Broussochalcone A (BCA) is a chalcone compound extracted from the cortex of Broussonetiapapyrifera (L.) Ventenat that exerts various effects, such as potent antioxidant, antiplatelet, and anticancer effects. However, the effects of BCA against cancers have been seldom studied. This study is aimed at demonstrating the apoptotic mechanisms of BCA in A498 and ACHN cells, which are two types of human renal cancer cell lines. MTT, cell counting, and colony formation assays indicated that BCA treatment inhibited cell viability and cell growth. Further, cell cycle analysis revealed that BCA induced cell cycle arrest at the G2/M phase. Annexin V/PI staining and TUNEL assays were performed to determine the apoptotic effects and DNA fragmentation after treatment with BCA. Based on western blot analysis, BCA induced the upregulation of cleaved PARP, FOXO3, Bax, p21, p27, p53, phosphorylated p53 (ser15, ser20, and ser46), and active forms of caspase-3, caspase-7, and caspase-9 proteins, but downregulated the proforms of the proteins. The expression levels of pAkt, Bcl-2, and Bcl-xL were also found to be downregulated. Western blot analysis of nuclear fractionation results revealed that BCA induced the nuclear translocation of FOXO3, which might be induced by DNA damage owing to the accumulation of reactive oxygen species (ROS). Elevated intracellular ROS levels were also found following BCA treatment. Furthermore, DNA damage was detected after BCA treatment using a comet assay. The purpose of this study was to elucidate the apoptotic effects of BCA against renal cancer A498 and ACHN cells. Collectively, our study findings revealed that the apoptotic effects of BCA against human renal cancer cells occur via the elevation of ROS level and activation of the FOXO3 signaling pathway.

Acetylshikonin is a shikonin derivative originated from Lithospermum erythrorhizon roots that exhibits various biological activities, including granulation tissue formation, promotion of inflammatory effects, and inhibition of angiogenesis. The anticancer effect of acetylshikonin was also investigated in several cancer cells; however, the effect against renal cell carcinoma (RCC) have not yet been studied. In this study, we aimed to investigate the anticarcinogenic mechanism of acetylshikonin in A498 and ACHN, human RCC cell lines. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide), cell counting, and colony forming assay showed that acetylshikonin induced cytotoxic and antiproliferative effects in a dose- and time-dependent manner. Cell cycle analysis and annexin V/propidium iodide (PI) double staining assay indicated the increase of subG1 phase and apoptotic rates. Also, DNA fragmentation was observed by using the TUNEL and comet assays. The intracellular ROS level in acetylshikonin-treated RCC was evaluated using DCF-DA. The ROS level was increased and cell viability was decreased in a dose- and time-dependent manner, while those were recovered when cotreated with NAC. Western blotting analysis showed that acetylshikonin treatment increased the expression of FOXO3, cleaved PARP, cleaved caspase-3, -6, -7, -8, -9, γH2AX, Bim, Bax, p21, and p27 while decreased the expressions of CYP2J2, peroxiredoxin, and thioredoxin-1, Bcl-2, and Bcl-xL. Simultaneously, nuclear translocation of FOXO3 and p27 was observed in cytoplasmic and nuclear fractionated western blot analysis. Acetylshikonin was formerly identified as a novel inhibitor of CYP2J2 protein in our previous study and it was evaluated that CYP2J2 was downregulated in acetylshikonin-treated RCC. CYP2J2 siRNA transfection augmented that apoptotic effect of acetylshikonin in A498 and ACHN via up-regulation of FOXO3 expression. In conclusion, we showed that the apoptotic potential of acetylshikonin against RCC is mediated via increase of intracellular ROS level, activation of FOXO3, and inhibition of CYP2J2 expressions. This study offers that acetylshikonin may be a considerable alternative therapeutic option for RCC treatment by targeting FOXO3 and CYP2J2.

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