Medicinal Plants Research Center, Yasuj University of Medical Sciences, Yasuj, Iran.
Department of Animal Science, Shiraz University, Shiraz, Iran.
Toxicon. 2024 Oct;249:108073. doi: 10.1016/j.toxicon.2024.108073. Epub 2024 Aug 15.
Cervical cancer is the fourth leading cause of cancer-related death in women worldwide. Microbial products are valuable sources of anti-cancer drugs. The aim of this study was to isolate secreted aspartyl proteinase protein from Candida tropicalis, investigate its inhibitory effect on human cervical cancer HeLa cells, and analyze the expression profiling of selected nuclear stem cell-associated transcription factors. The presence of secreted aspartyl proteinase protein was confirmed by the expression of SAP2 and SAP4 genes in C. tropicalis during the yeast-hyphae transition phase. The enzyme was purified and characterized using the aqueous two-phase system purification method, as well as proteolytic activity and the Bradford and micro-Kjeldahl methods, respectively. The in vitro anti-cancer properties of secreted aspartyl proteinase protein were evaluated by MTT assay, microscopic image analysis, nitric oxide (NO) scavenging activity assay, intracellular reactive oxygen species (ROS) production assay, and RT-qPCR. The isolated C. tropicalis secreted aspartyl proteinase protein exhibited proteinase activity with values ranging from 93.72 to 130.70 μg/mL and 89.88-127.72 μg/mL according to the Bradford and micro-Kjeldahl methods, respectively. Secreted aspartyl proteinase showed effective cytotoxicity in HeLa cell line leading to significant morphological changes. Additionally, it exhibited increased free radical scavenging activity compared to the untreated control group, as evidenced by nitrite inhibition. ROS production increased in HeLa cells exposed to secreted aspartyl proteinase. The expression levels of the nuclear stem cell-associated transcription factors octamer-binding transcription factor 4 (OCT4), sex determining region Y-box 2 (SOX2), and Nanog homeobox (NANOG) were significantly downregulated in the HeLa cells treated with secreted aspartyl proteinase. Secreted aspartyl proteinase protein may be a promising anti-cancer agent, as it effectively affects gene expression and may ultimately reduce the development and progression of cervical cancer. Targeting the genes related to nuclear stem cell-associated transcription factors may provide a novel amenable to cancer treatment.
宫颈癌是全球女性癌症相关死亡的第四大原因。微生物产物是抗癌药物的宝贵来源。本研究的目的是从热带假丝酵母中分离出分泌的天冬氨酸蛋白酶蛋白，研究其对人宫颈癌 HeLa 细胞的抑制作用，并分析选定的核干细胞相关转录因子的表达谱。在酵母-菌丝过渡阶段，通过 C. tropicalis 中 SAP2 和 SAP4 基因的表达来确认分泌的天冬氨酸蛋白酶蛋白的存在。使用双水相系统纯化法、蛋白酶活性以及 Bradford 和微量凯氏定氮法分别对酶进行了纯化和特性分析。通过 MTT 测定法、显微镜图像分析、一氧化氮 (NO) 清除活性测定法、细胞内活性氧 (ROS) 产生测定法和 RT-qPCR 评估了分泌的天冬氨酸蛋白酶蛋白的体外抗癌特性。分离的 C. tropicalis 分泌的天冬氨酸蛋白酶蛋白表现出蛋白酶活性，Bradford 和微量凯氏定氮法分别测定值范围为 93.72-130.70μg/mL 和 89.88-127.72μg/mL。分泌的天冬氨酸蛋白酶蛋白对 HeLa 细胞系表现出有效的细胞毒性，导致明显的形态变化。此外，与未经处理的对照组相比，它表现出更高的自由基清除活性，这表现为亚硝酸盐抑制。暴露于分泌的天冬氨酸蛋白酶的 HeLa 细胞中 ROS 产生增加。用分泌的天冬氨酸蛋白酶处理的 HeLa 细胞中，核干细胞相关转录因子八聚体结合转录因子 4（OCT4）、性别决定区 Y 框 2（SOX2）和 Nanog 同源框（NANOG）的表达水平显著下调。分泌的天冬氨酸蛋白酶蛋白可能是一种有前途的抗癌药物，因为它能有效影响基因表达，并可能最终减少宫颈癌的发展和进展。针对与核干细胞相关转录因子相关的基因可能为癌症治疗提供新的方法。
