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.

Figure 1

Metastatic OS cell lines and patient sarcomas display lower levels of intracellular Cu compared with nonmetastatic samples. (a) Human OS cell line triplicates (SaOS2, LM2, and LM7) were cultured under identical conditions and then collected for protein quantification and intracellular Cu analysis using atomic absorption spectrophotometry (AA). LM2 and LM7, which demonstrate increased metastatic potential, displayed significant decreases in intracellular Cu compared with parental SaOS2. (b) Deidentified patient information was used to separate sarcomas, including OS, that came from patients with metastatic disease and those with no signs of metastatic disease (n = 14). Tumors from patients with metastatic disease displayed significantly decreased Cu levels compared with tumors from nonmetastatic patients. The statistical significance of differences among the cell types was assessed using ordinary one-way ANOVA, while the difference between the two patient tumor groups was assessed using two-tailed Student's unpaired t-test. Data represent mean ± SD: ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 2

Blood plasma from humans and canines with metastatic sarcomas display significantly increased Cu levels. (a) Blood plasma samples from human sarcoma, including OS patients with metastatic disease, display significantly increased serum Cu levels compared with patients without metastatic disease (n = 24). (b) Blood plasma samples from canine OS patients display significantly increased Cu levels compared with plasma from healthy control canines (n = 6). The statistical significance difference between the two groups were assessed using two-tailed Student's unpaired t-test. Data represent mean ± SD: ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 3

Metastatic OS cell lines and patient tumors display increased ALDH expression compared with nonmetastatic samples. (a) ALDH gene expression was significantly increased in both LM2 and LM7 compared with SaOS2 cells. (b) In human patient-derived tumor cells, ALDH gene expression was significantly increased in tumor cells from patients with metastatic disease compared to patients without metastatic disease (n = 14). The statistical significance of differences among the treatment was assessed using ordinary one-way ANOVA, while the difference between metastatic and nonmetastatic was assessed using two-tailed Student's unpaired t-test. Data represent mean ± SD: ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 4

Selective cytotoxicity of Dis for metastatic OS cells compared with MSCs. After 24 h and 48 h of Dis treatment at the indicated concentrations, cytotoxicity was determined by MTT assay in LM2 cells (red bars) and MSCs (blue bars). LM2 cells were much more sensitive to the cytotoxic effects of Dis than MSCs. Dis treatment causes changes in apoptosis-related genes in OS cells but not human multipotential stromal cells (MSCs).

Figure 5

Dis treatment causes changes in apoptosis-related genes in OS cells but not human multipotential stromal cells (MSCs). After 4 h of Disulfiram (Dis) treatment at the concentration of 12.5 and 25 µM, ALDH, caspase 9, and caspase 3 expressions were checked by qRT-PCR in LM2 cells (red bars) and MSCs (blue bars). (a) Untreated LM2 cells expressed significantly greater ALDH expression compared with mesenchymal stem cells (MSCs, n = 3). (b) ALDH expression after 4 h of DIS treatment (n = 3). (c) Caspase 9 gene expression after 4 h of Dis treatment (n = 3). hPPIA was used as a housekeeping gene. The statistical significance of differences among the treatment was assessed using ordinary one-way ANOVA with Dunnett's multiple comparisons test, while the difference between the two cell types was assessed using two-tailed Student's unpaired t-test. Data represent mean ± SD: ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗∗p < 0.0001.

Figure 6

Dis treatment causes visible distress in LM2 cells but not MSCs. Representative images of both MSC and LM2 cells after 4 hours of treatment with Dis at the concentrations of 12.5 µM and 25 µM. LM2 appear smaller and display unhealthy signs like more cellular debris (blue arrows) and round dots inside which are probably apoptotic bodies (orange arrows). MSCs, even at high Dis concentration, remain healthy appearing.

Figure 7

Cu potentiated cytotoxicity of Dis reduces cell viability in both SaOS2 and highly metastatic LM7 cell lines. (a) SaOS2 (n = 6) and (b) LM7 (n = 6) cells were subjected to fold dilutions of Dox, Dis + CuCl₂, or Dis monotherapy over 24 hours. After treatment, cells were stained with PrestoBlue viability stain, and fluorescence intensity was evaluated to measure cell viability. The addition of CuCl₂ significantly decreased the cell viability in both LM7 and SaOS2 compared to monotherapy of Dis. Dis + Cu combination treatment resulted in increased cytotoxicity to LM7 compared to Dox monotherapy.

Figure 8

Combination treatment of Dox, Dis, and CuCl₂ effectively reduces viability of human OS cells and eliminates recovery in vitro. Viable cell counts of SaOS2 and LM7 were obtained using a Trypan Blue exclusion assay and a hemocytometer (colored bar graphs). After 24 h of treatment exposure, treated human OS cell lines were all recultured with fresh media without drug and monitored for recovery of cells; tan background shading indicates cells were able to grow after drug removal, and pink background shading indicates cells were unable to grow. (a) SaOS2 treated with Dox were unable to recover after a .05 µM dose, while LM7 was unable to recover after 1.2 µM. (b) SaOS2 and LM7 given Dis + CuCl₂ were both unable to recover after a 0.6 µM Dis dose. (c) LM7 given triple treatment with 0.1 μM Dis, 0.2 μM CuCl₂, and 0.1 μM Dox was unable to recover after drug removal.