Department of Biological Science, Gachon University, Seongnam 13120, Republic of Korea.
Department of Integrative Biotechnology, Sungkyunkwan University, Suwon 16419, Republic of Korea.
Oxid Med Cell Longev. 2021 Apr 13;2021:6647107. doi: 10.1155/2021/6647107. eCollection 2021.
Acetylshikonin, a naphthoquinone, is a pigment compound derived from Arnebia sp., which is known for its anti-inflammatory potential. However, its anticarcinogenic effect has not been well investigated. Thus, in this study, we focused on investigating its apoptotic effects against HCT-15 and LoVo cells, which are human colorectal cancer cells. MTT assay, cell counting assay, and colony formation assay have shown acetylshikonin treatment induced cytotoxic and antiproliferative effects against colorectal cancer cells in a dose- and time-dependent manner. DNA fragmentation was observed via terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Also, the increase of subG1 phase in cell cycle arrest assay and early/late apoptotic rates in annexin V/propidium iodide (PI) double staining assay was observed, which indicates an apoptotic potential of acetylshikonin against colorectal cancer cells. 2',7'-Dichlorofluorescin diacetate (DCF-DA) staining was used to evaluate reactive oxygen species (ROS) generation in acetylshikonin-treated colorectal cancer cells. Fluorescence-activated cell sorting (FACS) analysis showed that acetylshikonin induced an increase in reactive oxygen species (ROS) levels and apoptotic rate in a dose- and time-dependent manner in HCT-15 and LoVo cells. In contrast, cotreatment with N-acetyl cysteine (NAC) has reduced ROS generation and antiproliferative effects in colorectal cancer cells. Western blotting analysis showed that acetylshikonin treatment induced increase of cleaved PARP, H2AX, FOXO3, Bax, Bim, Bad, p21, p27, and active forms of caspase-3, caspase-7, caspase-9, caspase-6, and caspase-8 protein levels, while those of inactive forms were decreased. Also, the expressions of pAkt, Bcl-2, Bcl-xL, peroxiredoxin, and thioredoxin 1 were decreased. Furthermore, western blotting analysis of cytoplasmic and nuclear fractionated proteins showed that acetylshikonin treatment induced the nuclear translocation of FOXO3, which might result from DNA damage by the increased intracellular ROS level. This study represents apoptotic potential of acetylshikonin against colorectal cancer cells via translocation of FOXO3 to the nucleus and upregulation of ROS generation.
乙酰紫草素是一种萘醌类化合物，是一种来自紫草属植物的色素化合物，具有抗炎潜力。然而，其抗癌作用尚未得到充分研究。因此，在这项研究中，我们专注于研究其对人结肠癌细胞 HCT-15 和 LoVo 的凋亡作用。MTT 测定、细胞计数测定和集落形成测定表明，乙酰紫草素处理以剂量和时间依赖的方式诱导结肠癌细胞的细胞毒性和抗增殖作用。末端脱氧核苷酸转移酶 dUTP 缺口末端标记（TUNEL）测定观察到 DNA 片段化。此外，在细胞周期阻滞测定中观察到亚 G1 期的增加和在 Annexin V/碘化丙啶（PI）双重染色测定中早期/晚期凋亡率的增加，这表明乙酰紫草素对结肠癌细胞具有凋亡潜力。2'，7'-二氯荧光素二乙酸酯（DCF-DA）染色用于评估乙酰紫草素处理的结肠癌细胞中活性氧（ROS）的产生。荧光激活细胞分选（FACS）分析表明，乙酰紫草素以剂量和时间依赖的方式诱导 HCT-15 和 LoVo 细胞中 ROS 水平和凋亡率的增加。相比之下，用 N-乙酰半胱氨酸（NAC）共同处理可减少 ROS 的产生和结肠癌细胞的增殖作用。Western blot 分析表明，乙酰紫草素处理诱导裂解的 PARP、H2AX、FOXO3、Bax、Bim、Bad、p21、p27 和活性形式的 caspase-3、caspase-7、caspase-9、caspase-6 和 caspase-8 蛋白水平增加，而无活性形式则减少。此外，无活性形式的 pAkt、Bcl-2、Bcl-xL、过氧化物还原酶和硫氧还蛋白 1 的表达减少。此外，细胞质和核部分蛋白的 Western blot 分析表明，乙酰紫草素处理诱导 FOXO3 的核转位，这可能是由于细胞内 ROS 水平增加导致的 DNA 损伤。这项研究代表了乙酰紫草素通过 FOXO3 向核内易位和 ROS 生成的增加对结肠癌细胞的凋亡作用。
