Disulfiram’s Antineoplastic Effects on Ovarian Cancer
Author(s)
Youssef A. Rezk1,
Kun Yang2,
Shoumei Bai2,
Karen Mclean1,
Carolyn Johnston1,
R. Kevin Reynolds1,
Ronald J. Buckanovich1,2*
Affiliation(s)
1 Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI, USA. 2 Division of Hematology Oncology, Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA.
1 Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI, USA. 2 Division of Hematology Oncology, Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA.
ABSTRACT
Objective: Aldehyde dehydrogenase (ALDH) enzymatic activity identifies ovarian cancer stem-like cells. We investigated the antineoplastic activity of the ALDH inhibitor Disulfiram on bulk ovarian cancer cells and CD133+/ALDH+ cancer stem-like cells. Study Design: Ovarian cancer cell lines, human ovarian surface epithelial cells, and mesenchymal stem cells were treated with increasing concentrations of Disulfiram and/or Cisplatin in vitro. Treated cells were assessed for viability or FACS-analyzed for either percentage of ovarian cancer stem-like cells or induction of apoptosis. Disulfiram’s impact on cancer stem-like cells was tested in vitro using tumor sphere formation assays and in vivo using tumor initiation assays with in vitro-treated A2780 cells in NSG mice. Finally, Disulfiram’s in vivo activity was assessed versus CD133+/ALDH+ cell-initiated tumor xenografts. Results: Disulfiram demonstrated antineoplastic activity against multiple ovarian cancer cell lines. While Disulfiram had limited in vitro toxicity against human ovarian surface epithelial cells or mesenchymal stem cells (IC50 of ~15 μM and >30 μM, respectively), its antineoplastic activity against cell lines was comparable to Cisplatin (IC50 ~1.5 μM). Disulfiram-mediated cell death was due, at least in part, to induction of apoptosis. Disulfiram activity was additive with chemotherapy. Disulfiram demonstrated selective depletion of CD44+ cells but not the CD133+ cancer stem-like cells. Disulfiram had no therapeutic impact on tumor initiation studies or in vivo therapy of whole cell line or stem cell-initiated tumor xenografts. Conclusions: In biologically relevant concentrations, Disulfiram has clear antineoplastic activity against ovarian cancer cells in vitro. Disulfiram selectively depleted CD44+ but not CD133+ ovarian cancer stem-like cells in vitro. However, Disulfiram had no significant activity in vivo. Thus, improved and more selective ALDH inhibitors may be required to target ovarian cancer stem cells.
Objective: Aldehyde dehydrogenase (ALDH) enzymatic activity identifies ovarian cancer stem-like cells. We investigated the antineoplastic activity of the ALDH inhibitor Disulfiram on bulk ovarian cancer cells and CD133+/ALDH+ cancer stem-like cells. Study Design: Ovarian cancer cell lines, human ovarian surface epithelial cells, and mesenchymal stem cells were treated with increasing concentrations of Disulfiram and/or Cisplatin in vitro. Treated cells were assessed for viability or FACS-analyzed for either percentage of ovarian cancer stem-like cells or induction of apoptosis. Disulfiram’s impact on cancer stem-like cells was tested in vitro using tumor sphere formation assays and in vivo using tumor initiation assays with in vitro-treated A2780 cells in NSG mice. Finally, Disulfiram’s in vivo activity was assessed versus CD133+/ALDH+ cell-initiated tumor xenografts. Results: Disulfiram demonstrated antineoplastic activity against multiple ovarian cancer cell lines. While Disulfiram had limited in vitro toxicity against human ovarian surface epithelial cells or mesenchymal stem cells (IC50 of ~15 μM and >30 μM, respectively), its antineoplastic activity against cell lines was comparable to Cisplatin (IC50 ~1.5 μM). Disulfiram-mediated cell death was due, at least in part, to induction of apoptosis. Disulfiram activity was additive with chemotherapy. Disulfiram demonstrated selective depletion of CD44+ cells but not the CD133+ cancer stem-like cells. Disulfiram had no therapeutic impact on tumor initiation studies or in vivo therapy of whole cell line or stem cell-initiated tumor xenografts. Conclusions: In biologically relevant concentrations, Disulfiram has clear antineoplastic activity against ovarian cancer cells in vitro. Disulfiram selectively depleted CD44+ but not CD133+ ovarian cancer stem-like cells in vitro. However, Disulfiram had no significant activity in vivo. Thus, improved and more selective ALDH inhibitors may be required to target ovarian cancer stem cells.
