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Ataxia telangiectasia and Rad3-related (ATR) inhibition by VE-822 potently reversed 5-flourouracil resistance in colorectal cancer cells through targeting DNA damage response

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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.

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.

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Acknowledgements

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Funding

This study was funded by Student Research Committee, Urmia University of Medical Sciences (grant number: 215).

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Authors and Affiliations

  1. Solid Tumor Research Center, Cellular and Molecular Medicine Institute, Urmia University of Medical Sciences, Urmia, Iran

    Ainaz Mihanfar & Maryam Majidinia

  2. Immunology Department, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

    Faezeh Asghari

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  1. Ainaz Mihanfar
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  3. Maryam Majidinia
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Contributions

AM performed all research techniques; FA wrote the article; MM guided the present scientific team, wrote and revised the article. All the authors studied and approved the final manuscript.

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Correspondence to Maryam Majidinia.

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Mihanfar, A., Asghari, F. & Majidinia, M. Ataxia telangiectasia and Rad3-related (ATR) inhibition by VE-822 potently reversed 5-flourouracil resistance in colorectal cancer cells through targeting DNA damage response. Mol Biol Rep 51, 474 (2024). https://doi.org/10.1007/s11033-024-09431-7

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