Disulfiram reduces metastatic osteosarcoma tumor burden in an immunocompetent Balb/c or-thotopic mouse model

Jared Anthony Crasto, Mitchell Stephen Fourman, Alejandro Morales-Restrepo, Adel Mahjoub, Jonathan Brendan Mandell, Kavita Ramnath, Jessica C Tebbets, Rebecca J Watters, Kurt Richard Weiss, Jared Anthony Crasto, Mitchell Stephen Fourman, Alejandro Morales-Restrepo, Adel Mahjoub, Jonathan Brendan Mandell, Kavita Ramnath, Jessica C Tebbets, Rebecca J Watters, Kurt Richard Weiss

Abstract

Introduction: The overall survival rate of patients with osteosarcoma (OS) and pulmonary metastases has remained stagnant at 15-30% for several decades. Disulfiram (DSF) is an FDA-approved aldehyde dehydrogenase inhibitor that reduces the metastatic phenotype of OS cells in vitro. Here we evaluate its in vivo efficacy, as compared to doxorubicin chemotherapy, in a previously-validated orthotopic model of metastatic OS.

Results: All treatment groups displayed a significantly reduced quantitative OS metastatic burden compared with controls. The metastatic burden of Lo DSF-treated animals was equivalent to the DXR group. Ninety-five percent of control animals displayed evidence of metastatic disease, which was significantly greater than all treatment groups.

Discussion: Disulfiram treatment resulted in a reduced burden of OS metastatic disease compared with controls. This was statistically-equivalent to doxorubicin. No additive effect was observed between these two therapies.

Materials and methods: One-hundred twenty immunocompetent Balb/c mice received proximal tibia paraphyseal injections of 5 × 105 K7M2 murine OS cells. Therapy began three weeks after injection: saline (control), low-dose disulfiram (Lo DSF), high-dose disulfiram (Hi DSF), doxorubicin (DXR), Lo DSF + DXR, and Hi DSF + DXR. Transfemoral amputations were performed at 4 weeks. Quantitative metastatic tumor burden was measured using near-infrared indocyanine green (ICG) angiography.

Keywords: Akt; aldehyde dehydrogenase; bad; disulfiram; osteosarcoma.

Conflict of interest statement

CONFLICTS OF INTEREST The authors declare no potential conflicts of interest.

Figures

Figure 1. Quantitative Primary (left) and Metastatic…
Figure 1. Quantitative Primary (left) and Metastatic (right) Tumor Burden
All experimental groups displayed significant (p < 0.05) reductions in metastatic tumor burden compared to Saline-treated controls. Error bars depict 95% confidence interval. *signifies significant difference from Saline-treated mice (p < 0.01**p < 0.0001****) on comparison using One-Way ANOVA with Tukey’s post-test. No significant differences existed between DXR and the other treatment groups.
Figure 2. mRNA transcript expression analysis of…
Figure 2. mRNA transcript expression analysis of AKT Serine/Threonine Kinase 1 (Akt, left) and BCL2 Associated Agonist of Cell Death (Bad, right)
Fold change is compared to geometric mean of Rps17, Rpl30, and Nono expression levels of saline-treated mice primary tumor samples. Error bars depict 95% confidence interval. *signifies significant difference from Saline-treated mice (p < 0.01 **p < 0.0001****); *signifies significant difference from Doxorubicin-treated mice (p < 0.01**, p < 0.0001****) on comparison using Kruskal–Wallis analysis with Dunn’s multiple comparisons.
Figure 3. Regulation of Apoptosis in OS
Figure 3. Regulation of Apoptosis in OS
Phosphoinositide-3-Kinase (PI3K) enables AKT Serine/Threonine Kinase (Akt) to inhibit BCL2 Associated Agonist of Cell Death (Bad) and BCL2 Associated X Apoptosis Regulator (Bax), which both would otherwise facilitate apoptosis. Additionally, Phosphatase and Tensin Homolog (PTEN) inhibits PI3K’s activation of Akt. Furthermore, Akt also enables Nuclear Factor Kappa B (NF-κB) and Mechanistic Target of Rapamycin (mTOR), which both ultimately block apoptosis; mTOR does so by activating BCL2 Family Apoptosis Regulator (MCL-1).
Figure 4. Image depicting primary (left) and…
Figure 4. Image depicting primary (left) and metastatic lung (right) osteosarcoma as visualized by indocyanine green dye angiography
Quantitative measurements were performed with the SPY-Elite (Novadaq).