Cite this paper
Rezk, Y. , Yang, K. , Bai, S. , Mclean, K. , Johnston, C. , Reynolds, R. and Buckanovich, R. (2015) Disulfiram’s Antineoplastic Effects on Ovarian Cancer. Journal of Cancer Therapy, 6, 1196-1205. doi: 10.4236/jct.2015.614130.
Rezk, Y. , Yang, K. , Bai, S. , Mclean, K. , Johnston, C. , Reynolds, R. and Buckanovich, R. (2015) Disulfiram’s Antineoplastic Effects on Ovarian Cancer. Journal of Cancer Therapy, 6, 1196-1205. doi: 10.4236/jct.2015.614130.
References
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http://dx.doi.org/10.1200/JCO.2008.17.7436 [2] Ginestier, C., Hur, M.H., Charafe-Jauffret, E., Monville, F., Dutcher, J., Brown, M., et al. (2007) ALDH1 Is a Marker of Normal and Malignant Human Mammary Stem Cells and a Predictor of Poor Clinical Outcome. Cell Stem Cell, 1, 555-567.
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http://dx.doi.org/10.1158/0008-5472.CAN-10-3175 [4] Clarke, M.F., Dick, J.E., Dirks, P.B., Eaves, C.J., Jamieson, C.H., Jones, D.L., et al. (2006) Cancer Stem Cells—Perspectives on Current Status and Future Directions: AACR Workshop on Cancer Stem Cells. Cancer Research, 66, 9339-9344.
http://dx.doi.org/10.1158/0008-5472.CAN-06-3126 [5] Choi, Y., Ingram, P.N., Yang, K., Coffman, Y., Iyengar, M., Bai, S., et al. (2015) Identifying an Ovarian Cancer Hierarchy Regulated by BMP2. PNAS, In Press.
http://dx.doi.org/10.1073/pnas.1507899112 [6] Landen, C.N., Goodman, B., Katre, A.A., Steg, A.D., Nick, A.M., Stone, R.L., et al. (2010) Targeting Aldehyde Dehydrogenase Cancer Stem Cells in Ovarian Cancer. Molecular Cancer Therapeutics, 9, 3186-3199.
http://dx.doi.org/10.1158/1535-7163.MCT-10-0563 [7] Steg, A.D., Bevis, K.S., Katre, A.A., Ziebarth, A., Dobbin, Z.C., Alvarez, R.D., et al. (2012) Stem Cell Pathways Contribute to Clinical Chemoresistance in Ovarian Cancer. Clinical Cancer Research, 18, 869-881.
http://dx.doi.org/10.1158/1078-0432.CCR-11-2188 [8] Lindahl, R. (1992) Aldehyde Dehydrogenases and Their Role in Carcinogenesis. Critical Reviews in Biochemistry and Molecular Biology, 27, 283-335.
http://dx.doi.org/10.3109/10409239209082565 [9] Chen, D., Cui, Q.C., Yang, H. and Dou, Q.P. (2006) Disulfiram, a Clinically Used Anti-Alcoholism Drug and Copper-Binding Agent, Induces Apoptotic Cell Death in Breast Cancer Cultures and Xenografts via Inhibition of the Proteasome Activity. Cancer Research, 66, 10425-10433.
http://dx.doi.org/10.1158/0008-5472.CAN-06-2126 [10] Zhang, H., Chen, D., Ringler, J., Chen, W., Cui, Q.C., Ethier, S.P., et al. (2010) Disulfiram Treatment Facilitates Phosphoinositide 3-Kinase Inhibition in Human Breast Cancer Cells in Vitro and in Vivo. Cancer Research, 70, 3996-4004.
