Abstract：
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.
CONCLUSIONS: 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.
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.
CONCLUSIONS: 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.
摘要：
背景：VE-822是一种新型的ATR抑制剂，参与DNA损伤反应途径的关键激酶。已经研究了ATR抑制在逆转各种癌症类型的耐药性中的作用。因此,这项研究调查了VE-822抑制ATR对逆转大肠癌细胞系（Caco-2）中5-氟尿嘧啶（5-FU）耐药性的影响。
方法:用5-FU和VE-822单独和组合处理Caco-2和5-FU抗性Caco-2(Caco-2/5-FU)细胞。通过MTT测定和台盼蓝染色评估细胞增殖和活力。P-糖蛋白（P-gp）和多药耐药相关蛋白1（MRP1）活性通过Rhodamine123积累和摄取测定来测量。P-gp的mRNA水平,通过qRT-PCR测量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）。蛋白质印迹用于测量P-gp的蛋白质水平，MRP-1,γ-H2AX,细胞中的ATR和CHK1。通过ELISA测定8-氧代-2'-脱氧鸟苷(8-氧代-dG)水平。通过ELISA死亡测定法评估细胞凋亡，DAPI染色和乳酸脱氢酶(LDH)测定。
结果:与Caco-2细胞相比,Caco-2/5-FU细胞显示出更低水平的5-FU介导的增殖抑制。VE-822降低了5-FU对抗性细胞的IC50值。此外,用VE-822处理的耐药细胞中P-gp和MRP-1的表达水平和活性显着降低（P<0.05）。5-FU和VE-822的组合通过下调CHK1和ATR并上调γ-H2AX和8-氧代-dG来增加Caco-2/5-FU细胞的凋亡。
结论：同时使用5-FU和ATR抑制剂治疗耐药的结直肠癌细胞，VE-822被证明可有效逆转耐药性并通过靶向DNA损伤增强5-FU介导的抗癌作用。
方法:用5-FU和VE-822单独和组合处理Caco-2和5-FU抗性Caco-2(Caco-2/5-FU)细胞。通过MTT测定和台盼蓝染色评估细胞增殖和活力。P-糖蛋白（P-gp）和多药耐药相关蛋白1（MRP1）活性通过Rhodamine123积累和摄取测定来测量。P-gp的mRNA水平,通过qRT-PCR测量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）。蛋白质印迹用于测量P-gp的蛋白质水平，MRP-1,γ-H2AX,细胞中的ATR和CHK1。通过ELISA测定8-氧代-2'-脱氧鸟苷(8-氧代-dG)水平。通过ELISA死亡测定法评估细胞凋亡，DAPI染色和乳酸脱氢酶(LDH)测定。
结果:与Caco-2细胞相比,Caco-2/5-FU细胞显示出更低水平的5-FU介导的增殖抑制。VE-822降低了5-FU对抗性细胞的IC50值。此外,用VE-822处理的耐药细胞中P-gp和MRP-1的表达水平和活性显着降低（P<0.05）。5-FU和VE-822的组合通过下调CHK1和ATR并上调γ-H2AX和8-氧代-dG来增加Caco-2/5-FU细胞的凋亡。
结论：同时使用5-FU和ATR抑制剂治疗耐药的结直肠癌细胞，VE-822被证明可有效逆转耐药性并通过靶向DNA损伤增强5-FU介导的抗癌作用。
