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. 2021 Sep 2;10(9):2287.
doi: 10.3390/cells10092287.

Omega-3 Fatty Acids DHA and EPA Reduce Bortezomib Resistance in Multiple Myeloma Cells by Promoting Glutathione Degradation

Jing Chen  1 , Esther A Zaal  2   3 , Celia R Berkers  2   3 , Rob Ruijtenbeek  4 , Johan Garssen  1   5 , Frank A Redegeld  1
Affiliations

Omega-3 Fatty Acids DHA and EPA Reduce Bortezomib Resistance in Multiple Myeloma Cells by Promoting Glutathione Degradation

Jing Chen et al. Cells. .

Abstract

Multiple myeloma (MM) is a hematological malignancy that exhibits aberrantly high levels of proteasome activity. While treatment with the proteasome inhibitor bortezomib substantially increases overall survival of MM patients, acquired drug resistance remains the main challenge for MM treatment. Using a combination treatment of docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) and bortezomib, it was demonstrated previously that pretreatment with DHA/EPA significantly increased bortezomib chemosensitivity in MM cells. In the current study, both transcriptome and metabolome analysis were performed to comprehensively evaluate the underlying mechanism. It was demonstrated that pretreating MM cells with DHA/EPA before bortezomib potently decreased the cellular glutathione (GSH) level and altered the expression of the related metabolites and key enzymes in GSH metabolism, whereas simultaneous treatment only showed minor effects on these factors, thereby suggesting the critical role of GSH degradation in overcoming bortezomib resistance in MM cells. Moreover, RNA-seq results revealed that the nuclear factor erythroid 2-related factor 2 (NRF2)-activating transcription factor 3/4 (ATF3/4)-ChaC glutathione specific gamma-glutamylcyclotransferase 1 (CHAC1) signaling pathway may be implicated as the central player in the GSH degradation. Pathways of necroptosis, ferroptosis, p53, NRF2, ATF4, WNT, MAPK, NF-κB, EGFR, and ERK may be connected to the tumor suppressive effect caused by pretreatment of DHA/EPA prior to bortezomib. Collectively, this work implicates GSH degradation as a potential therapeutic target in MM and provides novel mechanistic insights into its significant role in combating bortezomib resistance.

Keywords: DHA; EPA; bortezomib; drug resistance; metabolome; multiple myeloma; omega-3 fatty acids; transcriptome.

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Conflict of interest statement

The authors declare that no conflicts of interest.

