The Effects Of Leflunomide On Cellular Proliferation And Apoptosis In Renal Carcinoma Cells

N-(4-trifluoromethyl-phenyl)-5-methylisoxazol-4-carboxamide(C12H9F3N2O2) is also named Leflunomide(LEF). It is an isoxazole derivative with a molecular weight of 270.2, which is an immunomodulatory drug used for the treatment of rheumatoid arthritis (RA).Leflunomide is rapidly converted in vivo to its pharmacologically active metabolite A771726, which is a potent non-cytotoxic inhibitor of the enzyme dihydroorotate dehydrogenase(DHODH). DHODH, a key enzyme in the de novo synthesis of uridine monophosphate UMP, thereby prevents DNA synthesis. T cells preferentially use this enzyme in pyrimidine synthesis. Meanwhile, A771726 has been reported to inhibit the activation of the NF-κB and the expression of adhesion molecules such as intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule 1(VCAM-1) and matrix metalloproteinase-1(MMP1) and so on, which is similar to the mechanism of antitumor drug action. We hypothesize that LEF could influence proliferation and apoptosis of renal cancer cells.The research contentTo investigate the effect of LEF on renal carcinoma cells, and further analyze the potential molecular mechanism.The research methodsIn this study, Caki-2 and 786-0 cells were treated with solutions of LEF(0,50,100 and 200μM) and cultured in varied duration of time(24,48 and 72 h). 1. The cell proliferation index was detected by MTS, EdU incorporation assay and Colony formation assay. Cell cycle progression was assessed by flow cytometry, and the expression of relative proteins was detected by Western Blotting.2. Apoptosis Assay, Western Blotting Assay and Transfection were used to detect apoptosis and autophagy of Caki-2.3. Western blotting, Immunofluorescence Assay and Real-time PCR were performed to investigate the expresson of β-catenin, c-Myc, Wnt3a, Wntl and Wnt5a. Western blotting and FCM were employed to determine whether inhibition of Wnt/β-catenin signaling pathway by an inhibitor of Wnt processing IWP-2 or changing the amount of P-catenin proteins could sensitize LEF in vitro. Furthermore, the mechanisms of LEF-mediated sensitization were investigated by using Gene Expression Microarray.The results of the study1. LEF inhibited cell growthMTS assays revealed that cell viability of Caki-2 and 786-0 dose-dependent decreased after exposuring to different concentrations of LEF(0-200μM) for 48 h, Caki-2 cells were more sensitive to LEF than 786-0 cells. Significant dose-dependent reduction in cell viability happened when Caki-2 cells were exposure to≥ 100 μM LEF. MTS assays also showed that LEF significantly inhibited cell growth in a time-dependent manner. EdU incorporation assay was used to detect DNA synthesis. The number of EdU positive cells significantly decreased in a dose-dependent manner after the treatment with different concentrations of LEF for 48 h. Colony formation assays showed that long-time treatment with LEF at concentrations exceeding 50 μM could inhibit the expansion of tumor clones from a single cell. LEF induced S-phase cell cycle arrest in Caki-2 cells. There was a decrease of cyclin A and CDK2 in Caki-2 cells after LEF administration. While p21 protein, the CDK inhibitor, was up-regulated.2. LEF induced cell apoptosis and autophagyLEF could induce apoptosis especially in 200 μM LEF group. Cleavage of Caspase-3 and PARP-1 was observed in 200 μM LEF group. The expression of the anti-apoptotic proteins Bcl-2 and APE/REF-1 were downregulated, while the expression of proapoptotic protein Bax was upregulated, and the expression of anti-apoptotic protein Bcl-xl was unaffected by LEF treatment at high concentrations. LEF could trigger autophagy in Caki-2 cells. LC3-GFP expression was predominantly diffuse in Caki-2 cells transfected with LC3-GFP plasmids in the absence of LEF. While, the accumulation of LC3 puncta in the cytoplasm was induced by LEF treatment. Unlike LEF-induced cell apoptosis,50μM LEF could induce autophagy in Caki-2 cells. There was elevation of LC3-Ⅱ and a decrease of P62 in protein levels in Caki-2 cells after treating with LEF.3. The potential molecular mechanism of LEF in RCC cells(1) High concentrations of LEF caused a remarkable decrease of β-catenin proteins rather than its mRNA expression. c-Myc was downregulated in mRNA and protein levels. β-catenin shuttled from the nucleus into the cytoplasm in Caki-2 cells by LEF treatment, which exhibited a speckled cytoplasmic distribution, representing the formation of β-catenin destruction complex. Nuclear export of P-catenin was a feature of canonical Wnt inhibition. All the datas indicated that LEF can inhibit the activation of Wnt/β-catenin pathway.(2) The degradation of β-catenin was greatly accelerated in Caki-2 cells subjected to a protein synthesis inhibitor, cycloheximide (CHX) upon LEF treatment. MG-132, an inhibitor of ubiquitin-proteasome system, but not autophagy inhibitor HCQ, could reverse LEF-induced β-catenin degradation. Thus, our results showed that the degradation of β-catenin protein in Caki-2 cells was happened via the ubiquitin-proteasome pathway upon LEF treatment. LEF at 100 and 200μM inhibited the phosphorylation of AKT kinase. Therefore, the inhibition of AKT induced β-catenin degradation.(3) The expression of Wnt3a was enhanced under LEF treatment. While the mRNA transcript of Wntl was slightly elevated by LEF, and Wnt5a level remained unchanged. IWP-2, an inhibitor of Wnt processing and secretion, enhanced the cytotoxic effect of LEF. LEF and IWP-2 could minimize the expression of β-catenin, c-Myc and cyclin D1 to the largest extent compared with single agents. So, LEF treatment can upregulate Wnt3a expression to counteract the anti-pro liferative and pro-apoptotic effects of LEF.(4) Gene expression microarray showed that the expression of Fzd10, a Wnt receptor, decreased by more than 800-fold after LEF treatment. The mRNA levels of Fzdl and Fzd2 were reduced by LEF.ConclusionLEF exerted its cytotoxicity through inducing growth arrest, autophagy and apoptosis. Canonical Wnt/β-catenin pathway was characterized as a pharmacological target of LEF in RCC cells. LEF treatment at high concentrations inhibited the phosphorylation of AKT kinase, induced the nucleo-cytoplasmic shuttling of β-catenin and subsequently promoted its proteasome-dependent proteolysis, inducing the decrease of c-Myc and cyclin D1. LEF treatment can change the expression of Wnt ligands and receptors in mRNA levels. Fzd10 and Fzd2 were repressed in Caki-2 cells by LEF treatment. Meanwhile, LEF treatment upregulated Wnt3a expression to counteract the antiproliferative and proapoptotic effects of LEF. Taken together, LEF treatment at high concentrations inhibited canonical Wnt/β-catenin pathway, which provided a potential therapy in treating renal carcinoma.