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.
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。