Figures

Figure 1
Figure 1
Transcriptomic profiling in response to DHA, EPA, or bortezomib in the OPM2 cell line. Cells were treated with 50 µM of DHA/EPA (6 h) or 10 nM of bortezomib (4 h), and then RNA-seq analysis were performed. (A) Number of significantly up- and down-regulated DEGs identified in different comparison groups. padj < 0.05. (B) Venn diagrams showing the numbers of overlapping and non-overlapping DEGs in three comparison groups. (C) Volcano plots summarizing the DEGs upon treatment with DHA (left), EPA (middle) or bortezomib (right). The seven overlapping DEGs from (B) were highlighted. Green, downregulated DEGs; red, upregulated DEGs. (D) KEGG pathway analysis of DEGs in treated cells compared to the control. The counts present the number of DEGs enriched in a particular pathway. Different colors represent padj values. Hierarchical clustering heatmaps depicting the levels of differentially expressed tumor suppressor genes (E) and oncogenes (F) from RNA-Seq analysis of the control and DHA/EPA/bortezomib-treated cells.
Figure 2
Figure 2
Simultaneous treatment with bortezomib and DHA or EPA altered tumor-associated gene expression in the OPM2 cell line. Cells were treated with 10 nM of bortezomib for 4 h in the presence of DHA/EPA (50 µM), and then RNA-seq analysis were performed. (A) Number of significantly up- and down-regulated DEGs identified in different comparison groups. padj < 0.05. DB, DHA, and bortezomib; EB, EPA, and bortezomib. (B) Venn diagrams showing the numbers of overlapping and non-overlapping DEGs in two comparison groups. (C) Volcano plots summarizing the DEGs upon treatment with bortezomib and DHA (left panel) or EPA (right panel). The six overlapping DEGs from (B) were highlighted. Green, downregulated DEGs; red, upregulated DEGs. (D) KEGG pathway analysis of DEGs in treated cells compared to the control. The counts present the number of DEGs enriched in a particular pathway. Different colors represent padj value. Left, DB vs Bort; right, EB vs Bort. Hierarchical clustering heatmaps depicting the levels of differentially expressed tumor suppressor genes (E) and oncogenes (F) from RNA-Seq analysis of bortezomib-, DB-, and EB-treated cells.
Figure 3
Figure 3
Pretreatment with DHA or EPA before bortezomib differentially regulated tumor-associated gene expression in the OPM2 cell line. Cells were pretreated with 50 µM of DHA or EPA for 0 and 2 h and treated with bortezomib (10 nM) for 4 h. Then, RNA-seq analysis was performed. (A) The number of significantly up- and down-regulated DEGs identified in different comparison groups. padj < 0.05. D2B/E2B, 2 h pretreatment with DHA/EPA plus 4 h treatment of bortezomib. (B) Venn diagrams showing the numbers of overlapping and non-overlapping DEGs in two comparison groups. (C) Volcano plots summarizing the DEGs upon treatment with D2B (left panel) or E2B (right panel). The top 10 overlapping DEGs from (B) were highlighted. Green, downregulated DEGs; red, upregulated DEGs. (D) KEGG pathway analysis of DEGs in treated cells compared to the control. The count presented the number of DEGs enriched in a particular pathway. Different colors represent padj value. Left, D2B vs Bort; right, E2B vs Bort. Hierarchical clustering heatmaps depicting the levels of differentially expressed tumor suppressor genes (E) and oncogenes (F) from RNA-Seq analysis of bortezomib, D2B and E2B-treated cells.
Figure 4
Figure 4
Transcriptomic and metabolomic analysis in the OPM2 cell line reveals the crucial role of GSH metabolism in increasing bortezomib chemosensitivity in MM. For metabolomic analysis, OPM2 cells were pretreated with 50 µM of DHA/EPA for 0 or 2 h, then bortezomib (10 nM) was added for 6 h treatment. (A) Metabolome pathway enrichment of 16 differentially regulated metabolites using MetaboAnalyst 5.0. The node color represents the p values, and the node size represents the pathway impact values. (B) The levels of GSH, oxidized GSH (GSSG) and NADPH upon different treatment. Heatmap analysis of the expression of the key enzymes involved in GSH metabolism (C), serine synthesis and metabolism (D) and folate cycle (E) in different conditions. (F) and (G) The levels of metabolites related to folate cycle and methionine cycle in different conditions. Data are presented as mean ± SD of three independent treatment. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001when compared with the control. (H) Graphical representation of the metabolic enzymes and pathways associated with the GSH cycle in OPM2 cells. Enzymes were highlighted in blue.
Figure 5
Figure 5
GSH metabolism plays a critical role in increasing bortezomib chemosensitivity in the bortezomib-resistant MM cell line. (A) RPMI8226-BTZ/100 was pretreated with 50 µM of DHA or EPA for 0 or 2 h and then incubated with bortezomib (200 nM) for 24 h. Apoptotic cells were determined by Annexin-V and PI staining. (B) Metabolome pathway enrichment of 24 differentially regulated metabolites using MetaboAnalyst 5.0. The node color represents the p values, and the node size represents the pathway impact values. (C) and (D) The levels of the indicated metabolites upon different treatment. Data are presented as mean ± SD of three independent treatment. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 when compared with the control.
Figure 6
Figure 6
CHAC1-mediated GSH depletion may be important for bortezomib chemosensitivity in MM cells increased by DHA/EPA pretreatment. (A) Heatmap analysis of the expression of ATF3, ATF4, NRF2, TAK, and TALDO1 in different conditions. (B) Summarizing scheme. Pretreating MM cells with DHA/EPA before bortezomib induces GSH degradation through activating the NRF2-ATF3/4-CHAC1 pathway, which eventually leads to cell death. Meanwhile, metabolic pathways of PPP, SSP, folate cycle, and methionine cycle may be activated to increase GSH synthesis to recover the cellular redox homeostasis during cell death.
Figure 7
Figure 7
Possible mechanism underlying the opposite effects of the different treatment schedules with DHA/EPA on bortezomib chemosensitivity in MM cells. (A) DHA or EPA may induce MM cell death through activating PTEN and p53 signaling pathways, p62-mediated autophagy, ferroptosis, and necroptosis, and inhibiting ERK, JNK, and EGFR signaling pathways. (B) Bortezomib-induced MM cell death may include blocking pro-survival pathways of WNT and EGFR and activating pathways of p53 and NRF2-ATF3/4-CHAC1 and p21-mediated cell cycle arrest. (C) Simultaneous treatment with bortezomib and DHA or EPA may decrease bortezomib chemosensitivity in MM cells through inhibiting PTEN and p53 pathways and activating CDK7-mediated cell cycle progression. (D) Pretreatment with DHA or EPA prior to bortezomib increase bortezomib sensitivity possibly through activating p53 and NRF2-ATF3/4-CHAC1 pathways, ferroptosis and necroptosis and inhibiting pathways of WNT, NF-κB, MAPK, ERK, and EGFR in MM cells.
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