Objective To explore the mechanism of total Panax japonicus saponins (TPJS) in inhibiting the proliferation of renal carcinoma cells.
Methods ACHN and A498 cells were cultured and treated with different concentrations of TPJS, the growth of ACHN and A498 cells was analyzed by cell morphology. The number of colonies formed was determined by colony formation test; the activity of Caspase Glo 3/7 was determined by luminescence meter; the cytotoxicity of TPJS was evaluated by the lactate dehydrogenase cytotoxicity test kit; cell counting kit was used to detect the effect of TPJS on the proliferation of renal cell carcinoma cells; apoptosis was detected with fluorescein isothiocyanate annexin V apoptosis detection kit; the western blot was used to detect the expression of apoptosis related protein Caspase 9, B cell lymphoma 2 (Bcl-2) and Bcl-2 like protein 4 (Bax) in renal cell carcinoma cells; the concentrations of angiotensin Ⅱ (AngⅡ), AngⅡ type 1 receptor (AT1R), vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2) in total protein extract were measured by enzyme-linked immunosorbent assay (ELISA), and the effects of AngⅡ, AT1R, VEGF and COX-2 on TPJS-induced cell growth inhibition and apoptosis were analyzed.
Results The result of morphological analysis showed that the untreated renal carcinoma cells grew well, while the cells treated with TPJS were distorted, rounded and apoptotic; the results of colony formation test showed that compared with the cells treated with 0 μmol/L TPJS, the colony formation decreased significantly after 6 hours of TPJS treatment, and TPJS treatment significantly increased cytotoxicity and Caspase 3/7 activity. The results of flow cytometry showed that TPJS was able to induce apoptosis of renal cell carcinoma cells after 24 hours of treatment, in addition, TPJS was able to up-regulate the levels of Caspase 9 and Bax, and down-regulate the level of Bcl-2. Compared with the cells treated with 0 μmol/L TPJS, the levels of AngII, AT1R, VEGF and COX-2 in both ACHN and A498 cell lines were significantly reduced in a dose-dependent manner after TPJS treatment. AT1R and VEGF were able to reverse the inhibitory effect of TPJS on cell growth, and the results of apoptosis test were similar to those of cell growth.
Conclusion TPJS may inhibit the proliferation and induce apoptosis of renal cell carcinoma cells through AT1R/VEGF pathway.
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