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Chloroquine prevents hypoxic accumulation of HIF-1α by inhibiting ATR kinase: implication in chloroquine-mediated chemosensitization of colon carcinoma cells under hypoxia

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Abstract

Background

Chloroquine (CQ) is an effective and safe antimalarial drug that is also used as a disease-modifying antirheumatic drug. Recent studies have shown that CQ can sensitize cancer cells to anti-cancer therapies.

Methods

In this study, we investigated the molecular mechanisms underlying CQ-mediated chemosensitization in human colon carcinoma cells.

Results

CQ prevented hypoxia-inducible factor (HIF)-1α protein induction in human colon carcinoma cells. CQ also suppressed HIF-1 activity, as represented by CQ inhibition of HIF-1-dependent luciferase activity and reduced induction of vascular endothelial growth factor. Under hypoxia, CQ restricted HIF-1α synthesis but did not affect HIF-1α transcription and protein stability. The hypoxic state activated ataxia telangiectasia and Rad3-related (ATR) kinase and increased the level of phosphorylated checkpoint kinase 1, a substrate of ATR kinase; however, this was prevented by CQ. An ATR kinase inhibitor suppressed the hypoxic induction of HIF-1α protein and was as effective as CQ. The cytotoxicity of 5-fluorouracil (5-FU), the first choice for the treatment of colorectal cancer, was attenuated under hypoxia. CQ enhanced the cytotoxicity of 5-FU treatment, which was mimicked by the transient transfection with HIF-1α siRNA.

Conclusions

Under hypoxia, CQ-mediated sensitization of colon carcinoma HCT116 cells to 5-FU involves HIF-1 inhibition via ATR kinase suppression.

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Data availability

The data presented in this study are available in article or supplementary material here.

Abbreviations

ATR:

Ataxia telangiectasia and Rad3 related

Chk1:

Checkpoint kinase 1

CHX:

Cycloheximide

CQ:

Chloroquine

DMEM:

Dulbecco’s modified Eagle’s medium

4E-BP1:

Eukaryotic initiation factor 4E-binding protein 1

ELISA:

Enzyme-linked immunosorbent assay

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

HIF:

Hypoxia-inducible factor

LD:

Lysosomal degradation

MTT:

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide

mTOR:

Mammalian target of rapamycin

NM:

Not measurable

n.s:

No significant

S6K:

Ribosomal protein S6 kinase β-1

SD:

Standard deviation

SDS-PAGE:

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

SiH:

HIF-1α siRNA

SiC:

Control siRNA

TBP:

TATA binding protein

VHL:

Von Hippel-Lindau

VEGF:

Vascular endothelial growth factor

3-MA:

3-Methyladenine

5-FU:

5-Fluorouracil

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Acknowledgements

This work was supported by a 2-Year Research grant from the Pusan National University.

Funding

This work was supported by a 2-year research grant from the Pusan National University.

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Authors and Affiliations

  1. College of Pharmacy, Pusan National University, Busan, 46241, Republic of Korea

    Changyu Kang, Sanghyun Ju, Jaejeong Kim & Yun** Jung

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  1. Changyu Kang
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Contributions

CK: investigation, formal analysis, data curation, and writing—original draft; SJ, and JK: validation and data curation; YJ: writing—review and editing, conceptualization, supervision, and funding acquisition.

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Correspondence to Yun** Jung.

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Kang, C., Ju, S., Kim, J. et al. Chloroquine prevents hypoxic accumulation of HIF-1α by inhibiting ATR kinase: implication in chloroquine-mediated chemosensitization of colon carcinoma cells under hypoxia. Pharmacol. Rep 75, 211–221 (2023). https://doi.org/10.1007/s43440-022-00441-5

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  • DOI: https://doi.org/10.1007/s43440-022-00441-5

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