Department of Beijing National Biochip Research Center Sub-Center in Ningxia, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, China.
Institute of Medical Sciences, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, China.
Mol Biol Rep. 2023 Aug;50(8):6543-6556. doi: 10.1007/s11033-023-08572-5. Epub 2023 Jun 20.
Colorectal cancer (CRC) is the most common primary malignancy. Recently, antineoplastic attributes of homoharringtonine (HHT) have attracted lots of attention. This study investigated the molecular target and underlying mechanism of HHT in the CRC process by using a cellular and animal models.
This study first detected the effects of HHT on the proliferation, cell cycle and apoptosis ability of CRC cells using CCK-8, Edu staining, flow cytometry and Western blotting assay. In vitro recovery experiment and in vivo tumorigenesis experiment were used to detect the targeted interaction between HHT and NKD1. After that, the downstream target and mechanism of action of HHT targeting NKD1 was determined using quantitative proteomics combined with co-immunoprecipitation/immunofluorescence assay.
HHT suppressed CRC cells proliferation by inducing cell cycle arrest and apoptosis in vitro and vivo. HHT inhibited NKD1 expression in a concentration and time dependent manner. NKD1 was overexpressed in CRC and its depletion enhanced the therapeutic sensitivity of HHT on CRC, which indicating that NKD1 plays an important role in the development of CRC as the drug delivery target of HHT. Furthermore, proteomic analysis revealed that PCM1 participated the process of NKD1-regulated cell proliferation and cell cycle. NKD1 interacted with PCM1 and promoted PCM1 degradation through the ubiquitin-proteasome pathway. The overexpression of PCM1 effectively reversed the inhibition of siNKD1 on cell cycle.
The present findings revealed that HHT blocked NKD1 expression to participate in inhibiting cell proliferation and inducing cell apoptosis, ultimately leading to obstruction of CRC development through NKD1/PCM1 dependent mechanism. Our research provide evidence for clinical application of NKD1-targeted therapy in improving HHT sensitivity for CRC treatment.
结直肠癌（CRC）是最常见的原发性恶性肿瘤。最近，海兔毒素（HHT）的抗肿瘤特性引起了广泛关注。本研究通过细胞和动物模型，研究了 HHT 在 CRC 过程中的分子靶标和作用机制。
本研究首先通过 CCK-8、Edu 染色、流式细胞术和 Western blot 检测 HHT 对 CRC 细胞增殖、细胞周期和凋亡能力的影响。体外恢复实验和体内肿瘤发生实验用于检测 HHT 与 NKD1 之间的靶向相互作用。之后，通过定量蛋白质组学结合共免疫沉淀/免疫荧光检测，确定 HHT 靶向 NKD1 的下游靶标和作用机制。
HHT 在体外和体内通过诱导细胞周期停滞和凋亡抑制 CRC 细胞增殖。HHT 呈浓度和时间依赖性抑制 NKD1 的表达。NKD1 在 CRC 中过表达，其缺失增强了 HHT 对 CRC 的治疗敏感性，表明 NKD1 作为 HHT 的药物递送靶点在 CRC 的发生发展中起重要作用。此外，蛋白质组学分析显示 PCM1 参与了 NKD1 调节的细胞增殖和细胞周期过程。NKD1 与 PCM1 相互作用，并通过泛素-蛋白酶体途径促进 PCM1 降解。PCM1 的过表达有效逆转了 siNKD1 对细胞周期的抑制作用。
本研究揭示了 HHT 通过阻断 NKD1 的表达参与抑制细胞增殖和诱导细胞凋亡，最终通过 NKD1/PCM1 依赖性机制阻止 CRC 的发展。我们的研究为 NKD1 靶向治疗在提高 HHT 敏感性以治疗 CRC 方面的临床应用提供了证据。
