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. 2023 Apr:60:102619.
doi: 10.1016/j.redox.2023.102619. Epub 2023 Feb 2.

ROS-lowering doses of vitamins C and A accelerate malignant melanoma metastasis

Muhammad Kashif  1 , Haidong Yao  2 , Sarah Schmidt  2 , Xue Chen  2 , Michelle Truong  2 , Elin Tüksammel  2 , Yiran Liu  2 , Martin O Bergo  3
Affiliations

ROS-lowering doses of vitamins C and A accelerate malignant melanoma metastasis

Muhammad Kashif et al. Redox Biol. 2023 Apr.

Abstract

Oxidative stress is a barrier of migration and metastasis for malignant melanoma cells. Consequently, reducing oxidative stress with the antioxidant N-acetylcysteine (NAC) stimulates melanoma cell migration in vitro and metastasis in vivo. However, it is not yet known whether the NAC effect is shared with other antioxidants. Here, we screened 104 redox-active compounds and identify 27 that increase migration of human malignant melanoma cells in two doses. Validation experiments in four cell lines and four drug doses resulted in a list of 18 compounds which were ranked based on their ability to increase migration and reduce ROS levels; vitamin C (VitC) ranked as number one, followed by the vitamin E analogue Trolox and several carotenoids and Vitamin A-related compounds. Four diet-relevant compounds from this list-VitC, β-carotene, retinyl palmitate, and canthaxanthin-were selected and found to accelerate metastasis in mice with BRAFV600E-driven malignant melanoma. Genomics analyses revealed that the transcription factor BACH1 is activated following antioxidant administration and knockout of Bach1 in mouse melanoma cells reduced lymph node and liver metastasis in xenograft mouse models. We conclude that a broad range of antioxidants accelerate melanoma migration and metastasis and that BACH1 is functionally linked to melanoma metastasis in vivo.

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Conflict of interest statement

Declaration of competing interest The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Vitamin C in ROS-lowering doses increases migration and invasion of NRAS- and BRAF-mutant human malignant melanoma cell lines. (A) Schematic of screening strategy. Redox compounds (n = 104) were incubated in two doses in malignant melanoma cell line SK-MEL-30, whose migration capacity was quantified using the IncuCyte ZOOM System. Based on migration profiles (shown in Supplementary Figs. 1 and 2) a subset of 27 migration-enhancing compounds were selected—based on their ability to increase migration in both doses—and next evaluated in four doses in four malignant melanoma cells lines for their ability to increase cell migration and reduce ROS levels. The 18 most potent migration-enhancers were ranked based on four parameters (shown in Table 1). Finally, 4 high-ranked diet-relevant compounds (Vitamin C, β-carotene, canthaxanthin, and retinyl palmitate) were selected and evaluated for their ability to influence cell invasion in vitro and malignant melanoma metastasis in vivo. See Material and Methods for further details. (B, C) Migration (B) and ROS levels (C) of NRAS-mutant cell line SK-MEL-30 following incubation with vitamin C for 24 h. (D, E) Migration (D) and ROS levels (E) of BRAF-mutant SK-MEL-3 cells incubated with vitamin C for 24 h. (F, G) Invasion of SK-MEL-30 (F) and SK-MEL-3 (G) cells incubated with vitamin C for 24 h. Data in panels B–G are expressed as the change (delta) in migration/invasion and ROS levels induced by VitC compared with DMSO control. Data are mean and standard error of quadruplicate analyses. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig. 2
Fig. 2
Common antioxidants increase authentic metastasis in mice with BRAFV600E-induced malignant melanoma; the increased invasiveness is strongly associated with BACH1. (A) Lymph node metastases in BrafCA/+Ptenfl/flTyr-Cre+/0 mice painted with tamoxifen at 2 days of age and randomized at weaning to receive: control food and drinking water; drinking water supplemented with vitamin C; or food supplemented with canthaxanthin, β-carotene, or retinyl palmitate. Each compound was administered in two different doses as indicated. (B) Analyses of RNAseq data from four human malignant melanoma cell lines incubated for 24 h with each of the four antioxidants from panel A. Shown are transcription factors whose motifs were most enriched in promoters of differentially expressed genes (DEGs). (C) Venn diagram showing the number and percentage DEGs in response to the four compounds shown in panel A. (D) LINCS database analysis showing genes that when overexpressed produced the highest connection (C) scores—i.e., the highest degree of gene expression signature (GES) similarity—compared with the GES observed in nine different human cancer cell lines (including melanoma) following incubation with ten different antioxidants (fraxetin, ascorbic acid, rutin, ascorbyl palmitate, vitexin, mesna, celastrol, seciosolariciresinol, esculin and coumaric acid). (E, F) Lymph node (E) and liver (F) metastases detected in mice injected intravenously with Bach1+/+ and Bach1−/− B16F10 mouse malignant melanoma cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
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