ABSTRACT
Renal ischemia/reperfusion (I/R) injury continues to be a complicated situation in clinical practice. Genistein, the main isoflavone found in soy products, is known to possess a wide spectrum of biochemical and pharmacological activities. However, the protective effect of genistein on renal I/R injury has not been well investigated. In the current study, we explore whether genistein exhibits its renal-protective effects through SIRT1 (Sirtuin 1) in I/R-induced mice model. We found the treatment of genistein significantly reduced renal I/R-induced cell death, simultaneously stimulating renal cell proliferation. Meanwhile, SIRT1 expression was up-regulated following the administration of genistein in renal region. Furthermore, pharmacological inhibition or shRNA-mediated depletion of SIRT1 significantly reversed the protective effect of genistein on renal dysfunction, cellular damage, apoptosis, and proliferation following I/R injury, suggesting an indispensible role of the increased SIRT1 expression and activity in this process. Meanwhile, the reduced p53 and p21 expression and increased PCNA (Proliferating Cell Nuclear Antigen) expression were blocked after the depletion of SIRT1 compared with the genistein treatment group in the renal I/R process. Hence, our results provided further experimental basis for the potential use of genistein for the treatment of kidney disease with deficiency of SIRT1 activity.
Subject(s)
Genistein/pharmacology , Kidney Diseases/drug therapy , Kidney/drug effects , Reperfusion Injury/drug therapy , Sirtuin 1/metabolism , Animals , Apoptosis/drug effects , Cell Proliferation/drug effects , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Disease Models, Animal , Kidney/physiopathology , Mice , Mice, Inbred BALB C , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism , Reperfusion Injury/complications , Sirtuin 1/genetics , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
OBJECTIVE: To investigate the correlation between methylation status and gene expression of APC (adenomatous polyposis coli) gene in HeLa, CaSki and SiHa cell lines of cervical carcinoma, and explore the effect of hydralazine on the transcription regulation of the 5'CpG island demethylation of APC gene and the proliferation and apoptosis of the cell lines. METHODS: Methylation status and the expression of APC gene were analyzed using methylated specific PCR, RT-PCR and FQ-PCR methods. The expression of beta-catenin protein which correlates closely with APC was detected by SP method after treatment with Hydralazine. MTT and FCM assays were used to observe the changes of proliferation activity and apoptosis of the cells after Hydralazine treatment. RESULTS: (1) APC gene was methylated or hemimethylated respectively in HeLa and CaSki cell lines, at the same time, APC gene was not methylated in SiHa cell. (2) After having been treated by 40 micromol/L Hydralazine for 72 hours, growth inhibitory ratios of HeLa, CaSki and SiHa cell lines were (52.12 +/- 3.78)%, (44.31 +/- 2.59)% and (47.73 +/- 4.73)% respectively, on the contrary, normal cell HECV's growth inhibitory ratio was only (27.18 +/- 0.79)%. APC gene in HeLa and CaSki cell lines which were treated by 40 micromol/L Hydralazine for 72 hours was demethylated and expressed positively, the expression of APC mRNA in HeLa, CaSki and SiHa cell lines increased to 10.35, 11.40 and 0.73 times respectively. (3) Hydralazine, when used at the concentration of 40 micromol/L for 72 hours, induced S phase and G2/M phase arrest and apoptosis in HeLa and CaSki cells. beta-catenin protein can be expressed in cell membrane after treatment with Hydralazine. CONCLUSION: APC gene methylation plays an important role in the carcinogenesis of cervical cells and can re-express after the treatment with Hydralazine which also could inhibit the growth of the cervical cancer cells.
