Human fibrosarcoma cells selected for ultra-high doxorubicin resistance, acquire trabectedin cross-resistance, remain sensitive to recombinant methioninase, and have increased c-MYC expression

Sei Morinaga^1*Qinghong Han²Kohei Mizuta²Byung Mo Kang²Michael Bouvet³Norio Yamamoto¹Katsuhiro Hayashi¹Hiroaki Kimura¹Shinji Miwa¹Kentaro Igarashi¹Takashi Higuchi¹Hiroyuki Tsuchiya¹Satoru Demura¹Robert M Hoffman^2*

• ¹Department of Orthopaedic Surgery, Graduate School of Medical Science Kanazawa University, Kanazawa, Japan
• ²AntiCancer Inc., San DIego, United States
• ³University of California San Diego, La Jolla, United States

Background: Doxorubicin is standard first-line chemotherapy for soft-tissue sarcoma (STS), yet the emergence of resistance severely limits its clinical efficacy. Developing novel strategies to overcome resistance are critical for improving soft-tissue sarcoma patient outcomes. Methods: An ultra-high doxorubicin-resistant (UHDR) HT1080 fibrosarcoma cell line was established through stepwise exposure to increasing doxorubicin concentrations over 5-months. Over the course of the five months, HT1080 cells were cultured in doxorubicin concentrations that increased stepwise from 8 nM to 15 µM, a 1875-fold increase. Cell viability assays for HT1080 and UHDR were performed using the WST-8 cell-viability agent. c-MYC expression was analyzed by Western blotting. Results: UHDR-HT1080 cells exhibited an 11.6-fold increase in doxorubicin resistance compared with parental HT1080 cells and displayed selective cross-resistance to trabectedin (8.9-fold), while remaining sensitive to rMETase. rMETase synergistically enhanced doxorubicin efficacy in UHDR cells. Western blotting demonstrated an 8.4-fold elevation in c-MYC expression in UHDR-HT1080 cells. Conclusion: The findings indicate that rMETase can overcome ultra-high doxorubicin resistance in fibrosarcoma cells, likely through targeting methionine addiction, a universal metabolic vulnerability of cancer. These results support the potential clinical application of methionine restriction therapy to treat doxorubicin-resistant STS.