Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Malaya, 50603 Kuala Lumpur, Malaysia.
Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.
Anticancer Agents Med Chem. 2020;20(9):1072-1086. doi: 10.2174/1871520620666200318100051.
Oxadiazoles, triazoles, and their respective precursors have been shown to exhibit various pharmacological properties, namely antitumour activities. Cytotoxic activity was reported for these compounds in various cancer cell lines.
In this study, we aim at investigating the mechanism of apoptosis by N-(4-chlorophenyl)-2-(4- (3,4,5-trimethoxybenzyloxy)benzoyl)-hydrazinecarbothioamide, a triazole precursor, henceforth termed compound P7a, in breast cancer cell line, MCF-7. We first screen a series of analogues containing (3,4,5-trimethoxybenzyloxy) phenyl moiety in breast cancer cell lines (MCF-7 and MDA-MB-231) to select the most cytotoxic compound and demonstrate a dose- and time-dependent cytotoxicity. Then, we unravel the mechanism of apoptosis of P7a in MCF-7 as well as its ability to cause cell cycle arrest.
Synthesis was performed as previously described by Kareem and co-workers. Cytotoxicity of analogues containing (3,4,5-trimethoxybenzyloxy)phenyl moiety against MCF-7 and MDA-MB-231 cell lines was evaluated using the MTS assay. Flow cytometric analyses was done using Annexin V/PI staining, JC-1 staining and ROS assay. The activity of caspases using a chemoluminescence assay and western blot analysis was conducted to study the apoptotic pathway induced by the compound in MCF-7 cells. Lastly, cell cycle analysis was conducted using flow cytometry.
Upon 48 hours of treatment, compound P7a inhibited the proliferation of human breast cancer cells with IC50 values of 178.92 ± 12.51μM and 33.75 ± 1.20μM for MDA-MB-231 and MCF-7, respectively. Additionally, compound P7a showed selectivity towards the cancer cell line, MCF-7 compared to the normal breast cell line, hTERT-HME1, an advantage against current anticancer drugs (tamoxifen and vinblastine). Flow cytometric analyses using different assays indicated that compound P7a significantly increased the proportion of apoptotic cells, increased mitochondria membrane permeabilisation and caused generation of ROS in MCF-7. In addition, cell cycle analysis showed that cell proliferation was arrested at the G1 phase in the MCF-7 cell line. Furthermore, upon treatment, the MCF-7 cell line showed increased activity of caspase-3/7, and caspase-9. Lastly, the western blot analysis showed the up-regulation of pro-apoptotic proteins along with up-regulation of caspase-7 and caspase-9, indicating that an intrinsic pathway of apoptosis was induced.
The results suggest that compound P7a could be a potential chemotherapeutic agent for breast cancer.
噁二唑、三唑及其各自的前体已被证明具有多种药理特性，即抗肿瘤活性。这些化合物在各种癌细胞系中表现出细胞毒性活性。
在这项研究中，我们旨在通过 N-(4-氯苯基)-2-(4-(3,4,5-三甲氧基苄氧基)苯甲酰基)肼甲硫酰胺，一种三唑前体，研究其在乳腺癌细胞系 MCF-7 中的细胞凋亡机制，简称化合物 P7a。我们首先在乳腺癌细胞系（MCF-7 和 MDA-MB-231）中筛选一系列含有（3,4,5-三甲氧基苄氧基）苯甲酰基的类似物，以选择最具细胞毒性的化合物，并证明其具有剂量和时间依赖性的细胞毒性。然后，我们揭示了 P7a 在 MCF-7 中诱导细胞凋亡的机制及其引起细胞周期停滞的能力。
合成如 Kareem 等人所述进行。使用 MTS 测定法评估含有（3,4,5-三甲氧基苄氧基）苯基部分的类似物对 MCF-7 和 MDA-MB-231 细胞系的细胞毒性。使用 Annexin V/PI 染色、JC-1 染色和 ROS 测定进行流式细胞术分析。使用化学发光测定法和 Western blot 分析研究化合物在 MCF-7 细胞中诱导的凋亡途径中的半胱天冬酶活性。最后，使用流式细胞术进行细胞周期分析。
经过 48 小时的治疗，化合物 P7a 抑制了人乳腺癌细胞的增殖，其 MDA-MB-231 和 MCF-7 的 IC50 值分别为 178.92±12.51μM 和 33.75±1.20μM。此外，与当前的抗癌药物（他莫昔芬和长春碱）相比，化合物 P7a 对乳腺癌细胞系 MCF-7 具有选择性，而对正常乳腺细胞系 hTERT-HME1 则没有选择性。使用不同测定法的流式细胞术分析表明，化合物 P7a 显著增加了凋亡细胞的比例，增加了线粒体膜通透性并在 MCF-7 中引起了 ROS 的产生。此外，细胞周期分析表明，细胞增殖在 MCF-7 细胞系中被阻滞在 G1 期。此外，经处理后，MCF-7 细胞系中 caspase-3/7 和 caspase-9 的活性增加。最后，Western blot 分析显示促凋亡蛋白上调，同时 caspase-7 和 caspase-9 上调，表明诱导了内在的细胞凋亡途径。
结果表明，化合物 P7a 可能是一种有潜力的乳腺癌化疗药物。
