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Broussochalcone A Induces Apoptosis in Human Renal Cancer Cells via ROS Level Elevation and Activation of FOXO3 Signaling Pathway

ABSTRACT: 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.

SUBMITTER: Lee H

PROVIDER: S-EPMC8566040 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:OBJECTIVES:SL4, a chalcone-based compound, exhibits clearly inhibitory effects on HIF-1 and has been shown to effectively suppress tumour invasion and angiogenesis in vitro and in vivo. Here, studies were conducted to determine SL4's anti-apoptotic effects and its underlying mechanisms, in human cancer cells. MATERIALS AND METHODS:Cytotoxicity, apoptotic induction and its involved mechanisms of SL4 were investigated using normal cells, cancer cells and mouse xenograft models. The role of reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK) signalling in SL4-induced apoptosis was explored by manipulating specific scavenger or signalling inhibitors, in cultured cells. RESULTS:SL4 significantly inhibited cell population growth of human cancer cell lines but exhibited lower cytotoxicity against normal cells. In addition, SL4 effectively induced apoptosis of Hep3B and MDA-MB-435 cells by activating procaspase-8, -9 and -3, and down-regulating expression levels of XIAP, but did not affect HIF-1 apoptosis-related targets, Survivin and Bcl-XL. Further study showed that SL4 also reduced mitochondrial membrane potential and promoted generation of ROS. ROS generation and apoptotic induction by SL4 were blocked by NAC, a scavenger of ROS, suggesting SL4-induced apoptosis via ROS accumulation. We also found that MAPKs, JNK and p38, but not ERK1/2, to be critical mediators in SL4-induced apoptosis. SP600125 and SB203580, specific inhibitors of JNK kinase and p38 kinase, significantly retarded apoptosis induced by SL4. Moreover, anti-oxidant NAC blocked activation of JNK and p38 induced by SL4, indicating that ROS may act as upstream signalling of JNK and p38 activation. It is noteworthy that animal studies revealed dramatic reduction (49%) in tumour volume after 11 days SL4 treatment. CONCLUSIONS:These data demonstrate that SL4 induced apoptosis in human cancer cells through activation of the ROS/MAPK signalling pathway, suggesting that it may be a novel lead compound, as a cancer drug candidate, with polypharmacological characteristics.

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Broussochalcone A Induces Apoptosis in Human Renal Cancer Cells via ROS Level Elevation and Activation of FOXO3 Signaling Pathway

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