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Free docosahexaenoic acid promotes ferroptotic cell death via lipoxygenase dependent and independent pathways in cancer cells

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European Journal of Nutrition Aims and scope Submit manuscript

Abstract

Purpose

Ferroptosis is a form of regulated cell death that has the potential to be targeted as a cancer therapeutic strategy. But cancer cells have a wide range of sensitivities to ferroptosis, which limits its therapeutic potential. Accumulation of lipid peroxides determines the occurrence of ferroptosis. However, the type of lipid involved in peroxidation and the mechanism of lipid peroxide accumulation are less studied.

Methods

The effects of fatty acids (10 μM) with different carbon chain length and unsaturation on ferroptosis were evaluated by MTT and LDH release assay in cell lines derived from prostate cancer (PC3, 22RV1, DU145 and LNCaP), colorectal cancer (HT-29), cervical cancer (HeLa) and liver cancer (HepG2). Inhibitors of apoptosis, necroptosis, autophagy and ferroptosis were used to determine the type of cell death. Then the regulation of reactive oxygen species (ROS) and lipid peroxidation by docosahexaenoic acid (DHA) was measured by HPLC–MS and flow cytometry. The avtive form of DHA was determined by siRNA mediated gene silencing. The role of lipoxygenases was checked by inhibitors and gene silencing. Finally, the effect of DHA on ferroptosis-mediated tumor killing was verified in xenografts.

Results

The sensitivity of ferroptosis was positively correlated with the unsaturation of exogenously added fatty acid. DHA (22:6 n-3) sensitized cancer cells to ferroptosis-inducing reagents (FINs) at the highest level in vitro and in vivo. In this process, DHA increased ROS accumulation, lipid peroxidation and protein oxidation independent of its membrane receptor, GPR120. Inhibition of long chain fatty acid-CoA ligases and lysophosphatidylcholine acyltransferases didn't affect the role of DHA. DHA-involved ferroptosis can be induced in both arachidonate lipoxygenase 5 (ALOX5) negative and positive cells. Down regulation of ALOX5 inhibited ferroptosis, while overexpression of ALOX5 promoted ferroptosis.

Conclusion

DHA can effectively promote ferroptosis-mediated tumor killing by increasing intracellular lipid peroxidation. Both ALOX5 dependent and independent pathways are involved in DHA-FIN induced ferroptosis. And during this process, free DHA plays an important role.

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Data availability

All data generated or analyzed during this study are included in this manuscript.

Abbreviations

PUFA:

Polyunsaturated fatty acid

DHA:

Docosahexaenoic acid

FIN:

Ferroptosis-inducing reagent

ROS:

Reactive oxygen species

LPO:

Lipid peroxide

GPX4:

Glutathione peroxidase 4

ALOX:

Arachidonate lipoxygenase

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Acknowledgements

This research was supported by the National Key Research and Development Program of China (2017YFD0400200), the National Natural Science Foundation of China Grants No. 31471128 and 31771539, Key Research and Development Program of Jiangsu Province (BE2018624), and the National First-class Discipline Program of Food Science and Technology (JUFSTR20180101).

Funding

This research was supported by the National Key Research and Development Program of China (2017YFD0400200), the National Natural Science Foundation of China Grants No. 31471128 and 31771539, Key Research and Development Program of Jiangsu Province (BE2018624), and the National First-class Discipline Program of Food Science and Technology (JUFSTR20180101).

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Author notes

  1. Kai Shan, and Ninghan Feng have contributed equally to this work.

Authors and Affiliations

  1. Wuxi School of Medicine, Jiangnan University, Wuxi, 214122, Jiangsu Province, China

    Kai Shan, Doudou Zhu, Hongyan Qu, Guoling Fu, Jiaqi Li, Jing Cui, Heyan Chen, Rong Wang, Yumin Qi & Yong Q. Chen

  2. School of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu Province, China

    Kai Shan, Hongyan Qu, Guoling Fu, Jiaqi Li, Jing Cui, Heyan Chen, Rong Wang, Yumin Qi & Yong Q. Chen

  3. Department of Urology, Wuxi No. 2 People’s Hospital, Wuxi, 214000, Jiangsu Province, China

    Ninghan Feng

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All animals were cared for and sacrificed according to the protocol approved by the ACUC of Jiangnan University.