References

    1. Harrison DJ, Schwartz CL. Osteogenic Sarcoma: Systemic Chemotherapy Options for Localized Disease. Curr Treat Options Oncol. 2017;18:24. doi: 10.1007/s11864-017-0464-2.
    1. Luetke A, Meyers PA, Lewis I, Juergens H. Osteosarcoma treatment - where do we stand? A state of the art review. Cancer Treat Rev. 2014;40:523–32. doi: 10.1016/j.ctrv.2013.11.006.
    1. Bielack S, Carrle D, Casali PG, Group EGW Osteosarcoma: ESMO clinical recommendations for diagnosis, treatment and follow-up. Ann Oncol. 2009;20:137–9. doi: 10.1093/annonc/mdp154.
    1. Kempf-Bielack B, Bielack SS, Jurgens H, Branscheid D, Berdel WE, Exner GU, Gobel U, Helmke K, Jundt G, Kabisch H, Kevric M, Klingebiel T, Kotz R, et al. Osteosarcoma relapse after combined modality therapy: an analysis of unselected patients in the Cooperative Osteosarcoma Study Group (COSS) J Clin Oncol. 2005;23:559–68. doi: 10.1200/JCO.2005.04.063.
    1. Chi SN, Conklin LS, Qin J, Meyers PA, Huvos AG, Healey JH, Gorlick R. The patterns of relapse in osteosarcoma: the Memorial Sloan-Kettering experience. Pediatr Blood Cancer. 2004;42:46–51. doi: 10.1002/pbc.10420.
    1. Bielack SS, Kempf-Bielack B, Delling G, Exner GU, Flege S, Helmke K, Kotz R, Salzer-Kuntschik M, Werner M, Winkelmann W, Zoubek A, Jurgens H, Winkler K. Prognostic factors in high-grade osteosarcoma of the extremities or trunk: an analysis of 1,702 patients treated on neoadjuvant cooperative osteosarcoma study group protocols. J Clin Oncol. 2002;20:776–90. doi: 10.1200/JCO.2002.20.3.776.
    1. Abdullah LN, Chow EK. Mechanisms of chemoresistance in cancer stem cells. Clin Transl Med. 2013;2:3. doi: 10.1186/2001-1326-2-3.
    1. Khan IN, Al-Karim S, Bora RS, Chaudhary AG, Saini KS. Cancer stem cells: a challenging paradigm for designing targeted drug therapies. Drug Discov Today. 2015;20:1205–16. doi: 10.1016/j.drudis.2015.06.013.
    1. Clark DW, Palle K. Aldehyde dehydrogenases in cancer stem cells: potential as therapeutic targets. Ann Transl Med. 2016;4:518. doi: 10.21037/atm.2016.11.82.
    1. Greco N, Schott T, Mu X, Rothenberg A, Voigt C, McGough RL, 3rd, Goodman M, Huard J, Weiss KR. ALDH Activity Correlates with Metastatic Potential in Primary Sarcomas of Bone. J Cancer Ther. 2014;5:331–8. doi: 10.4236/jct.2014.54040.
    1. Jiao Y, Hannafon BN, Ding WQ. Disulfiram’s Anticancer Activity: Evidence and Mechanisms. Anticancer Agents Med Chem. 2016;16:1378–84.
    1. Cho HJ, Lee TS, Park JB, Park KK, Choe JY, Sin DI, Park YY, Moon YS, Lee KG, Yeo JH, Han SM, Cho YS, Choi MR, et al. Disulfiram suppresses invasive ability of osteosarcoma cells via the inhibition of MMP-2 and MMP-9 expression. J Biochem Mol Biol. 2007;40:1069–76.
    1. Mu X, Isaac C, Schott T, Huard J, Weiss K. Rapamycin Inhibits ALDH Activity, Resistance to Oxidative Stress, and Metastatic Potential in Murine Osteosarcoma Cells. Sarcoma. 2013;2013:480713. doi: 10.1155/2013/480713.
