Establishment Of P53 High Throughput Screening Model & Studies Of Effects Of 42-5 On Renal Insufficiency And Its Mechanisms Of Action

The growth suppressor protein p53 is a key regulator of the cell cycle and cell proliferation. Usually, level of p53 in the cell is low due to the short half life of the protein. However, in cases of DNA damage, including those induced by genotoxic anticancer drugs and environmental exposures, p53 is stabilized and induces a G1- or G2-phase arrest of the cell cycle or even causes cell death. Wild type p53 is one of main targets for many anticancer drugs to take action, including ADM, VP-16 and Taxol. Designing efficient ways to induce the expression of p53 in tumor expressing wild type p53 is therefore key issue in anticancer drug research.However, the role of wild type p53 in cancer treatment is not limited to its involvement in killing tumor cells. The wild type p53 gene is highly expressed in several normal tissues, including lymphoid and hematopoietic organs, intestinal epithelia, and the testis, and it is these tissues that are damaged by anticancer therapy. P53-dependent apoptosis occurs in sensitive tissues shortly after gamma irradiation. More than 50% of human primary tumors have lost the wild type p53 suppressor gene and instead express high level of mutant p53 protein. P53-dependented apoptosis is invalid when cure these turners with the above antitumor drugs, and the side effects associated with the activation of p53 become relative serious in normal tissues. Thus it may be an appropriate target for therapeutic suppression to reduce the damage to normal tissue. This approach would be applicable only for tumors that lack functional p53.In order to obtain an experimental tool for the analysing expression of wild type p53 with different compounds, we created a plasmid named p53-luc-pcDNA3.0 carrying a reporter luciferase gene under the control of a p53-responsive promoter with p53-binding sequences of human p21 promotor. The choice of the construct was based on its efficient and specific p53-dependent transcriptional activation. The plasmid was transfected into a human breast cancer cell line (MCF-7) which expressing wild type p53 to constructe wild type p53-specific reporter system. The reconstructed human breast cancer cells, termed p53R-MCF-7 cells can be used to screening p53 inducer or inhibitor through bioluminescent. We tested 9 conventional chemotherapeutic agents in vitro. ADM, Taxol and 5-FU activated p53 activity by about 50ï¼…or above 50ï¼…under a concentration of 10^-7mol/L. The kidney disease, especially chronic kidney disease (including all kinds of chronic nephritis, diabetic nephropathy and hypertensive nephropathy) is one of the common chronic diseases that are hard to cure. No matter what pathogeny, pathological changes of renal tissue is always advanced to the end-stage glomerular sclerosis and/or interstitial fibrosis from the early-stage changes. Recent studies show that transforming growth factor-β(TGF-β) and Angiotensinâ…¡(Angâ…¡) are two key factors which relate to renal fibrosis. Angâ…¡increases the accumulation of extracellular matrix proteins through the induction of fibrogenic cytokines like transforming growth factor-β1(TGF-β1). TGF-β1, part of a multimember protein super-family with overlapping functions, stimulates the production of extracellular matrix proteins like collagens and fibronectin as part of a wound repair response to injury, reduces collagenase production, facilitates matrix assembly, inhibits matrix degradation, finally leads to renal scarring.To define TGF-β(one of the important factors that relate to renal fibrosis) as a target in vitro, by purposeful design, synthesis, screening, structure modification, optimization and repeated pharmacodynamics confirmation in vivo, we regard 42-5 as a new structure compound which has the specific effects on renal insufficiency. It is a coumarin derivative synthesized by professor Shiping Xu and Ping Xie. This study aimed to evaluate its protective effects against renal insufficiency and to clarify the mechanism of this protective action. Preliminary study was also made in its toxicological characteristics. Main results cound be summarized as follows:(1) Protection of 42-5 on the cisplatin-caused toxicity in renal cells In vitro, 42-5 was found to protect from damage of rat renal mesangial cells (rMC) and human kidney tubule epithelial cell (HKC) induced by low concentration cisplatin. MTT assay showed the inhibition ratios of rMC induced by cisplatin were lower in incubation with 33μmol/L or 100μml/L 42-5 compared with incubation with cisplatin (0.12—1μmol/L) alone. The inhibition ratios of HKC induced by cisplatin were lower in incubation with 2μmol/L, 10μmol/L or 50μmol/L 42-5 compared with incubation with cisplatin (0.12—2.5μmol/L) alone too.