http://dx.doi.org/10.1158/0008-5472.CAN-09-3752 [11] Yip, N.C., Fombon, I.S., Liu, P., Brown, S., Kannappan, V., Armesilla, A.L., et al. (2011) Disulfiram Modulated ROS-MAPK and NF-κB Pathways and Targeted Breast Cancer Cells with Cancer Stem Cell-Like Properties. British Journal of Cancer, 104, 1564-1574.
http://dx.doi.org/10.1038/bjc.2011.126 [12] Wang, W., McLeod, H.L. and Cassidy, J. (2003) Disulfiram-Mediated Inhibition of NF-κB Activity Enhances Cytotoxicity of 5-Fluorouracil in Human Colorectal Cancer Cell Lines. International Journal of Cancer, 104, 504-511.
http://dx.doi.org/10.1002/ijc.10972 [13] Lin, J., Haffner, M.C., Zhang, Y., Lee, B.H., Brennen, W.N., Britton, J., et al. (2011) Disulfiram Is a DNA Demethylating Agent and Inhibits Prostate Cancer Cell Growth. Prostate, 71, 333-343.
http://dx.doi.org/10.1002/pros.21247 [14] Morrison, B.W., Doudican, N.A., Patel, K.R. and Orlow, S.J. (2010) Disulfiram Induces Copper-Dependent Stimulation of Reactive Oxygen Species and Activation of the Extrinsic Apoptotic Pathway in Melanoma. Melanoma Research, 20, 11-20.
http://dx.doi.org/10.1097/CMR.0b013e328334131d [15] Raha, D., Wilson, T.R., Peng, J., Peterson, D., Yue, P., Evangelista, M., et al. (2014) The Cancer Stem Cell Marker Aldehyde Dehydrogenase Is Required to Maintain a Drug-Tolerant Tumor Cell Subpopulation. Cancer Research, 74, 3579-3590.
http://dx.doi.org/10.1158/0008-5472.CAN-13-3456 [16] Coffman, L., Mooney, C., Lim, J., Bai, S., Silva, I., Gong, Y., et al. (2013) Endothelin Receptor-A Is Required for the Recruitment of Antitumor T Cells and Modulates Chemotherapy Induction of Cancer Stem Cells. Cancer Biology & Therapy, 14, 184-192.
http://dx.doi.org/10.4161/cbt.22959 [17] Vaccari, A., Ferraro, L., Saba, P., Ruiu, S., Mocci, I., Antonelli, T., et al. (1998) Differential Mechanisms in the Effects of Disulfiram and Diethyldithiocarbamate Intoxication on Striatal Release and Vesicular Transport of Glutamate. Journal of Pharmacology and Experimental Therapeutics, 285, 961-967. [18] Yourick, J.J. and Faiman, M.D. (1991) Disulfiram Metabolism as a Requirement for the Inhibition of Rat Liver Mitochondrial Low Km Aldehyde Dehydrogenase. Biochemical Pharmacology, 42, 1361-1366.
http://dx.doi.org/10.1016/0006-2952(91)90446-C [19] Hart, B.W. and Faiman, M.D. (1992) In Vitro and in Vivo Inhibition of Rat Liver Aldehyde Dehydrogenase by S-Methyl N, N-Diethylthiolcarbamate Sulfoxide, a New Metabolite of Disulfiram. Biochemical Pharmacology, 43, 403-406.
http://dx.doi.org/10.1016/0006-2952(92)90555-W [20] Madan, A. and Faiman, M.D. (1994) Diethyldithiocarbamate Methyl Ester Sulfoxide, an Inhibitor of Rat Liver Mitochondrial Low Km Aldehyde Dehydrogenase and Putative Metabolite of Disulfiram. Alcoholism: Clinical and Experimental Research, 18, 1013-1017.
http://dx.doi.org/10.1111/j.1530-0277.1994.tb00075.x [21] Lipsky, J.J., Shen, M.L. and Naylor, S. (2001) In Vivo Inhibition of Aldehyde Dehydrogenase by Disulfiram. Chemico-Biological Interactions, 130-132, 93-102.
