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Cited by 20Enzyme function and inhibitionCancer, Hypoxia, and MetabolismMetabolism, Diabetes, and Cancer

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Drug Repurposing as an Antitumor Agent: Disulfiram-Mediated Carbonic Anhydrase 12 and Anion Exchanger 2 Modulation to Inhibit Cancer Cell Migration

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Hwang, Soyoung, Shin, Dong Min, Hong, Jeong Hee·2019·Molecules
DOI 10.3390/molecules24183409PMC PMC6767608
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Abstract

Disulfiram has been used in the treatment of alcoholism and exhibits an anti-tumor effect. However, the intracellular mechanism of anti-tumor activity of Disulfiram remains unclear. In this study, we focused on the modulatory role of Disulfiram via oncogenic factor carbonic anhydrase CA12 and its associated transporter anion exchanger AE2 in lung cancer cell line A549. The surface expression of CA12 and AE2 were decreased by Disulfiram treatment with a time-dependent manner. Disulfiram treatment did not alter the expression of Na+-bicarbonate cotransporters, nor did it affect autophagy regulation. The chloride bicarbonate exchanger activity of A549 cells was reduced by Disulfiram treatment in a time-dependent manner without change in the resting pH level. The expression and activity of AE2 and the expression of CA12 were also reduced by Disulfiram treatment in the breast cancer cell line. An invasion assay and cell migration assay revealed that Disulfiram attenuated the invasion and migration of A549 cells. In conclusion, the attenuation of AE2 and its supportive enzyme CA12, and the inhibitory effect on cell migration by Disulfiram treatment in cancer cells provided the molecular evidence supporting the potential of Disulfiram as an anticancer agent.

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Crossref · 10.3390/molecules24183409

