Mihanfar Ainaz, Asghari Faezeh, Majidinia Maryam
Solid Tumor Research Center, Cellular and Molecular Medicine Research Institute, Urmia University of Medical Sciences, Urmia, Iran.
Immunology Department, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Drug Res (Stuttg). 2025 Feb;75(2):66-75. doi: 10.1055/a-2499-3067. Epub 2025 Jan 13.
WEE1 is a key tyrosine kinase involved in the cell cycle regulation with potent anticancer effects in various cancer types including colorectal cancer. Recent studies have focused on the potential of combinational inhibition of Ataxia Telangiectasia and Rad-3-related protein (ATR) and WEE1 in increasing apoptosis in cancer cells. Therefore, this study investigates the effects of inhibiting WEE1, by employing AZD1775, on colorectal cancer cells' susceptibility to VE-822-induced DNA damage and apoptosis.SW-480 and HT-29 cells were treated with AZD1775 and VE-822, alone and in combination. MTT assay was used to assess cell proliferation and viability. The mRNA levels of ATR, checkpoint kinase 1 (CHK1), WEE1, ribonucleotide reductase (RR) catalytic subunit M1 (RRM1) and RRM2 were measured by qRT-PCR. Cellular γ-(H2A histone family member X) H2AX levels were measured by Western blot. Analyses were conducted using ELISA to assess 8-Oxo-2'-deoxyguanosine (8-oxo-dG) levels. Lactate dehydrogenase (LDH) and ELISA death assays were used to assess apoptosis.The SW-480 and HT-29 cells have low proliferation rate when treated with VE-822 and AZD1775. The IC50 value for VE-822 was 1.3 μM and 1.6 μM in SW480 and HT-29, respectively. Also, this value for AZD1775 in SW480 was 140 nM and in HT-29 was 185 nM. The expression levels of ATR, CHK1, WEE1, RRM1, and RRM2 were significantly downregulated in both cell lines treated with combination of VE-822 and AZD1775 (P<0.05). DNA damage markers, including γ-H2AX and 8-oxo-dG were upregulated in these cells. Simultaneous treatment with VE-822 and AZD177 increased apoptosis capacity of both cell lines.The inhibition of WEE1 via AZD1775 potentiated the anticancer effects of ATR inhibitor, VE-822, in combating colorectal cancer via targeting DNA damage.
WEE1是一种关键的酪氨酸激酶，参与细胞周期调控，对包括结直肠癌在内的多种癌症类型具有强大的抗癌作用。最近的研究集中在联合抑制共济失调毛细血管扩张症和Rad-3相关蛋白（ATR）与WEE1以增加癌细胞凋亡的潜力。因此，本研究通过使用AZD1775来研究抑制WEE1对结直肠癌细胞对VE-822诱导的DNA损伤和凋亡敏感性的影响。
将SW-480和HT-29细胞单独及联合用AZD1775和VE-822处理。采用MTT法评估细胞增殖和活力。通过qRT-PCR检测ATR、检查点激酶1（CHK1）、WEE1、核糖核苷酸还原酶（RR）催化亚基M1（RRM1）和RRM2的mRNA水平。通过蛋白质免疫印迹法检测细胞γ-（H2A组蛋白家族成员X）H2AX水平。使用ELISA进行分析以评估8-氧代-2'-脱氧鸟苷（8-氧代-dG）水平。采用乳酸脱氢酶（LDH）和ELISA死亡检测法评估细胞凋亡。
用VE-822和AZD1775处理时，SW-480和HT-29细胞的增殖率较低。VE-822在SW480和HT-29中的IC50值分别为1.3μM和1.6μM。此外，AZD1775在SW480中的该值为140 nM，在HT-29中为185 nM。在同时用VE-822和AZD1775处理的两种细胞系中，ATR、CHK1、WEE1、RRM1和RRM2的表达水平均显著下调（P<0.05）。在这些细胞中，包括γ-H2AX和8-氧代-dG在内的DNA损伤标志物上调。同时用VE-822和AZD1775处理可增加两种细胞系的凋亡能力。
通过AZD1775抑制WEE1可增强ATR抑制剂VE-822通过靶向DNA损伤对抗结直肠癌的抗癌作用。
