ALDH1A1 Gene Expression and Cellular Copper Levels between Low and Highly Metastatic Osteosarcoma Provide a Case for Novel Repurposing with Disulfiram and Copper

Abstract

Aldehyde dehydrogenase 1A1 (ALDH) is a cancer stem cell marker highly expressed in metastatic cells. Disulfiram (Dis) is an FDA-approved antialcoholism drug that inhibits ALDH and has been studied as a candidate for drug repurposing in multiple neoplasia. Dis cytotoxicity in cancer cells has been shown to be copper-dependent, in part due to Dis’s ability to function as a bivalent metal ion chelator of copper (Cu). The objectives of this research were to test ALDH expression levels and Cu concentrations in sarcoma patient tumors and human osteosarcoma (OS) cell lines with differing metastatic phenotypes. We also sought to evaluate Dis + Cu combination therapy in human OS cells. Intracellular Cu was inversely proportional to the metastatic phenotype in human OS cell lines (SaOS2 > LM2 > LM7). Nonmetastatic human sarcoma tumors demonstrated increased Cu concentrations compared with metastatic tumors. qPCR demonstrated that ALDH expression was significantly increased in highly metastatic LM2 and LM7 human OS cell lines compared with low metastatic SaOS2. Tumor cells from sarcoma patients with metastatic disease displayed significantly increased ALDH expression compared with tumor cells from patients without metastatic disease. Serum Cu concentration in canine OS versus normal canine patients demonstrated similar trends. Dis demonstrated selective cytotoxicity compared with human multipotential stromal cells (MSCs): Dis-treated OS cells demonstrated increased apoptosis, whereas MSCs did not. CuCl2 combined with Dis and low-dose doxorubicin resulted in a superior cytotoxic effect in both SaOS2 and LM7 cell lines. In summary, ALDH gene expression and Cu levels are altered between low and highly metastatic human OS cells, canine samples, and patient tumors. Our findings support the feasibility of a repurposed drug strategy for Dis and Cu in combination with low-dose anthracycline to specifically target metastatic OS cells.

Antioxidant 1 copper chaperone gene expression and copper levels in dog osteosarcoma patients

“…ALDH is responsible for metabolizing aldehydes into carboxylic acids which may permit cancer cells to resist the oxidative stress imparted by chemotherapeutic agents 28–31 . We also observed that metastatic human OS tumours have reduced copper concentrations compared with non‐metastatic OS tumours 32 . Conversely, OS patients with metastases displayed increased levels of blood serum copper levels compared with patients who did not develop metastases 32 …”

“…Dog tumour, bone, and serum samples were processed and stored as described above. Copper concentrations were determined using a PerkinElmer Analyst 600 atomic absorption spectrophotometer adjusted to detect copper (324.8 nm) as previously described 32,51 . The final copper concentrations were calculated as total μg/ml.…”

“…Copper concentrations were determined using a PerkinElmer Analyst 600 atomic absorption spectrophotometer adjusted to detect copper (324.8 nm) as previously described. 32,51 The final copper concentrations were calculated as total μg/ml. Intracellular bone and tumour copper concentrations were calculated as (final sample concentration of copper [ng/mL]/(total sample protein concentration [mg/ml])) and recorded as ng copper/mg protein.…”

“…[28][29][30][31] We also observed that metastatic human OS tumours have reduced copper concentrations compared with non-metastatic OS tumours. 32 Conversely, OS patients with metastases displayed increased levels of blood serum copper levels compared with patients who did not develop metastases. 32 Copper is an essential trace nutrient metal involved in multiple cellular processes.…”

“…32 Conversely, OS patients with metastases displayed increased levels of blood serum copper levels compared with patients who did not develop metastases. 32 Copper is an essential trace nutrient metal involved in multiple cellular processes. Ingested copper is absorbed in the stomach and small intestine 33 and transported to the liver via portal circulation followed by redistribution to extrahepatic tissues.…”