Shikonin induces apoptosis and autophagy via downregulation of pyrroline-5-carboxylate reductase1 in hepatocellular carcinoma cells

Shikonin(SK) is a natural small molecule naphthoquinone compound, which has anti-cancer activity in various human malignant tumors. Pyrroline-5-carboxylate reductase 1(PYCR1) is involved in tumorigenesis and regulates various cellular processes, including growth, invasion, migration, and apoptosis. However, the effect of SK and PYCR1 on apoptosis and autophagy in hepatocellular carcinoma are unclear. Our goal is to determine the internal molecular mechanism of the interaction between SK and PYCR1 and its role in the occurrence and development of liver cancer. The CCK8 assay, wound healing assay, and transwell assays show that SK and siPYCR1(gene silence PYCR1) inhibited the malignant phenotype of HCC cells, including cell viability, colony formation, migration, and invasion, respectively. The flow cytometry assays and immunofluorescence show that SK and siPYCR1 activated apoptosis and autophagy, respectively. SK induces apoptosis and autophagy in a dose-dependent manner. In addition, HCC cells were transfected with small interference fragment PYCR1 siRNA to construct siPYCR1 and SK single treatment group and co-treatment group to verify the interaction between SK and PYCR1. The Western blot identified that PI3K/Akt/mTOR signal pathway protein expression was significantly downregulated in HCC cells treated with SK and siPYCR1 together. Collectively, SK may induce apoptosis and autophagy by reducing the expression of PYCR1 and suppressing PI3K/Akt/mTOR. Thus, SK may be a promising antineoplastic drug in Hepatocellular carcinoma (HCC). SK downregulating PYCR1 might supply a theoretical foundation for the potential therapeutic application in hepatocellular carcinoma.

紫草素（Shikonin，SK）是一种天然小分子萘醌类化合物，在多种人类恶性肿瘤中展现出抗癌活性。吡咯啉-5-羧酸还原酶1（Pyrroline-5-carboxylate reductase 1，PYCR1）参与肿瘤发生过程，并调控包括细胞增殖、侵袭、迁移及凋亡在内的多种细胞生物学过程。目前尚不明确紫草素与PYCR1在肝细胞癌（hepatocellular carcinoma，HCC）中对细胞凋亡与自噬的调控作用。本研究旨在阐明紫草素与PYCR1相互作用的内在分子机制，及其在肝癌发生发展中的作用。
CCK8检测法、划痕愈合实验与Transwell（transwell）实验结果显示，紫草素与PYCR1基因沉默小干扰RNA（siPYCR1）可分别抑制肝癌细胞的恶性表型，具体包括降低细胞活力、削弱集落形成、迁移及侵袭能力。流式细胞术检测与免疫荧光实验结果表明，紫草素与siPYCR1可分别激活细胞凋亡与自噬；且紫草素诱导细胞凋亡与自噬的作用呈剂量依赖性。
此外，本研究通过向肝癌细胞中转染PYCR1小干扰RNA片段，构建了siPYCR1单处理组、紫草素单处理组与联合处理组，以验证紫草素与PYCR1之间的相互作用。蛋白质印迹法（Western blot）检测结果显示，联合使用紫草素与siPYCR1处理肝癌细胞后，其PI3K/Akt/mTOR信号通路相关蛋白的表达量显著下调。
综上，紫草素可通过下调PYCR1的表达并抑制PI3K/Akt/mTOR信号通路，进而诱导肝癌细胞发生凋亡与自噬。因此，紫草素有望成为肝细胞癌领域极具潜力的抗肿瘤药物；而紫草素下调PYCR1的作用机制，可为其在肝细胞癌中的潜在临床治疗应用提供理论依据。