    1. Fourman MS, Mahjoub A, Mandell JB, Yu S, Tebbets JC, Crasto JA, Alexander PE, Weiss KR. Quantitative Primary Tumor Indocyanine Green Measurements Predict Osteosarcoma Metastatic Lung Burden in a Mouse Model. Clin Orthop Relat Res. 2018;476:479–87. doi: 10.1007/s11999.0000000000000003.
    1. Fourman MS, Gersch RP, Levites HA, Phillips BT, Bui DT. Is There a Right Way to Interpret SPY? Normalization of Indocyanine Green Angiography Readings in a Burn Model. Plast Reconstr Surg. 2015;136:128e–30e. doi: 10.1097/PRS.0000000000001380.
    1. Yang Y, Han L, He Z, Li X, Yang S, Yang J, Zhang Y, Li D, Yang Y, Yang Z. Advances in limb salvage treatment of osteosarcoma. J Bone Oncol. 2018;10:36–40. doi: 10.1016/j.jbo.2017.11.005.
    1. Lipshultz SE, Franco VI, Miller TL, Colan SD, Sallan SE. Cardiovascular disease in adult survivors of childhood cancer. Annu Rev Med. 2015;66:161–76. doi: 10.1146/annurev-med-070213-054849.
    1. Cappetta D, Rossi F, Piegari E, Quaini F, Berrino L, Urbanek K, De Angelis A. Doxorubicin targets multiple players: A new view of an old problem. Pharmacol Res. 2018;127:4–14. doi: 10.1016/j.phrs.2017.03.016.
    1. Hamilton SN, Carlson R, Hasan H, Rassekh SR, Goddard K. Long-term Outcomes and Complications in Pediatric Ewing Sarcoma. Am J Clin Oncol. 2017;40:423–428. doi: 10.1097/COC.0000000000000176.
    1. Oeffinger KC, Mertens AC, Sklar CA, Kawashima T, Hudson MM, Meadows AT, Friedman DL, Marina N, Hobbie W, Kadan-Lottick NS, Schwartz CL, Leisenring W, Robison LL, et al. Chronic health conditions in adult survivors of childhood cancer. N Engl J Med. 2006;355:1572–82. doi: 10.1056/NEJMsa060185.
    1. Sirimangkalakitti N, Chamni S, Suwanborirux K, Chanvorachote P. Renieramycin M Attenuates Cancer Stem Cell-like Phenotypes in H460 Lung Cancer Cells. Anticancer Res. 2017;37:615–21. doi: 10.21873/anticanres.11355.
    1. Shima H, Yamada A, Ishikawa T, Endo I. Are breast cancer stem cells the key to resolving clinical issues in breast cancer therapy. Gland Surg. 2017;6:82–8. doi: 10.21037/gs.2016.08.03.
    1. Yu L, Zhu B, Wu S, Zhou L, Song W, Gong X, Wang D. Evaluation of the correlation of vasculogenic mimicry, ALDH1, KiSS-1, and MACC1 in the prediction of metastasis and prognosis in ovarian carcinoma. Diagn Pathol. 2017;12:23. doi: 10.1186/s13000-017-0612-9.
    1. Chen X, Li Q, Liu X, Liu C, Liu R, Rycaj K, Zhang D, Liu B, Jeter C, Calhoun-Davis T, Lin K, Lu Y, Chao HP, et al. Defining a Population of Stem-like Human Prostate Cancer Cells That Can Generate and Propagate Castration-Resistant Prostate Cancer. Clin Cancer Res. 2016;22:4505–16. doi: 10.1158/1078-0432.ccr-15-2956.
    1. Li W, Liu J, Zou D, Cai X, Wang J, Wang J, Zhu L, Zhao L, Ou R, Xu Y. Exploration of bladder cancer molecular mechanisms based on miRNA-mRNA regulatory network. Oncol Rep. 2017;37:1461–8. doi: 10.3892/or.2017.5433.