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Figure 1 Surface protein expression of CA12 protein was decreased by DSF treatment. (A) Analysis of CA12 mRNA expression at rising concentrations from 0.1–4 μM DSF for 48 h (n = 5). (B) Protein expression of CA12 at indicated DSF concentration. (C) Analysis of relative CA12 intensity (n = 3, * p < 0.05). (D) Immunostained images of CA12 (green), nucleus (DAPI, blue), and cell morphology (TPM-T, gray) following 2 μM DSF treatment. The scale bars represent 20 μm. (E) Time-dependent changes in surface expression of CA12 in the presence of 2 μM DSF in A549 cells. The β-actin and input CA12 blots were used as loading controls. (F) Analysis of relative intensity of CA12 for input and biotinylated (Bio) blot. Bars represent mean ± SEM (n = 4, * p < 0.05).
Figure 1 Surface protein expression of CA12 protein was decreased by DSF treatment. (A) Analysis of CA12 mRNA expression at rising concentrations from 0.1–4 μM DSF for 48 h (n = 5). (B) Protein expression of CA12 at indicated DSF concentration. (C) Analysis of relative CA12 intensity (n = 3, * p < 0.05). (D) Immunostained images of CA12 (green), nucleus (DAPI, blue), and cell morphology (TPM-T, gray) following 2 μM DSF treatment. The scale bars represent 20 μm. (E) Time-dependent changes in surface expression of CA12 in the presence of 2 μM DSF in A549 cells. The β-actin and input CA12 blots were used as loading controls. (F) Analysis of relative intensity of CA12 for input and biotinylated (Bio) blot. Bars represent mean ± SEM (n = 4, * p < 0.05).CC BY · PubMed Central
Figure 2 Surface protein expression of AE2 protein was decreased by DSF treatment. (A) Analysis of AE2 mRNA expression at rising concentrations from 0.1–4 μM DSF for 48 h (n = 5). (B) Protein expression of AE2 at indicated DSF concentration. (C) Analysis of relative AE2 intensity (n = 3, * p < 0.05, *** p < 0.001). (D) Immunostained images of AE2 (red), nucleus (DAPI, blue) and merge of cell morphology (TPM-T, gray) with and without DSF treatment. The scale bars represent 20 μm. (E) The surface expression of AE2 after treatment with 2 μM DSF. The β-actin and input AE2 blots were used as loading controls. (F) Analysis of relative intensity of AE2 for input and biotinylated (Bio) blot. Bars represent mean ± SEM (n = 3, ** p < 0.01). (G) Protein expressions of NBCe1 and NBCn1. (H) Analysis of relative intensity of NBCe1 and NBCn1 blots. Bars represent mean ± SEM. (I) Protein expressions of LC3B and p62. The β-actin blot was used as a loading control. (J) Analysis of relative intensity of LC3B and p62 blots. Bars represent mean ± SEM.
Figure 2 Surface protein expression of AE2 protein was decreased by DSF treatment. (A) Analysis of AE2 mRNA expression at rising concentrations from 0.1–4 μM DSF for 48 h (n = 5). (B) Protein expression of AE2 at indicated DSF concentration. (C) Analysis of relative AE2 intensity (n = 3, * p < 0.05, *** p < 0.001). (D) Immunostained images of AE2 (red), nucleus (DAPI, blue) and merge of cell morphology (TPM-T, gray) with and without DSF treatment. The scale bars represent 20 μm. (E) The surface expression of AE2 after treatment with 2 μM DSF. The β-actin and input AE2 blots were used as loading controls. (F) Analysis of relative intensity of AE2 for input and biotinylated (Bio) blot. Bars represent mean ± SEM (n = 3, ** p < 0.01). (G) Protein expressions of NBCe1 and NBCn1. (H) Analysis of relative intensity of NBCe1 and NBCn1 blots. Bars represent mean ± SEM. (I) Protein expressions of LC3B and p62. The β-actin blot was used as a loading control. (J) Analysis of relative intensity of LC3B and p62 blots. Bars represent mean ± SEM.CC BY · PubMed Central
Figure 3 DSF decreased CBE activity in A549 cells. (A) CBE activity of A549 cells with and without treatment with 2 μM DSF (blue trace at 24 h, red trace at 48 h, and control, black trace). Average traces are represented. (B) Bars indicate the means ± SEM (n = 4, ** p < 0.01). (C) The bars show resting intracellular pH (pHi) level of A549 cells represented as the means ± SEM. (D) The calibration curve of A549 cells (pH = 6.97-log(5.84-R/R-2.53)).
Figure 3 DSF decreased CBE activity in A549 cells. (A) CBE activity of A549 cells with and without treatment with 2 μM DSF (blue trace at 24 h, red trace at 48 h, and control, black trace). Average traces are represented. (B) Bars indicate the means ± SEM (n = 4, ** p < 0.01). (C) The bars show resting intracellular pH (pHi) level of A549 cells represented as the means ± SEM. (D) The calibration curve of A549 cells (pH = 6.97-log(5.84-R/R-2.53)).CC BY · PubMed Central
Figure 4 DSF also decreased the expressions of AE2 and CA12 and CBE activity in breast cancer cell lines. (A) The protein expressions of AE2 and CA12 with and without treatment of 2 μM DSF in MDA-MB-231 and MCF-7 cells. The β-actin was used as loading control. (B) Analysis of relative intensity of AE2 and CA12 blots in MDA-MB-231 and MCF-7 cells. Bars represent mean ± SEM (n = 3, * p < 0.05, ** p < 0.01). (C) CBE activity of MDA-MB-231 cells treated with 2 μM DSF (yellow trace) at 24 h and the cells not treated with DSF (Control, gray trace). Average traces are represented. The bar graph indicates CBE activity of MDA-MB-231 cells with and without DSF and the means ± SEM (n = 4, *** p < 0.001). (D) The bars show resting pHi level of MDA-MB-231 cells which indicate the means ± SEM. (E) CBE activity of MCF-7 cells treated with 2 μM DSF (yellow trace) at 24 h and the cells not treated with DSF (Control, gray trace). Average traces are represented. The bar graph indicates CBE activity of MCF-7 cells with and without DSF and the means ± SEM (n = 4, * p < 0.05). (F) The bars show resting pHi level of MCF-7 cells which indicate the means ± SEM.
Figure 4 DSF also decreased the expressions of AE2 and CA12 and CBE activity in breast cancer cell lines. (A) The protein expressions of AE2 and CA12 with and without treatment of 2 μM DSF in MDA-MB-231 and MCF-7 cells. The β-actin was used as loading control. (B) Analysis of relative intensity of AE2 and CA12 blots in MDA-MB-231 and MCF-7 cells. Bars represent mean ± SEM (n = 3, * p < 0.05, ** p < 0.01). (C) CBE activity of MDA-MB-231 cells treated with 2 μM DSF (yellow trace) at 24 h and the cells not treated with DSF (Control, gray trace). Average traces are represented. The bar graph indicates CBE activity of MDA-MB-231 cells with and without DSF and the means ± SEM (n = 4, *** p < 0.001). (D) The bars show resting pHi level of MDA-MB-231 cells which indicate the means ± SEM. (E) CBE activity of MCF-7 cells treated with 2 μM DSF (yellow trace) at 24 h and the cells not treated with DSF (Control, gray trace). Average traces are represented. The bar graph indicates CBE activity of MCF-7 cells with and without DSF and the means ± SEM (n = 4, * p < 0.05). (F) The bars show resting pHi level of MCF-7 cells which indicate the means ± SEM.CC BY · PubMed Central
Figure 5 DSF reduced cancer cell invasion and migration in acidic microenvironment. (A) Time dependent representative images of A549 cells, invading (4, 24, 48, and 72 h) towards agarose spots containing PBS (pH 6.5) with and without DSF treatment. The red dotted lines indicate the direction of invasion across the boundary of the agarose spot shown as a dashed curve. The yellow dotted lines indicate the lineage of cells that migrated into the agarose spots. (B) The schematic representation of agarose spot assay. (C) Analysis of invasion range per hour. Bars indicate the means ± SEM (n = 4, ** p < 0.01). (D) A549 cell migration was measured by transwell-membrane migration assay incubated with indicated conditions (bottom plates; pH 6.5, upper chamber; pretreated with 2 μΜ DSF at 48 h). Immunofluorescence staining of nucleus (DAPI, blue) was analyzed for the evaluation of cell migration. The scale bars represent 50 μm. (E) Analysis of total intensity of DAPI for A549 migration. Bars represent mean ± SEM (n = 3, * p < 0.05).
Figure 5 DSF reduced cancer cell invasion and migration in acidic microenvironment. (A) Time dependent representative images of A549 cells, invading (4, 24, 48, and 72 h) towards agarose spots containing PBS (pH 6.5) with and without DSF treatment. The red dotted lines indicate the direction of invasion across the boundary of the agarose spot shown as a dashed curve. The yellow dotted lines indicate the lineage of cells that migrated into the agarose spots. (B) The schematic representation of agarose spot assay. (C) Analysis of invasion range per hour. Bars indicate the means ± SEM (n = 4, ** p < 0.01). (D) A549 cell migration was measured by transwell-membrane migration assay incubated with indicated conditions (bottom plates; pH 6.5, upper chamber; pretreated with 2 μΜ DSF at 48 h). Immunofluorescence staining of nucleus (DAPI, blue) was analyzed for the evaluation of cell migration. The scale bars represent 50 μm. (E) Analysis of total intensity of DAPI for A549 migration. Bars represent mean ± SEM (n = 3, * p < 0.05).CC BY · PubMed Central

