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. 2022 Sep 1;30(5):465-472.
doi: 10.4062/biomolther.2022.061. Epub 2022 Jun 17.

Anti-Tumor Effect of IDF-11774, an Inhibitor of Hypoxia-Inducible Factor-1, on Melanoma

Nan-Hyung Kim  1 , Jong Heon Jeong  1 , Yu Jeong Park  1 , Hui Young Shin  1 , Woo Kyoung Choi  1 , Kyeong Lee  2 , Ai-Young Lee  1
Affiliations

Anti-Tumor Effect of IDF-11774, an Inhibitor of Hypoxia-Inducible Factor-1, on Melanoma

Nan-Hyung Kim et al. Biomol Ther (Seoul). .

Abstract

Melanoma is one of the most aggressive skin cancers. Hypoxia contributes to the aggressiveness of melanoma by promoting cancer growth and metastasis. Upregulation of cyclin D1 can promote uncontrolled cell proliferation in melanoma, whereas stimulation of cytotoxic T cell activity can inhibit it. Epithelial mesenchymal transition (EMT) plays a critical role in melanoma metastasis. Hypoxia-inducible factor-1α (HIF-1α) is a main transcriptional mediator that regulates many genes related to hypoxia. CoCl2 is one of the most commonly used hypoxia-mimetic chemicals in cell culture. In this study, inhibitory effects of IDF-11774, an inhibitor of HIF-1α, on melanoma growth and metastasis were examined using cultured B16F10 mouse melanoma cells and nude mice transplanted with B16F10 melanoma cells in the presence or absence of CoCl2-induced hypoxia. IDF-11774 reduced HIF-1α upregulation and cell survival, but increased cytotoxicity of cultured melanoma cells under CoCl2-induced hypoxia. IDF-11774 also reduced tumor size and local invasion of B16F10 melanoma in nude mice along with HIF-1α downregulation. Expression levels of cyclin D1 in melanoma were increased by CoCl2 but decreased by IDF-11774. Apoptosis of melanoma cells and infiltration of cytotoxic T cells were increased in melanoma after treatment with IDF-11774. EMT was stimulated by CoCl2, but restored by IDF- 11774. Overall, IDF-11774 inhibited the growth and metastasis of B16F10 melanoma via HIF-1α downregulation. The growth of B16F10 melanoma was inhibited by cyclin D1 downregulation and cytotoxic T cell stimulation. Metastasis of B16F10 melanoma was inhibited by EMT suppression.

Keywords: HIF-1α inhibitor; IDF11774; Inhibition of melanoma growth and metastasis.

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Figures

Fig. 1
Fig. 1
IDF-11774 reduces CoCl2-induced HIF-1α upregulation in cultured B16F10 melanoma cells. (A) Western blot analysis for HIF-1α protein expression in three kinds of cultured melanoma cells. (HS936T, MNT1, and B16F10) and primary cultured normal human epidermal melanocytes (MC). (B, C) MTT assay for cell viability and LDH release for cytotoxicity (B) and Western blot analysis for HIF-1α protein expression in B16F10 cells treated with different concentrations (0, 1. 25, 2.5, 5, and 10 mM) of IDF-11774 for 48 h. (D, E) Western blot analysis for relative ratios of HIF-1α levels (D) and representative immunofluorescent staining using anti-HIF-1α antibody in B16F10 treated with IDF-11774 in the absence or presence of CoCl2 for 48 h. Nuclei were counter-stained with Hoechst 33258 (Bar=0.05 mm) (E). (F) MTT assay and LDH release in B16F10 treated with IDF-11774 in the absence or presence of CoCl2 for 48 h. β-Actin was used as an internal control for Western blot analysis. Intensities of immunofluorescence staining were measured using a Wright Cell Imaging Facility ImageJ software. Data in the graph represent mean ± SD of relative values compared to non-treated control from four independent experiments. *p<0.05, **p<0.01.
Fig. 2
Fig. 2
IDF-11774 decreases size and local invasion of B16F10 melanoma in nude mice along with HIF-1α downregulation. (A) B16F10 melanoma in nude mice treated without (control) or with 60 mg/kg/day of IDF-11774. Data in the graph represent mean ± SD of relative melanoma volumes of mice treated with different doses of IDF-11774 compared to non-treated control, which were measured once every two or three days in five mice of each group. (B, C) Light microscopy after H&E staining (B) and representative immunofluorescent staining using anti-HIF-1α antibody (C) of B16F10 melanoma in nude mice treated without (control) or with 60 mg/kg/day of IDF-11774. Intensities of immunofluorescence staining were measured using a Wright Cell Imaging Facility ImageJ software. Nuclei were counter-stained with Hoechst 33258 (Bar=0.05 mm). Data in the graph represent mean ± SD of relative values compared to non-treated control from four independent experiments. *p<0.05, **p<0.01.
Fig. 3
Fig. 3
IDF-11774 decreases cyclin D1 expression but increases apoptosis and cytotoxic T-cell infiltration in B16F10 melanoma. (A) Representative immunofluorescent staining of B16F10 melanoma in nude mice treated without (control) or with 60 mg/kg/day of IDF-11774 using anti-cyclin D1 antibody. (B) Western blot analysis for relative ratios of cyclin D1 levels in cultured B16F10 treated with IDF-11774 in the absence or presence of CoCl2 for 48 h. (C) Representative TUNEL assay of B16F10 melanoma in nude mice treated without (control) or with 60 mg/kg/day of IDF-11774. (D) Western blot analysis for relative ratios of active caspase-3 levels in cultured B16F10 treated with IDF-11774 in the absence or presence of CoCl2 for 48 h. (E) Representative immunofluorescent staining of B16F10 melanoma in nude mice treated without (control) or with 60 mg/kg/day of IDF-11774 using anti-CD8 antibody. β-Actin was used as an internal control for Western blot analysis. Nuclei were counter-stained with Hoechst 33258 (Bar=0.05 mm). Intensities of immunofluorescence staining were measured using Wright Cell Imaging Facility ImageJ software. Data in the graph represent mean ± SD of relative values compared to non-treated control from four independent experiments. *p<0.05, **p< 0.01.
Fig. 4
Fig. 4
IDF-11774 downregulates EMT in B16F10 melanoma. (A, B) Western blot analysis of E-cadherin, N-cadherin and SNAIL levels (A) and representative immunofluorescent staining using anti-N-cadherin or anti-SNAIL antibody (B) in cultured B16F10 treated with IDF-11774 in the absence or presence of CoCl2 for 48 h. (C, D) Representative immunofluorescent staining of B16F10 melanoma in nude mice treated without (control) or with 60 mg/kg/day of IDF-11774 using anti-N-cadherin (C) or anti-SNAIL antibody (D). β-Actin was used as an internal control for Western blot analysis. Nuclei were counter-stained with Hoechst 33258 (Bar=0.05 mm). Intensities of immunofluorescence staining were measured using a Wright Cell Imaging Facility ImageJ software. Data in the graph represent mean ± SD of relative values compared to non-treated control from four independent experiments. *p<0.05, **p<0.01.
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