    1. Shiozaki A, Kudou M, Ichikawa D, Fujiwara H, Shimizu H, Ishimoto T, Arita T, Kosuga T, Konishi H, Komatsu S, Okamoto K, Marunaka Y, Otsuji E. Esophageal cancer stem cells are suppressed by tranilast, a TRPV2 channel inhibitor. J Gastroenterol. 2018;53:197–207. doi: 10.1007/s00535-017-1338-x.
    1. Petrachi T, Romagnani A, Albini A, Longo C, Argenziano G, Grisendi G, Dominici M, Ciarrocchi A, Dallaglio K. Therapeutic potential of the metabolic modulator phenformin in targeting the stem cell compartment in melanoma. Oncotarget. 2017;8:6914–28. doi: 10.18632/oncotarget.14321.
    1. Gasparetto M, Pei S, Minhajuddin M, Khan N, Pollyea DA, Myers JR, Ashton JM, Becker MW, Vasiliou V, Humphries KR, Jordan CT, Smith CA. Targeted therapy for a subset of acute myeloid leukemias that lack expression of aldehyde dehydrogenase 1A1. Haematologica. 2017;102:1054–65. doi: 10.3324/haematol.2016.159053.
    1. Mutschler J, Grosshans M, Soyka M, Rosner S. Current Findings and Mechanisms of Action of Disulfiram in the Treatment of Alcohol Dependence. Pharmacopsychiatry. 2016;49:137–41. doi: 10.1055/s-0042-103592.
    1. Leggio L, Lee MR. Treatment of Alcohol Use Disorder in Patients with Alcoholic Liver Disease. Am J Med. 2017;130:124–34. doi: 10.1016/j.amjmed.2016.10.004.
    1. Xiao Y, Chen D, Zhang X, Cui Q, Fan Y, Bi C, Dou QP. Molecular study on copper-mediated tumor proteasome inhibition and cell death. Int J Oncol. 2010;37:81–7.
    1. Tawari PE, Wang Z, Najlah M, Tsang CW, Kannappan V, Liu P, McConville C, He B, Armesilla AL, Wang W. The cytotoxic mechanisms of disulfiram and copper(ii) in cancer cells. Toxicol Res (Camb) 2015;4:1439–42. doi: 10.1039/c5tx00210a.
    1. Huang J, Campian JL, Gujar AD, Tran DD, Lockhart AC, DeWees TA, Tsien CI, Kim AH. A phase I study to repurpose disulfiram in combination with temozolomide to treat newly diagnosed glioblastoma after chemoradiotherapy. J Neurooncol. 2016;128:259–66. doi: 10.1007/s11060-016-2104-2.
    1. Li X, Lu Y, Liang K, Liu B, Fan Z. Differential responses to doxorubicin-induced phosphorylation and activation of Akt in human breast cancer cells. Breast Cancer Research. 2005;7:R589–R97. doi: 10.1186/bcr1259.
    1. Piguet AC, Semela D, Keogh A, Wilkens L, Stroka D, Stoupis C, St-Pierre MV, Dufour JF. Inhibition of mTOR in combination with doxorubicin in an experimental model of hepatocellular carcinoma. J Hepatol. 2008;49:78–87. doi: 10.1016/j.jhep.2008.03.024.
    1. West KA, Sianna Castillo S, Dennis PA. Activation of the PI3K/Akt pathway and chemotherapeutic resistance. Drug Resistance Updates. 2002;5:234–48. doi: 10.1016/S1368-7646(02)00120-6.
    1. Meng F, Liu L, Chin PC, D’Mello SR. Akt is a downstream target of NF-kappa B. J Biol Chem. 2002;277:29674–80. doi: 10.1074/jbc.M112464200.
    1. Yip NC, Fombon IS, Liu P, Brown S, Kannappan V, Armesilla AL, Xu B, Cassidy J, Darling JL, Wang W. Disulfiram modulated ROS-MAPK and NFkappaB pathways and targeted breast cancer cells with cancer stem cell-like properties. Br J Cancer. 2011;104:1564–74. doi: 10.1038/bjc.2011.126.
