VE-822 通过靶向 DNA 损伤反应抑制毛细血管扩张共济失调症突变和 Rad3 相关蛋白（ATR），可有效逆转结直肠癌细胞对 5-氟尿嘧啶的耐药性。
Ataxia telangiectasia and Rad3-related (ATR) inhibition by VE-822 potently reversed 5-flourouracil resistance in colorectal cancer cells through targeting DNA damage response.
机构信息
Solid Tumor Research Center, Cellular and Molecular Medicine Institute, Urmia University of Medical Sciences, Urmia, Iran.
Immunology Department, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
出版信息
Mol Biol Rep. 2024 Mar 29;51(1):474. doi: 10.1007/s11033-024-09431-7.
BACKGROUND
VE-822 is a novel inhibitor of ATR, a key kinase involved in the DNA damage response pathway. The role of ATR inhibition in reversing drug resistance in various cancer types has been investigated. Therefore, this study investigated the effects of ATR inhibition by VE-822 on reversing 5-fluorouracil (5-FU) resistance in colorectal cancer cell line (Caco-2).
METHODS
Caco-2 and 5-FU resistance Caco-2 (Caco-2/5-FU) cells were treated with 5-FU and VE-822, alone and in combination. Cell proliferation and viability were assessed by MTT assay and Trypan Blue staining. P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) activities were measured by Rhodamine123 accumulation and uptake assay. The mRNA levels of P-gp, MRP-1, ataxia telangiectasia and Rad3-related (ATR) and checkpoint kinase 1 (CHK1) were measured by qRT-PCR. Western blot was used to measure the protein levels of P-gp, MRP-1, γ-H2AX, ATR and CHK1 in cells. 8-Oxo-2'-deoxyguanosine (8-oxo-dG) levels were determined via ELISA. Apoptosis was evaluated by ELISA death assay, DAPI staining and lactate dehydrogenase (LDH) assay.
RESULTS
The Caco-2/5-FU cells showed lower levels of 5-FU mediated proliferation inhibition in comparison to Caco-2 cells. VE-822 decreased the IC50 value of 5-FU on resistant cells. In addition, the expression levels and activity of P-gp and MRP-1 were significantly decreased in resistant cells treated with VE-822 (P < 0.05). The combination of 5-FU and VE-822 increased apoptosis in Caco-2/5-FU cells by downregulating CHK1 and ATR and upregulating γ-H2AX and 8-oxo-dG.
CONCLUSION
The simultaneous treatment of resistant colorectal cancer cells with 5-FU and ATR inhibitor, VE-822, was demonstrated to be effective in reversing drug resistance and potentiating 5-FU mediated anticancer effects via targeting DNA damage.
背景
VE-822 是一种新型的 ATR 抑制剂，ATR 是参与 DNA 损伤反应途径的关键激酶。ATR 抑制在逆转各种癌症类型的药物耐药性方面的作用已被研究。因此，本研究探讨了 ATR 抑制剂 VE-822 对逆转结直肠癌细胞系（Caco-2）中 5-氟尿嘧啶（5-FU）耐药性的影响。
方法
用 5-FU 和 VE-822 单独或联合处理 Caco-2 和 5-FU 耐药 Caco-2（Caco-2/5-FU）细胞。通过 MTT 检测和台盼蓝染色评估细胞增殖和活力。通过罗丹明 123 积累和摄取测定法测量 P-糖蛋白（P-gp）和多药耐药相关蛋白 1（MRP1）的活性。通过 qRT-PCR 测量 P-gp、MRP-1、共济失调毛细血管扩张症和 Rad3 相关（ATR）和检查点激酶 1（CHK1）的 mRNA 水平。通过 Western blot 测量细胞中 P-gp、MRP-1、γ-H2AX、ATR 和 CHK1 的蛋白水平。通过 ELISA 测定 8-氧-2'-脱氧鸟苷（8-oxo-dG）水平。通过 ELISA 死亡测定、DAPI 染色和乳酸脱氢酶（LDH）测定评估细胞凋亡。
结果
与 Caco-2 细胞相比，Caco-2/5-FU 细胞对 5-FU 介导的增殖抑制作用较低。VE-822 降低了耐药细胞中 5-FU 的 IC50 值。此外，VE-822 处理耐药细胞后 P-gp 和 MRP-1 的表达水平和活性显著降低（P<0.05）。5-FU 和 VE-822 的联合应用通过下调 CHK1 和 ATR 以及上调 γ-H2AX 和 8-oxo-dG 增加了 Caco-2/5-FU 细胞的凋亡。
结论
同时用 5-FU 和 ATR 抑制剂 VE-822 处理耐药结直肠癌细胞，通过靶向 DNA 损伤，被证明在逆转耐药性和增强 5-FU 介导的抗癌作用方面是有效的。
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