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Metadata

Authors
Hwang, S., Shin, D. M., & Hong, J. H.
Affiliations
  • Gachon University (KR)
  • Yonsei University (KR)
Corresponding author
  • Shin, Dong Min
  • Hong, Jeong Hee
ORCID
Venue
Molecules
Publisher
MDPI AG
Published
2019-09-19
Volume
24
Issue
18
Pages
3409
ISSN
1420-3049 (ISSN-L)
DOI
10.3390/molecules24183409
PMC
PMC6767608
OpenAlex
W2973928408
Cited by
20
References
31
Citations by year
2026: 1 · 2025: 1 · 2024: 3 · 2023: 3 · 2022: 4 · 2021: 3 · 2020: 5
FWCI
0.99
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecules (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • YUHSpace (Yonsei University Medical Library) (submittedVersion, cc-by-nc-nd)
Funders
the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) and the grant from the Gachon University Gil Medical Center project (NRF-2019R1F1A1046785 (JHH), NRF-2016R1A5A2008630 (DMS), FRD 2018-07 (JHH))
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme function and inhibition
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer, Hypoxia, and Metabolism
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolism, Diabetes, and Cancer
Keywords
disulfiram, drug repurposing, AE2, tumor cell migration, tumor invasion, lung cancer, breast cancer, chloride/bicarbonate exchanger, anticancer activity, A549 cells
MeSH terms
Antineoplastic Agents, Carbonic Anhydrases, Cell Membrane, Cell Movement, Disulfiram, Humans, Neoplasm Invasiveness, Neoplasms, Chloride-Bicarbonate Antiporters, Cell Line, Tumor, Drug Repositioning, Tumor Microenvironment

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