http://dx.doi.org/10.1016/S0009-2797(00)00225-8 [22] Vasiliou, V. and Nebert, D.W. (2005) Analysis and Update of the Human Aldehyde Dehydrogenase (ALDH) Gene Family. Human Genomics, 2, 138-143. [23] Moreb, J.S., Ucar, D., Han, S., Amory, J.K., Goldstein, A.S., Ostmark, B., et al. (2012) The Enzymatic Activity of Human Aldehyde Dehydrogenases 1A2 and 2 (ALDH1A2 and ALDH2) Is Detected by Aldefluor, Inhibited by Diethylaminobenzaldehyde and Has Significant Effects on Cell Proliferation and Drug Resistance. Chemico-Biological Interactions, 195, 52-60.
http://dx.doi.org/10.1016/j.cbi.2011.10.007 [24] Marchitti, S.A., Orlicky, D.J., Brocker, C. and Vasiliou, V. (2010) Aldehyde Dehydrogenase 3B1 (ALDH3B1): Immunohistochemical Tissue Distribution and Cellular-Specific Localization in Normal and Cancerous Human Tissues. Journal of Histochemistry and Cytochemistry, 58, 765-783.
http://dx.doi.org/10.1369/jhc.2010.955773 [25] Rosen, J.M. and Jordan, C.T. (2009) The Increasing Complexity of the Cancer Stem Cell Paradigm. Science, 324, 1670-1673.
http://dx.doi.org/10.1126/science.1171837 [26] Sládek, N.E. (2003) Human Aldehyde Dehydrogenases: Potential Pathological, Pharmacological, and Toxicological Impact. Journal of Biochemical and Molecular Toxicology, 17, 7-23.
http://dx.doi.org/10.1002/jbt.10057 [27] Moore, S.A., Baker, H.M., Blythe, T.J., Kitson, K.E., Kitson, T.M. and Baker, E.N. (1998) Sheep Liver Cytosolic Aldehyde Dehydrogenase: The Structure Reveals the Basis for the Retinal Specificity of Class 1 Aldehyde Dehydrogenases. Structure, 6, 1541-1551.
http://dx.doi.org/10.1016/S0969-2126(98)00152-X
[1] Boman, B.M. and Wicha, M.S. (2008) Cancer Stem Cells: A Step toward the Cure. Journal of Clinical Oncology, 26, 2795-2799.
http://dx.doi.org/10.1200/JCO.2008.17.7436 [2] Ginestier, C., Hur, M.H., Charafe-Jauffret, E., Monville, F., Dutcher, J., Brown, M., et al. (2007) ALDH1 Is a Marker of Normal and Malignant Human Mammary Stem Cells and a Predictor of Poor Clinical Outcome. Cell Stem Cell, 1, 555-567.
http://dx.doi.org/10.1016/j.stem.2007.08.014 [3] Silva, I.A., Bai, S., McLean, K., Yang, K., Griffith, K., Thomas, D., et al. (2011) Aldehyde Dehydrogenase in Combination with CD133 Defines Angiogenic Ovarian Cancer Stem Cells That Portend Poor Patient Survival. Cancer Research, 71, 3991-4001.
http://dx.doi.org/10.1158/0008-5472.CAN-10-3175 [4] Clarke, M.F., Dick, J.E., Dirks, P.B., Eaves, C.J., Jamieson, C.H., Jones, D.L., et al. (2006) Cancer Stem Cells—Perspectives on Current Status and Future Directions: AACR Workshop on Cancer Stem Cells. Cancer Research, 66, 9339-9344.
http://dx.doi.org/10.1158/0008-5472.CAN-06-3126 [5] Choi, Y., Ingram, P.N., Yang, K., Coffman, Y., Iyengar, M., Bai, S., et al. (2015) Identifying an Ovarian Cancer Hierarchy Regulated by BMP2. PNAS, In Press.
