Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand; Cell-Based Drug and Health Product Development Research Unit, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand.
Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
Phytomedicine. 2021 May;85:153534. doi: 10.1016/j.phymed.2021.153534. Epub 2021 Mar 11.
Lung cancer is a leading fatal malignancy due to the high incidence of treatment failure. Dysfunction of the tumor suppressor p53 contributes to cancer initiation, progression, and therapeutic resistance. Targeting MDM2, a negative regulator of p53, has recently attracted interest in cancer drug research as it may restore tumor suppressive function.
The present study aimed to investigate the effect of 3,4-dihydroxy-5,4'-dimethoxybibenzyl (DS-1) on targeting MDM2 and restoring p53 function in lung cancer cells.
The efficacy of DS-1 alone or in combination with cisplatin in lung cancer cells was determined by MTT, nuclear staining, and annexin V/PI assay. The expression of apoptosis-related proteins was determined by western blot analysis. To evaluate the role of DS-1 on the stabilization and degradation of p53, cycloheximide chasing assay and immunoprecipitation were conducted, and the active form of p53 was investigated by immunofluorescent staining assay. To confirm and demonstrate the site interaction between DS-1 and the MDM2 protein, in silico computational analysis was performed.
DS-1 exhibited a cytotoxic effect and sensitized lung cancer cells to cisplatin-induced apoptosis. DS-1 caused a significant increase in the cellular level of p53 protein, while the active form of p53 (phosphorylation at Ser15) was unaltered. DS-1 treatment in combination with cisplatin could enhance activated p-p53 (Ser15) and p53 downstream signaling (Bax, Bcl-2, and Akt), leading to a higher level of apoptosis. Immunoprecipitation analysis revealed that DS-1 decreased the p53-ubiquitin complex, a prerequisite step in p53 proteasomal degradation. Molecular docking simulation further evidenced that DS-1 interacts with MDM2 within the p53-binding domain by carbon-hydrogen bond interaction at Lys27, π-alkyl interactions at Ile37 and Leu30, and van der Waals interactions at Ile75, Val51, Val69, Phe67, Met38, Tyr43, Gly34, and Phe31. Treatment by DS-1 and cisplatin in patient-derivated primary lung cancer cells showed consistent effects by increasing cisplatin sensitivity.
Our findings provide evidence that DS-1 is an MDM2 inhibitor and its underlying mechanism involves MDM2 binding and p53 induction, which may benefit the development of this compound for lung cancer treatment.
肺癌是一种主要的致命恶性肿瘤，其治疗失败率很高。肿瘤抑制因子 p53 的功能障碍导致癌症的发生、发展和治疗耐药性。作为癌症药物研究的热点，靶向负调控 p53 的 MDM2，可能恢复肿瘤抑制功能。
本研究旨在探讨 3,4-二羟基-5,4'-二甲氧基二苯并基（DS-1）对肺癌细胞中靶向 MDM2 和恢复 p53 功能的影响。
通过 MTT、核染色和 Annexin V/PI 检测，测定 DS-1 单独或与顺铂联合对肺癌细胞的疗效。通过 Western blot 分析测定凋亡相关蛋白的表达。为了评估 DS-1 对 p53 稳定和降解的作用，进行了环已酰亚胺追踪试验和免疫沉淀试验，并通过免疫荧光染色试验研究了 p53 的活性形式。为了确认和证明 DS-1 与 MDM2 蛋白之间的位点相互作用，进行了计算分析。
DS-1 表现出细胞毒性作用，并使肺癌细胞对顺铂诱导的细胞凋亡敏感。DS-1 显著增加了细胞内 p53 蛋白水平，而 p53 的活性形式（Ser15 磷酸化）不变。DS-1 联合顺铂治疗可增强激活的 p-p53（Ser15）和 p53 下游信号（Bax、Bcl-2 和 Akt），导致更高水平的凋亡。免疫沉淀分析显示，DS-1 降低了 p53-泛素复合物，这是 p53 蛋白酶体降解的必要步骤。分子对接模拟进一步证明，DS-1 通过碳氢键相互作用在 Lys27 处、π-烷基相互作用在 Ile37 和 Leu30 处以及范德华相互作用在 Ile75、Val51、Val69、Phe67、Met38、Tyr43、Gly34 和 Phe31 处与 MDM2 结合于 p53 结合域内相互作用。在源自患者的原发性肺癌细胞中，DS-1 和顺铂联合治疗通过增加顺铂敏感性表现出一致的效果。
我们的研究结果提供了证据表明，DS-1 是一种 MDM2 抑制剂，其作用机制涉及 MDM2 结合和 p53 诱导，这可能有利于该化合物在肺癌治疗中的开发。
