Reduced argininosuccinate synthetase expression in refractory sarcomas: Impacts on therapeutic potential and drug resistance
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Youngji Kim1,2,3,5, Eisuke Kobayashi1, Daisuke Kubota1,3, Yoshiyuki Suehara3, Kenta Mukaihara3, Keisuke Akaike3, Ayumu Ito4,5, Kazuo Kaneko3, Hirokazu Chuman1, Akira Kawai1, Shigehisa Kitano2,5
1Division of Musculoskeletal Oncology, National Cancer Center Hospital, Tokyo, Japan
2Department of Experimental Therapeutics, National Cancer Center Hospital, Tokyo, Japan
3Department of Orthopaedic Surgery, Juntendo University School of Medicine, Tokyo, Japan
4Department of Hematopoietic Stem Cell Transplantation, National Cancer Center Hospital, Tokyo, Japan
5Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Tokyo, Japan
Eisuke Kobayashi, email: [email protected]
Keywords: argininosuccinate synthetase, bone and soft-tissue sarcoma, drug-resistance, metabolic error, P-glycoprotein
Received: May 23, 2016 Accepted: September 15, 2016 Published: September 23, 2016
Background: Treating drug-resistant sarcomas remains a major challenge. The present study aimed to identify a novel therapy for drug-resistant sarcomas based on metabolic errors involving argininosuccinate synthetase1 (ASS1).
Results: ASS1 expression was reduced in Dox-resistant sarcoma cells. Immunohistochemistry and real-time PCR showed an inverse correlation between ASS1 and P-gp expressions. The inhibition of cellular proliferation with G1-arrest was shown to lead to autophagy with arginine deprivation. In addition, the combination of an autophagy inhibitor plus arginine deprivation was more effective than arginine deprivation alone. In cells with suppressed ASS1 expression, P-gp expression was upregulated as compared to that in negative controls.
Discussion: These results indicate that the reduced ASS1 expression in Dox-resistant sarcomas may contribute to drug resistance in association with the expression of P-gp. ASS1 deficiency is a potential target for novel drug therapies. The combination of arginine-deprivation therapy and an autophagy inhibitor may have anti-tumor effects in refractory sarcomas.
Methods: We assessed the expressions of ASS1 and P-glycoprotein (P-gp) in clinical specimens and cell lines of osteosarcoma (KHOS), doxorubicin (Dox)-resistant osteosarcoma (KHOSR2), epithelioid sarcomas (ES-X and VAESBJ) and alveolar soft part sarcoma (ASPS-KY). Each cell line was cultured in arginine-containing and arginine-free media. Cell growth was assessed using an XTT assay and flow cytometry. We analyzed the induction of autophagy in arginine-free medium. Moreover, we assessed the expression of P-gp after suppressing ASS1 in Dox-sensitive cells (MCF-7 and KHOS) and after transfecting ASS1 into Dox-resistant cells (ES-X, VAESBJ, ASPS-KY and KHOSR2).
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