http://dx.doi.org/10.1073/pnas.1507899112 [6] Landen, C.N., Goodman, B., Katre, A.A., Steg, A.D., Nick, A.M., Stone, R.L., et al. (2010) Targeting Aldehyde Dehydrogenase Cancer Stem Cells in Ovarian Cancer. Molecular Cancer Therapeutics, 9, 3186-3199.
http://dx.doi.org/10.1158/1535-7163.MCT-10-0563 [7] Steg, A.D., Bevis, K.S., Katre, A.A., Ziebarth, A., Dobbin, Z.C., Alvarez, R.D., et al. (2012) Stem Cell Pathways Contribute to Clinical Chemoresistance in Ovarian Cancer. Clinical Cancer Research, 18, 869-881.
http://dx.doi.org/10.1158/1078-0432.CCR-11-2188 [8] Lindahl, R. (1992) Aldehyde Dehydrogenases and Their Role in Carcinogenesis. Critical Reviews in Biochemistry and Molecular Biology, 27, 283-335.
http://dx.doi.org/10.3109/10409239209082565 [9] Chen, D., Cui, Q.C., Yang, H. and Dou, Q.P. (2006) Disulfiram, a Clinically Used Anti-Alcoholism Drug and Copper-Binding Agent, Induces Apoptotic Cell Death in Breast Cancer Cultures and Xenografts via Inhibition of the Proteasome Activity. Cancer Research, 66, 10425-10433.
http://dx.doi.org/10.1158/0008-5472.CAN-06-2126 [10] Zhang, H., Chen, D., Ringler, J., Chen, W., Cui, Q.C., Ethier, S.P., et al. (2010) Disulfiram Treatment Facilitates Phosphoinositide 3-Kinase Inhibition in Human Breast Cancer Cells in Vitro and in Vivo. Cancer Research, 70, 3996-4004.
http://dx.doi.org/10.1158/0008-5472.CAN-09-3752 [11] Yip, N.C., Fombon, I.S., Liu, P., Brown, S., Kannappan, V., Armesilla, A.L., et al. (2011) Disulfiram Modulated ROS-MAPK and NF-κB Pathways and Targeted Breast Cancer Cells with Cancer Stem Cell-Like Properties. British Journal of Cancer, 104, 1564-1574.
http://dx.doi.org/10.1038/bjc.2011.126 [12] Wang, W., McLeod, H.L. and Cassidy, J. (2003) Disulfiram-Mediated Inhibition of NF-κB Activity Enhances Cytotoxicity of 5-Fluorouracil in Human Colorectal Cancer Cell Lines. International Journal of Cancer, 104, 504-511.
http://dx.doi.org/10.1002/ijc.10972 [13] Lin, J., Haffner, M.C., Zhang, Y., Lee, B.H., Brennen, W.N., Britton, J., et al. (2011) Disulfiram Is a DNA Demethylating Agent and Inhibits Prostate Cancer Cell Growth. Prostate, 71, 333-343.
http://dx.doi.org/10.1002/pros.21247 [14] Morrison, B.W., Doudican, N.A., Patel, K.R. and Orlow, S.J. (2010) Disulfiram Induces Copper-Dependent Stimulation of Reactive Oxygen Species and Activation of the Extrinsic Apoptotic Pathway in Melanoma. Melanoma Research, 20, 11-20.
http://dx.doi.org/10.1097/CMR.0b013e328334131d [15] Raha, D., Wilson, T.R., Peng, J., Peterson, D., Yue, P., Evangelista, M., et al. (2014) The Cancer Stem Cell Marker Aldehyde Dehydrogenase Is Required to Maintain a Drug-Tolerant Tumor Cell Subpopulation. Cancer Research, 74, 3579-3590.
http://dx.doi.org/10.1158/0008-5472.CAN-13-3456 [16] Coffman, L., Mooney, C., Lim, J., Bai, S., Silva, I., Gong, Y., et al. (2013) Endothelin Receptor-A Is Required for the Recruitment of Antitumor T Cells and Modulates Chemotherapy Induction of Cancer Stem Cells. Cancer Biology & Therapy, 14, 184-192.