(2) Effects of 42-5 on renal insufficiency of ARF induced by cisplatin, 5/6 nephrectomy and diabetic induced by STZ in rats.In vivo, different renal insufficiency models including ARF induced by cisplatin, 5/6 nephrectomy and diabetic induced by STZ in rats were characterized with renal dysfunction (BUN and Scr increase), decrease of body weight and the development of proteinuria. Morphological examination showed renal tubule epithelial cell necrosis in acute failure model (ARF induced by cisplatin in rats) and glomerulosclerosis and interstitial fibrosis in chronic renal insufficiency model (5/6 nephrectomy and diabetic induced by STZ in rats). 42-5 administration exerted functional and morphological protection against ARF induced by cisplatin, 5/6 nephrectomy and diabetic induced by STZ in rats. BUN, Scr and urine protein were 237.10±24.34 mg/dl, 4.41±0.73mg/dl and 5.65±3.43 mg/24hr in rats 4 days following cisplatin injection compared with control values of 27.98±4.01 mg/dl, 1.14±0.56mg/dl and 1.24±0.72 mg/24hr in ARF induced by cisplatin in rats. Treatment with 42-5(30mg/kg) resulted in a significant reduction in BUN(125.84±67.06 mg/dl), Scr(2.83±1.16 mg/dl)and urine protein (2.06±1.41 mg/24hr)that after 7 day of treatment. BUN, Scr and urine protein were 46.66±9.38 mg/dl, 1.65±0.27mg/dl and 105.38±38.61 mg/24hr in rats 16 wk following surgery compared with control values of 25.34±4.52 mg/dl, 0.98±0.36mg/dl and 11.49±5.12 mg/24h in 5/6 nephrectomy rats. Treatment with 42-5(30mg/kg) resulted in a significant reduction in BUN(37.38±6.67 mg/dl), Scr(1.07±0.40 mg/dl)and urine protein (29.21±13.70 mg/24h)that after 12 wk of treatment. Glu, BUN, Scr and urine protein were 494.35±45.89 mg/dl, 45.64±6.74 mg/dl, 2.15±0.42mg/dl and 14.13±5.10mg/24hr in rats 16 wk following STZ injection compared with control values of 62.68±8.62mg/dl, 22.31±1.46 mg/dl, 1.41±0.21mg/dl and 8.62±1.71mg/24hr in diabetic induced by STZ in rats. Treatment with 42-5(20mg/kg) resulted in a significant reduction in Glu (332.44±84.06mg/di), BUN (32.84±4.67mg/dl), Scr (1.72±0.34 mg/dl) and urine protein (7.41±2.04mg/24hr)that after 14wk of treatment. Morphological examination showed that 42-5 can attenuate the renal tubule epithelial cell necrosis in ARF induced by cisplatin in rats and glomerular sclerosis and interstitial fibrosis in rats with chronic renal disease.(3) Main mechanisms of the protective actionIn vitro mechanism research demonstrated that the activity of 42-5 correlated with inhibiting apoptosis of HKC, transdifferentiation of HKC, lipid peroxidation, and TGF-β₁ receptor binding, increasing matrix metalloproteinase activity, uPA mRNA and matrix metalloproteinase-2(MMP-2) mRNA expression. In vivo mechanism research demonstrated that the activity of 42-5 correlated with inhibiting plasma renin activity, TGF-β₁ mRNA expression, decreasing serum TGF-β₁ and Angâ…¡level, inhibiting synthesis of TGF-β₁, Fibronectin, and Collagenâ…£in kidney tissue.(4) Preliminary study on toxicological characteristics of 42-5Primary safety assay showed that 42-5 had not cytotoxicity on tested different cell lines. Acute toxicity test showed MTD＞5g in mouse orally once. Using His^- type Salmonella Typhimurium TA97, TA98, TA100 and TA102, mutagenesis of 42-5 was determined at concentration of 0.05, 0.5, 5.0, 50.0, 500.0μg/plate with S9 or without S9, according to Ames (1983) revised method. The results showed that the number of colony formation of Salmonella Typhimurium TA97, TA98, TA100 and TA102 induced by 42-5 did not increase by 2 fold. The frequency of micronucleated polychromatic erythrocytes (PCE) in bone marrow cells in mice, induced by 42-5 was not increase either. It suggests that 42-5 has no mutagenesis.In summary, It was found in this study that 42-5, a coumarin derivative, could protect the kidney from several renal insufficiency including models induced by cisplatin, 5/6 nephrectomy and diabetic induced by STZ in rats. Mechanism research indicated TGF-β₁ and Angâ…¡were two main targets which 42-5 take actions and it had no definite mutagenesis. It suggests that 42-5 may be a candidate to develop new drug used to cure renal insufficiency.