宫颈癌是全球女性癌症相关死亡的第四大原因。微生物产品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分泌型天冬氨酰蛋白酶蛋白，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-菌丝转变阶段热带假丝酵母中 SAP2 和 SAP4 基因的表达证实了分泌型天冬氨酰蛋白酶蛋白的存在。分别使用水性两相系统纯化方法以及蛋白水解活性以及 Bradford 和微量凯氏定氮法对该酶进行纯化和表征。通过MTT测定、显微图像分析、一氧化氮（NO）清除活性测定、细胞内活性氧（ROS）产生测定和RT-qPCR评估分泌型天冬氨酰蛋白酶蛋白的体外抗癌特性。根据 Bradford 法和微量凯氏定氮法，分离的热带念珠菌分泌的天冬氨酰蛋白酶蛋白的蛋白酶活性值分别为 93.72 至 130.70 μg/mL 和 89.88 至 127.72 μg/mL。分泌的天冬氨酰蛋白酶在 HeLa 细胞系中表现出有效的细胞毒性，导致显着的形态变化。此外，与未处理的对照组相比，它表现出更高的自由基清除活性，亚硝酸盐抑制证明了这一点。接触分泌型天冬氨酰蛋白酶的 HeLa 细胞中 ROS 产量增加。在分泌型天冬氨酰处理的 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-box 2 (SOX2) 和 Nanog 同源框 (NANOG) 的表达水平显着下调蛋白酶。分泌型天冬氨酰蛋白酶蛋白可能是一种有前途的抗癌剂，因为它能有效影响基因表达，并最终可能减少宫颈癌的发生和进展。靶向与核干细胞相关转录因子相关的基因可能会提供一种新的癌症治疗方法。版权所有 © 2024。由 Elsevier Ltd 出版。

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 to 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.Copyright © 2024. Published by Elsevier Ltd.