    1. Li Y, Fu SY, Wang LH, Wang FY, Wang NN, Cao Q, Wang YT, Yang JY, Wu CF. Copper improves the anti-angiogenic activity of disulfiram through the EGFR/Src/VEGF pathway in gliomas. Cancer Lett. 2015;369:86–96. doi: 10.1016/j.canlet.2015.07.029.
    1. Tardito S, Bassanetti I, Bignardi C, Elviri L, Tegoni M, Mucchino C, Bussolati O, Franchi-Gazzola R, Marchio L. Copper binding agents acting as copper ionophores lead to caspase inhibition and paraptotic cell death in human cancer cells. J Am Chem Soc. 2011;133:6235–42. doi: 10.1021/ja109413c.
    1. Liu P, Brown S, Goktug T, Channathodiyil P, Kannappan V, Hugnot JP, Guichet PO, Bian X, Armesilla AL, Darling JL, Wang W. Cytotoxic effect of disulfiram/copper on human glioblastoma cell lines and ALDH-positive cancer-stem-like cells. British Journal of Cancer. 2012;107:1488–97. doi: 10.1038/bjc.2012.442.
    1. Matak P, Zumerle S, Mastrogiannaki M, El Balkhi S, Delga S, Mathieu JR, Canonne-Hergaux F, Poupon J, Sharp PA, Vaulont S, Peyssonnaux C. Copper deficiency leads to anemia, duodenal hypoxia, upregulation of HIF-2alpha and altered expression of iron absorption genes in mice. PLoS One. 2013;8:e59538. doi: 10.1371/journal.pone.0059538.
    1. Prohaska JR, Lukasewycz OA. Immunological Consequences of Copper Deficiency in Mice. In: Sorenson JRJ, editor. Inflammatory Diseases and Copper: The Metabolic and Therapeutic Roles of Copper and Other Essential Metalloelements in Humans. Humana Press. 1982. p. 599.
    1. Jiang JX, Keating JJ, Jesus EM, Judy RP, Madajewski B, Venegas O, Okusanya OT, Singhal S. Optimization of the enhanced permeability and retention effect for near-infrared imaging of solid tumors with indocyanine green. Am J Nucl Med Mol Imaging. 2015;5:390–400.
    1. Yardeni T, Eckhaus M, Morris HD, Huizing M, Hoogstraten-Miller S. Retro-orbital injections in mice. Lab animal. 2011;40:155–60. doi: 10.1038/laban0511-155.
    1. Cole HA, Ichikawa J, Colvin DC, O'Rear L, Schoenecker JG. Quantifying intra-osseous growth of osteosarcoma in a murine model with radiographic analysis. J Orthop Res. 2011;29:1957–62. doi: 10.1002/jor.21474.
    1. Khanna C, Prehn J, Yeung C, Caylor J, Tsokos M, Helman L. An orthotopic model of murine osteosarcoma with clonally related variants differing in pulmonary metastatic potential. Clin Exp Metastasis. 2000;18:261–71.
    1. Khanna C, Khan J, Nguyen P, Prehn J, Caylor J, Yeung C, Trepel J, Meltzer P, Helman L. Metastasis-associated differences in gene expression in a murine model of osteosarcoma. Cancer Res. 2001;61:3750–9.
    1. Mu X, Isaac C, Greco N, Huard J, Weiss K. Notch Signaling is Associated with ALDH Activity and an Aggressive Metastatic Phenotype in Murine Osteosarcoma Cells. Front Oncol. 2013;3:143. doi: 10.3389/fonc.2013.00143.

Source: PubMed

Sponsors en medewerkers

Medische omstandigheden

Geneesmiddelinterventies

3
Abonneren