Radiosensitization effect of emodin derivatives was explored. Radiosensitization molecular mechanism is studied on nasopharyngeal carcinoma cells CNE-1 and comfirmed the mechanisms responsible for mitochondria-mediatedMethod (1) Hydroxyl structure of Emodin was modified through the methylation, acylation method. Physical and chemical properties of emodin derivatives was determined with IR, NMR. (2) The growth inhibition rate of CNE-1 cells was detected by MTT method. (3) Cloning survival assay was used for studing the radiosensitizing effect of emodin derivatives and HE was observed cells morphological changes in radiosensitization process.(4) CNE-1 cells ultrastructure changes during radiotherapy sensitization process by emodin derivative was observed electronmicroscope.(5) mitochondria membrane potential changes of CNE-1 cells in radiosensitization process was assayed by rhodamine 123 under laser scanning confocal microscope.(6) ATP6, ATP8, CtyB mitochondrial genes expression were detected through fluorescence quantitative PCR.Results (1) Four emodin derivatives were obtained.They are GXHSWAQ-2, GXHSWAQ-3, GXHSWAQ-4, GXHSWAQ-5, respectively.(2) MTT results show that growth inhibition rate of CNE-1 cells was less than 10% when the emodin derivatives GXHSWAQ-1, GXHSWAQ-2 concentration was 12.5μg/ml.(3) There were effects radiosensitivity when CNE-1 cells was exposed to GXHSWAQ-1, GXHSWAQ-2 in no cytotoxic effects concentration (10 ug/ml). The sensitization enhancement ration(SER) was 1.36 and 1.46, respectively.(4) After CNE-1 cells exposed in high doses GXHSWAQ-1200μg/ml and 50μg/ml respectively,the HE results showed that cells vacuolation, volume increases, membrane appear disintegration. It seems the cells was oncosis.(5) There were not obvious ultrastructural changes in 10μg/ml emodin derivatives GXHSWAQ-1 and GXHSWAQ-2 by Transmission electron microscopy. The cells demonstrated volume expansion, membrane permeability, integrity, sabotage, cell dissolve, cytoplasmic leakage after 100μg/ml GXHSWAQ-1 treatment. The volumn cells of radiosensitization groups were increased and the mitochondria swelling, mitochondria ridge swelling.(6) Fluorescence intensity was 40.632 in control group after rhodamine 123 stained, Fluorescence intensity was 21.390 for GXHSWAQ-1 group and 26.560 for GXHSWAQ-2 group after treatment 16h. Fluorescence intensity was 36.534 for 2Gy radiation after 16h. Fluorescence intensity was 26.526 for 8Gy radiation after 16h. Fluorescence intensity was 14.730 and 19.136 in GXHSWAQ-1 and 2 GXHSWAQ radiosensitization group after radiation 16h,respectively. Compare withcontrol group, fluorescence intensity was down in drug alone group (P<0.01). The radiosensitization group compared with control group fluorescence intensity was dramatic decline after radiation 16h (P<0.01). Fluorescence intensity of radiosensitization group was lower than that of drug alone group and radiate alone group(P<0.01).(7) The expression of ATP6 mitochondrial gene was dramatic increased compare with the control group (P<0.01) and had the dose dependent. Expression of ATP8, CtyB mitochondrial gene was relatively diminished (P<0.01) and had the dose dependent.Conclusion (1) There are radiosensitizing effect on CNE-1 cells after exposure GXHSWAQ-1 and GXHSWAQ-2 in the no-cytotoxic concentrations.(2) The Radiosensitization mechanism of emodin derivatives GXHSWAQ-1 and GXHSWAQ-2 may be decreased Mitochondrial membrane potential, raised mitochondrial gene of ATP6 expression, downed regulation ATP8 and CtyB mitochondrial gene expression, induced cells oncosis.