Dihydromyricetin reverses MRP2-induced multidrug resistance by preventing NF-κB-Nrf2 signaling in colorectal cancer cell
Dihydromyricetin reverses MRP2-induced multidrug resistance by preventing NF-κB-Nrf2 signaling in colorectal cancer cell
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二氢杨梅素通过阻止结直肠癌细胞中的 NF-κB-Nrf2 信号传导逆转 MRP2 诱导的多药耐药性
DOI：
10.1016/j.phymed.2020.153414
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发表时间：
2021-01-16
期刊：
影响因子：
7.9
通讯作者：
Li, Qi
中科院分区：
文献类型：
--
作者：
Wang, Ziyuan;Sun, Xiaoting;Li, Qi
背景：二氢杨梅素（DMY）是一种来自中草药葡萄属黑葡萄（Vitis heyneana）叶的天然黄酮类化合物，已被证明具有通过抑制结直肠癌（CRC）细胞中的Nrf 2/MRP 2信号传导来对抗化学抗性的潜力。目的：本研究旨在探讨DMY对多药耐药（multidrug resistance，MDR）的影响，并阐明其作用机制。用DMY（50 μ M）或MK-571（50 μ M）加奥沙利铂（OXA）（10 μ M）或长春新碱（VCR）（10 μ M）处理细胞48小时。在体内，我们使用BALB/c小鼠作为CRC异种移植小鼠模型。BALB/c小鼠腹腔注射DMY（100 mg/kg）、OXA（5 mg/kg）及DMY（100 mg/kg）+OXA（5 mg/kg），每周2次，共4周。流式细胞仪检测细胞凋亡。免疫细胞化学法检测Nrf 2和NF-κ B B/p65的表达。采用Western blot、qRT-PCR和报告基因分析来测量蛋白和基因转录水平（MRP 2、Nrf 2、NF-.B/p65）。此外，使用染色质免疫沉淀（ChIP）分析来研究NF-.B/p65的内源性启动子占用。结果：DMY通过抑制HCT 116/OXA和HCT 8/VCR细胞株MRP 2的表达及其启动子活性，恢复了OXA和VCR的化疗敏感性。DMY还能抑制NF-κ B/p65的表达，减少NF-κ B/p65向细胞核的转位，从而抑制MRP 2表达所必需的Nrf 2信号通路。过表达NF-κ B/p65降低DMY的逆转作用。此外，NF-κ B/p65通过直接结合其特异性启动子区域并激活其转录来调节Nrf 2的表达。结论：本研究为DMY提高结直肠癌化疗敏感性提供了一种新的机制。DMY、NF-κ B B或Nrf 2的下游信号也可能成为治疗CRC的潜在靶点。
Backgroud: Dihydromyricetin (DMY), a natural flavonoid compound from the leaves of the Chinese medicinal herb Vitis heyneana, has been shown to have the potential to combat chemoresistance by inhibiting Nrf2/MRP2 signaling in colorectal cancer (CRC) cells. However, the precise underlying molecular mechanism and its therapeutic target are not well understood.Purpose: Our study aims to investigate the effects of DMY on multidrug resistance (MDR), and elucidate the underlying mechanisms.Study design: In vitro, HCT116/OXA and HCT8/VCR cells were employed as our MDR models. The cells were treated with DMY (50 mu M) or MK-571 (50 mu M) plus oxaliplatin (OXA) (10 mu M) or vincristine (VCR) (10 mu M) for 48 h. In vivo, we used BALB/c mice as a CRC xenograft mouse model. BALB/c mice were given DMY (100 mg/kg), OXA (5 mg/kg) and DMY (100 mg/kg) combined with OXA (5 mg/kg) via intraperitoneal route every 2 days per week for 4 weeks.Methods: We used MTT and colony forming assays to detect DMY's ability to reverse MDR. Flow cytometric analysis was used to detect apoptosis. Immunocytochemistry was used to detect the localization of Nrf2 and NF-kappa B/p65. Western blot, qRT-PCR and reporter gene assays were employed to measure the protein and gene transcriptional levels (MRP2, Nrf2, NF-.B/p65). Moreover, chromatin immunoprecipitation (ChIP) assay was used to investigate the endogenous promoter occupancy of NF-.B/p65. Finally, immunohistochemistry and TUNEL staining were used to detect protein expression and apoptosis in vivo.Results: DMY restored chemosensitivity (OXA and VCR) by inhibiting both MRP2 expression and its promoter activity in HCT116/OXA and HCT8/VCR cell lines. Furthermore, DMY could inhibit NF-kappa B/p65 expression, reducing NF-kappa B/p65 translocation to the nucleus to silence Nrf2 signaling, which is necessary for MRP2 expression. Overexpressing NF-kappa B/p65 expression reduced the reversal effect of DMY. In addition, NF-kappa B/p65 regulated Nrf2 expression by directly binding to its specific promoter region and activating its transcription. Finally, we proved that the combination of OXA and DMY has a synergistic tumor suppression effect in vivo.Conclusion: Our study provided a novel mechanism of DMY boosted chemosensitivity in human CRC. The downstream signals of DMY, NF-kappa B or Nrf2 could also be potential targets for the treatment of CRC.