Department of Biochemistry, A.T. Still University, 800 West Jefferson Street, Kirksville, MO 63501, USA.
Department of Physiology Kirksville College of Osteopathic Medicine, A.T. Still University, 800 West Jefferson Street, Kirksville, MO 63501, USA.
Anticancer Agents Med Chem. 2023;23(16):1880-1887. doi: 10.2174/1871520623666230701000338.
Melanoma treatment is highly resistant to current chemotherapeutic agents. Due to its resistance towards apoptotic cell death, non-apoptotic cell death pathways are sought after.
We investigated a Chinese herbal medicine, shikonin, and its effect on B16F10 melanoma cells in vitro.
Cell growth of B16F10 melanoma cells treated with shikonin was analyzed using an MTT assay. Shikonin was combined with necrostatin, an inhibitor of necroptosis; caspase inhibitor; 3-methyladenine, an inhibitor of autophagy; or N-acetyl cysteine, an inhibitor of reactive oxygen species. Flow cytometry was used to assess types of cell death resulting from treatment with shikonin. Cell proliferation was also analyzed utilizing a BrdU labeling assay. Monodansylcadaverine staining was performed on live cells to gauge levels of autophagy. Western blot analysis was conducted to identify specific protein markers of necroptosis including CHOP, RIP1, and pRIP1. MitoTracker staining was utilized to identify differences in mitochondrial density in cells treated with shikonin.
Analysis of MTT assays revealed a large decrease in cellular growth with increasing shikonin concentrations. The MTT assays with necrostatin, 3-methyladenine, and N-acetyl cysteine involvement, suggested that necroptosis, autophagy, and reactive oxygen species are a part of shikonin's mechanism of action. Cellular proliferation with shikonin treatment was also decreased. Western blotting confirmed that shikonin-treated melanoma cells increase levels of stress-related proteins, e.g., CHOP, RIP, pRIP.
Our findings suggest that mainly necroptosis is induced by the shikonin treatment of B16F10 melanoma cells. Induction of ROS production and autophagy are also involved.
黑色素瘤治疗对目前的化疗药物具有高度抗性。由于其对细胞凋亡的抗性，因此寻求非细胞凋亡的细胞死亡途径。
我们研究了一种中药紫草素及其对体外 B16F10 黑色素瘤细胞的作用。
用 MTT 法分析紫草素处理的 B16F10 黑色素瘤细胞的细胞生长。将紫草素与坏死抑制剂 necrostatin、半胱天冬酶抑制剂、自噬抑制剂 3-甲基腺嘌呤或活性氧抑制剂 N-乙酰半胱氨酸联合使用。用流式细胞术评估紫草素处理后导致的细胞死亡类型。还利用 BrdU 标记测定法分析细胞增殖。用单丹磺酰尸胺染色法对活细胞进行染色，以评估自噬水平。通过 Western blot 分析鉴定坏死包括 CHOP、RIP1 和 pRIP1 的特异性蛋白标记物。用 MitoTracker 染色鉴定用紫草素处理的细胞中线粒体密度的差异。
MTT 分析表明，随着紫草素浓度的增加，细胞生长大量减少。涉及 necrostatin、3-甲基腺嘌呤和 N-乙酰半胱氨酸的 MTT 分析表明，坏死、自噬和活性氧是紫草素作用机制的一部分。紫草素处理后的细胞增殖也减少了。Western blot 证实，紫草素处理的黑色素瘤细胞增加应激相关蛋白（如 CHOP、RIP、pRIP）的水平。
我们的研究结果表明，紫草素处理 B16F10 黑色素瘤细胞主要诱导坏死。还涉及 ROS 产生和自噬的诱导。
