(W1130-04-27) The Effect of MO-OH-Nap Tropolone on Osteosarcoma Cellular Metabolism

Purpose: Osteosarcoma (OS) is the most common malignant form of bone cancer. OS primarily occurs in children and teens with an overall 5-year survival rate of ~70% [1]. Metastatic disease is the most important prognostic factor. Current treatment strategy includes a combination of surgery and chemotherapy[2]. Through previous studies, it has been observed that malignant and metastatic OS cells have an overall increase in the metabolic flux compared to non-metastatic OS cells. Furthermore, there is an increase in the expression of proteins associated with central carbon metabolism (glycolysis and TCA cycle) in malignant and metastatic OS cells[3]. Tropolones are naturally occurring seven-membered aromatic compounds that have been shown to have anti-bacterial, anti-fungal and anti-cancer properties[4, 5]. Currently, there is an increased interest in tropolone alkaloids especially in the field of cancer biology due to the wide ranged pharmacological activity of tropolones. Studies from our collaborators have shown that MO-OH-Nap tropolone has been shown to induce apoptosis in cancer cells through induction of cleaved caspases[6]. MO-OH-Nap Tropolone (MO-OH-Nap) is an a-substituted novel small molecule that belongs to the naturally occurring tropolone family. Our preliminary studies have revealed that MO-OH-Nap induces concentration-dependent cytotoxic effect in human OS cell lines[7]. However, the exact mechanism by which MO-OH-Nap exerts its anti-cancer effects have not yet been fully elucidated. This project aims to discover the role of MO-OH-Nap in OS cellular metabolism using LC-MS/MS and targeted metabolomics. Through this study, we can understand the metabolic changes, differentially expressed metabolites, and altered metabolic pathways that are associated with MO-OH-Nap treatment in OS and in turn find novel therapies to sensitize OS.

Methods: Two human OS cells lines., 143B and SaOS, were utilized for all experiments. 24 hours after plating, cells were treated with 10 µM MO-OH-Nap and control cells treated with DMSO alone (three biological replicates). 48 hours post MO-OH-Nap treatment, the cells were harvested for metabolite isolation in 80% methanol. Scraped cell samples were precipitated and centrifuged followed by drying the supernatants using nitrogen gas. Dried extracts were resuspended followed by running the samples on Shimadzu 8060 NX (Shimadzu Scientific Inc., Columbia, MD, USA) using previously developed and validated LC-MS/MS assays that accounts for ~200 metabolites. Method validation followed FDA’s guidance for industry – Bioanalytical method validation [May 2018]. Metabolite concentrations and peak areas were integrated using Lab Solutions software and normalized to the respective cell counts. The normalized values were further analyzed using Metaboanalyst’s 5.0 (www.metaboanalyst.ca>). Student’s t-test (p < 0.05 was considered significant) was utilized to compare the differences between the control and treatment groups.

Results: MO-OH-Nap treatment led to distinct metabolic alterations that were shown in the principal component analysis (PCA) plot (Fig.1). A total of 57 metabolites (out of ~200) showed statistically significant changes upon treatment with MO-OH-Nap (Fig.2). Differentially altered metabolites were used to perform pathway analysis which predicted pyrimidine metabolism and purine metabolism as the top altered pathways (Fig.3). Data shown for 143B cell line. Similar results were observed for SaOS cell line.

Conclusion: Distinct alterations of the metabolome were observed upon treatment with MO-OH-Nap. Following MO-OH-Nap treatment we observed downregulation of key metabolites. Through the pathway analysis plot, it is observed that MO-OH-Nap treatment targets pyrimidine metabolism and could thus lead to reduced cancer cell survival. Numerous studies have shown that pyrimidine metabolism is highly upregulated across different cancers[8]. Our study is the first to show that MO-OH-Nap alters pyrimidine metabolism. Through this study, we have identified a previously unknown mechanism by which MO-OH-Nap exerts its anti-cancer effects in osteosarcoma.

References: 1. Anderson, M.E., Update on Survival in Osteosarcoma. Orthop Clin North Am, 2016. 47(1): p. 283-92.
2. Rothzerg, E., A.L. Pfaff, and S. Koks, Innovative approaches for treatment of osteosarcoma. Exp Biol Med (Maywood), 2022. 247(4): p. 310-316.
3. Fritsche-Guenther, R., et al., Progression-Dependent Altered Metabolism in Osteosarcoma Resulting in Different Nutrient Source Dependencies. Cancers (Basel), 2020. 12(6).
4. Budihas, S.R., et al., Selective inhibition of HIV-1 reverse transcriptase-associated ribonuclease H activity by hydroxylated tropolones. Nucleic Acids Res, 2005. 33(4): p. 1249-56.
5. Trust, T.J., Antibacterial activity of tropolone. Antimicrob Agents Chemother, 1975. 7(5): p. 500-6.
6. Haney, S.L., et al., Novel tropolones induce the unfolded protein response pathway and apoptosis in multiple myeloma cells. Oncotarget, 2017. 8(44): p. 76085-76098.
7. Haney, S.L., et al., Tropolone-induced effects on the unfolded protein response pathway and apoptosis in multiple myeloma cells are dependent on iron. Leuk Res, 2019. 77: p. 17-27.
8. Mullen, N.J. and P.K. Singh, Nucleotide metabolism: a pan-cancer metabolic dependency. Nat Rev Cancer, 2023. 23(5): p. 275-294.

Acknowledgements: The authors declare no conflict of interest. This work was partially supported by Hyundai Hope on Wheels Young Investigator Grant(JBF)

Figure.1 Principal Component Analysis (PCA) plot showing the distinct metabolic alterations upon treatment with 10 UM MO-OH-Nap. Red circles indicate MO-OH-Nap treated cells and green circles indicate DMSO treated controls (n=3)

Figure.2 Heat map representing the differentially altered metabolites upon treatment with 10 UM MO-OH-Nap. Red class indicate MO-OH-Nap treated cells and green class indicate DMSO treated controls (n=3)

Figure.3 Pathway enrichment analysis for human KEGG pathways shows pyrimidine and purine metabolism as the top altered pathways upon 10 UM MO-OH-Nap treatment.