| 1．Objective Renal cell carcinoma is common malignant tumor in urinary system. Up to date, surgery remains the only effective method of treatment of primary renal cancer. The traditional modern management of advanced solid tumors has been with cytotoxic agents, despite the remarkable advances realized with other tumors, renal cancer has remained refractory to these agents and radiotherapy, chemotherapy involving more than 4000 patients with cancer of kidney was found a total response rate of 6%. So, it is necessary for us to explore the more effective chemotherapy for patients with renal carcinoma. In nineties last century, As2O3 was successfully used to cure the acute promyelocytic leukemia (APL) in our country, and its anti-cancer effect immediately attracted the great interest of scientists in the world. Now, the studies on anti-tumor effect of As2O3 have been undertaken not only in hemological tumor, but also in solid tumor. However, there have been few reports about the effect of As2O3 in renal cancer. In orde to provide the theoretical basis for clinical application of As2O3 in therapy of the kidney cancer, the inhibiting effects of As2O3 on human renal cell carcinoma 786-0 cells and its mechanisms were studied in this paper. 2．Methods and ResultsPart One Effect of As2O3 on the proliferation inhibition of 786-0 cells were analysed by cell culture, light microscopy, electronic microscopy, MTT and tumor colony formation, and the changes of protein expression of PCNA and ki-67 were studied by immunocytochemistry. The result showed that ≥2.0μmol/L As2O3 could effectively inhibit the cell proliferation of 786-0(P <0.01) , decrease it's mitosis index (P <0.01), down-regulate expression of PCNA and ki-67 protein in cells(P <0.05), which was the time and dose-dependent. Part Two In this study, The change of 786-0 cell cycle was measured by cell culture flow cytometric assay, immunocytochemistry and RT-PCR.We found that treatment with 2.0μmol/L or 5 μmol/L As2O3 for 24h, 48h and 72h, could obviously induce a G2/M and G0/G1 phase arrest in 786-0 cells, decreased protein expression of cyclinA, B1, D1, E (P <0.05) and increased activities of CDKI genes p16, p21WAF! and p27 CIPI (P <0.01), the expression of Egr-1 mRNA was observed 786-0 cells treated with As2O3 at half an hour, increased to a peak at 4 hours , and then decreased quickly.Part Three This study was to explored the onset of apoptosis in 786-0 cells treated with As2O3 by cell morphology, DNA gel electrophoresis, flow cytometric assay and TUNEL, immunocytochemistry and RT-PCR. The characteristic apoptotic morphological changes and DNA fragmentation were observed after treated with ≥2.0μmol/L As2O3 for 48h, and FCM revealed that 2.0μmol/L As2O3 can induce a sub-G1 peak in 786-0 cells after treated with As2O3 for 24h. The expression of p53 gene was up-regulatied (P <0.05), but bcl-2, c-myc and c-fos were down regulated (P <0.05).Part Four The changes of VEGF,nm-23,MMP-9,TIMP-2 protein expression were studied by immunocytochemistry , and TGF-β1, TβR-I and TβR-II mRNA were analysed by RT-PCR in 786-0 cells treated with As2O3. Results showed that 2.0μmol/L As2O3 could effectively inhibit the protein expression of VEGF and MMP-9 in 786-0 cells (P <0.05), but increase nm-23 and TIMP-1 protein expression (P <0.05). In this study, absence of expression of transforming growth factor-β type II receptor mRNA was found in 786-0 cells, but we could not detected the change of mRNA expression of transforming growth factor-β1 and transforming growth factor-β type II receptor in 786-0 cells treated with As2O3 for 72 h.Part Five In this study, we investigated NF-κB DNA binding activity, p65 mRNA, p65, IKKα/β,IκBαand ICAM-1 protein in reanl cell cancer tissues and human renal cell carcinoma 786-0 cells by immunocytochemistry, in situ hybridization, Western blot and EMSA. Results showed that in renal cell cancer tissues, NF-κB DNA binding activity, p65, IKKα/βand ICAM-1 protein were increased , compared to normal...
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