Inthi Pitsinee, Pandith Hataichanok, Kongtawelert Prachya, Subhawa Subhawat, Banjerdpongchai Ratana
Department of Biochemistry, Chiang Mai University, 110 Inthawaroros Road., Sripoom, Muang, Chiang Mai, 50200, Thailand.
Department of Biology, Chiang Mai University, 239 Huaykaew Road, Suthep, Muang, Chiang Mai, 50200, Thailand.
Heliyon. 2023 Jul 28;9(8):e18755. doi: 10.1016/j.heliyon.2023.e18755. eCollection 2023 Aug.
Thunb. (HCT) is a perennial plant used in traditional Thai medicine for many centuries. This study aimed to investigate the antiproliferative effect of the hexane fraction, which has not been explored before. HCT ethanol extract (crude extract) was sequentially fractionated to obtain a hexane (H) fraction. GC-MS was used to determine the phytochemicals. The H fraction consisted of lipids, mainly α-linolenic acid and some terpenoids. MTT assay was used to determine the cytotoxic effects of H fraction in MCF-7, MDA-MB-231, NIH3T3 and PBMCs. The mode of cell death and cell cycle analysis were determined by flow cytometry. The mechanisms of cell death were defined by mitochondrial transmembrane potential (MTP) reduction and activation of caspase-3, -8 and -9. The expression levels of the Bcl-2 family, cell cycle-related, endoplasmic reticulum (ER) stress-associated proteins; and Akt/ERK signaling molecules were investigated by immunoblotting. The H fraction was toxic to MDA-MB-231 more than MCF-7 cells but not to NIH3T3 and PBMCs. The growth of MDA-MB-231 cells was inhibited through apoptosis. MTP was disrupted whereas caspase-3, -8 and -9 were activated. The expression of pro-apoptotic Bax and Bak was upregulated, while Bid and anti-apoptotic Bcl-xL proteins were downregulated. Cyclin D1 and CDK4 levels were downregulated. The cell cycle was arrested at G1. Moreover, GRP78 and CHOP elevation indicated ER stress-mediated pathway. The expression ratio of pAkt/Akt and pERK/ERK were reduced. Taken together, the molecular mechanisms of MDA-MB-231 cell apoptosis were via intrinsic/extrinsic pathways, cell cycle arrest, ER stress and abrogation of Akt/ERK survival pathways. According to the most current research, the H fraction may be used as an adjuvant in the BC treatment; however, before the anticancer strategy can be applied to patients, it is important to determine each active compound's effects in cell lines and when compared with a combined mixture.
刺蒺藜（HCT）是一种多年生植物，在传统泰国医学中已使用了数百年。本研究旨在探究此前未被研究过的己烷馏分的抗增殖作用。对HCT乙醇提取物（粗提取物）进行顺序分级分离以获得己烷（H）馏分。采用气相色谱 - 质谱联用（GC-MS）法测定植物化学物质。H馏分由脂质组成，主要是α-亚麻酸和一些萜类化合物。采用MTT法测定H馏分对MCF-7、MDA-MB-231、NIH3T3和外周血单个核细胞（PBMCs）的细胞毒性作用。通过流式细胞术确定细胞死亡模式和细胞周期分析。通过线粒体跨膜电位（MTP）降低以及半胱天冬酶-3、-8和-9的激活来确定细胞死亡机制。通过免疫印迹法研究Bcl-2家族、细胞周期相关、内质网（ER）应激相关蛋白的表达水平；以及Akt/ERK信号分子。H馏分对MDA-MB-231细胞的毒性比对MCF-7细胞大，但对NIH3T3和PBMCs无毒性。MDA-MB-231细胞的生长通过凋亡受到抑制。MTP被破坏，而半胱天冬酶-3、-8和-9被激活。促凋亡蛋白Bax和Bak的表达上调，而Bid和抗凋亡蛋白Bcl-xL下调。细胞周期蛋白D1和细胞周期蛋白依赖性激酶4（CDK4）水平下调。细胞周期停滞在G1期。此外，葡萄糖调节蛋白78（GRP78）和C/EBP同源蛋白（CHOP）升高表明内质网应激介导的途径。pAkt/Akt和pERK/ERK的表达比值降低。综上所述，MDA-MB-231细胞凋亡的分子机制是通过内源性/外源性途径、细胞周期停滞、内质网应激以及Akt/ERK存活途径的废除。根据最新研究，H馏分可作为乳腺癌（BC）治疗的辅助药物；然而，在将抗癌策略应用于患者之前，确定每种活性化合物在细胞系中的作用以及与混合混合物相比时的作用非常重要。