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394_2022_2940_MOESM1_ESM.tif

Supplementary file1 (TIF 25311 kb) A. Cell viability, as determined by MTT assays, after treatment of cells with erastin, RSL3, or glutamate and fatty acids for 48 h. The results are expressed as percentages relative to the control cells (untreated with either erastin or fatty acid) and correspond to the means ± S.D.(n=3). ANOVA (Tukey test) was performed. Asterisks marked the statistical difference between the addition group and the non-addition group of Fer-1 under the same treatment. *, P<0.05; **, P<0.01; ***, P<0.001. B. Cell viability of non-cancer cells, as determined by MTT assays, after treatment of six cells with erastin, RSL3, or glutamate and fatty acids for 48 h. Results are expressed as percentages relative to the control cells (untreated with either erastin or fatty acid) and correspond to the means ± S.D. of three independent experiments. C. Viability of cells treated with fish oil, erastin and RSL3 was measured using MTT assay. Results are expressed as percentages relative to the control and correspond to the means ± S.D. of three independent experiments. D. PC3 survival rate with different DHA concentration was determined by the MTT assay (n=3). ANOVA (Tukey test) was performed. Compared with the group without DHA addition, the statistical difference was marked with asterisks. ***, P<0.001.

394_2022_2940_MOESM2_ESM.tif

Supplementary file2 (TIF 14616 kb) A. Activation of caspase 3 was checked in ferroptosis and apoptosis. B. PC3 was treated with indicated conditions for 10 h, then flow cytometry was performed. FSC and SSC were used to evaluate cell survival. Gate R1 marked dead cells. C. Intracellular ROS in PC3 cells with different treatments for 2h and 6h. Fluorescence intensity are measured by a flow cytometry and shown as mean±S.D. D. Rols of different ROS inducers on cell viability. Data were shown as the means ± S.D. of three independent experiments. ANOVA with Tukey test was performed, and P < 0.05 was considered significant. Groups with significant differences were shown with different characters.

394_2022_2940_MOESM3_ESM.docx

Supplementary file3 (DOCX 23418 kb) A. HPLC-MS based free AA and DHA analysis was performed in PC3 upon indicated treatment. Fatty acids were determined by high resolution mass spectrometry. B. HPLC-MS based analysis of peroxidized AA and DHA was performed in PC3 upon indicated treatment. Peroxidized fatty acids were determined by high resolution mass spectrometry. C. HPLC-MS based lipidomics analysis was performed in PC3 upon indicated treatment. Target phospholipids were shown. The chromatogram showed the target lipids in different groups. Mass spectrometry was used for qualitative analysis. D. Peroxidized PE (18:0/22:6) and PE (18:0/20:4) were detected by HPLC-MS. Peroxidized PE (18:0/22:6) and PE (18:0/20:4) were determined by high resolution mass spectrometry. E. Western blot was performed to check the expression of ALOX5 in indicated cell lines (THP-1 was primed with PMA for 24h). Role of siALOX5 and zileuton were explored in different cell lines by MTT assay (n=3). F. Viability of cells treated with ALOX5 or FLAP inhibitor during ferroptosis was measured by the MTT assay (n=3). The T-test was performed and asterisks marked the statistical difference. *, P<0.05.

Supplementary file4 (TIF 21 kb)

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Shan, K., Feng, N., Zhu, D. et al. Free docosahexaenoic acid promotes ferroptotic cell death via lipoxygenase dependent and independent pathways in cancer cells. Eur J Nutr 61, 4059–4075 (2022). https://doi.org/10.1007/s00394-022-02940-w

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  • DOI: https://doi.org/10.1007/s00394-022-02940-w

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