http://dx.doi.org/10.4161/cbt.22959 [17] Vaccari, A., Ferraro, L., Saba, P., Ruiu, S., Mocci, I., Antonelli, T., et al. (1998) Differential Mechanisms in the Effects of Disulfiram and Diethyldithiocarbamate Intoxication on Striatal Release and Vesicular Transport of Glutamate. Journal of Pharmacology and Experimental Therapeutics, 285, 961-967. [18] Yourick, J.J. and Faiman, M.D. (1991) Disulfiram Metabolism as a Requirement for the Inhibition of Rat Liver Mitochondrial Low Km Aldehyde Dehydrogenase. Biochemical Pharmacology, 42, 1361-1366.
http://dx.doi.org/10.1016/0006-2952(91)90446-C [19] Hart, B.W. and Faiman, M.D. (1992) In Vitro and in Vivo Inhibition of Rat Liver Aldehyde Dehydrogenase by S-Methyl N, N-Diethylthiolcarbamate Sulfoxide, a New Metabolite of Disulfiram. Biochemical Pharmacology, 43, 403-406.
http://dx.doi.org/10.1016/0006-2952(92)90555-W [20] Madan, A. and Faiman, M.D. (1994) Diethyldithiocarbamate Methyl Ester Sulfoxide, an Inhibitor of Rat Liver Mitochondrial Low Km Aldehyde Dehydrogenase and Putative Metabolite of Disulfiram. Alcoholism: Clinical and Experimental Research, 18, 1013-1017.
http://dx.doi.org/10.1111/j.1530-0277.1994.tb00075.x [21] Lipsky, J.J., Shen, M.L. and Naylor, S. (2001) In Vivo Inhibition of Aldehyde Dehydrogenase by Disulfiram. Chemico-Biological Interactions, 130-132, 93-102.
http://dx.doi.org/10.1016/S0009-2797(00)00225-8 [22] Vasiliou, V. and Nebert, D.W. (2005) Analysis and Update of the Human Aldehyde Dehydrogenase (ALDH) Gene Family. Human Genomics, 2, 138-143. [23] Moreb, J.S., Ucar, D., Han, S., Amory, J.K., Goldstein, A.S., Ostmark, B., et al. (2012) The Enzymatic Activity of Human Aldehyde Dehydrogenases 1A2 and 2 (ALDH1A2 and ALDH2) Is Detected by Aldefluor, Inhibited by Diethylaminobenzaldehyde and Has Significant Effects on Cell Proliferation and Drug Resistance. Chemico-Biological Interactions, 195, 52-60.
http://dx.doi.org/10.1016/j.cbi.2011.10.007 [24] Marchitti, S.A., Orlicky, D.J., Brocker, C. and Vasiliou, V. (2010) Aldehyde Dehydrogenase 3B1 (ALDH3B1): Immunohistochemical Tissue Distribution and Cellular-Specific Localization in Normal and Cancerous Human Tissues. Journal of Histochemistry and Cytochemistry, 58, 765-783.
http://dx.doi.org/10.1369/jhc.2010.955773 [25] Rosen, J.M. and Jordan, C.T. (2009) The Increasing Complexity of the Cancer Stem Cell Paradigm. Science, 324, 1670-1673.
http://dx.doi.org/10.1126/science.1171837 [26] Sládek, N.E. (2003) Human Aldehyde Dehydrogenases: Potential Pathological, Pharmacological, and Toxicological Impact. Journal of Biochemical and Molecular Toxicology, 17, 7-23.
http://dx.doi.org/10.1002/jbt.10057 [27] Moore, S.A., Baker, H.M., Blythe, T.J., Kitson, K.E., Kitson, T.M. and Baker, E.N. (1998) Sheep Liver Cytosolic Aldehyde Dehydrogenase: The Structure Reveals the Basis for the Retinal Specificity of Class 1 Aldehyde Dehydrogenases. Structure, 6, 1541-1551.
http://dx.doi.org/10.1016/S0969-2126(98)00152-X