Altered pH gradient at the plasma membrane of osteosarcoma cells is a key mechanism of drug resistance
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Sofia Avnet1, Silvia Lemma1, Margherita Cortini1, Paola Pellegrini2, Francesca Perut1, Nicoletta Zini3,4, Katsuyuki Kusuzaki5, Tokuhiro Chano6, Giulia Grisendi7, Massimo Dominici7, Angelo De Milito2, Nicola Baldini1,8
1Orthopaedic Pathophysiology and Regenerative Medicine Unit, Istituto Ortopedico Rizzoli, Bologna, Italy
2Department of Oncology-Pathology, Cancer Center Karolinska, Karolinska Institute, Stockholm, Sweden
3CNR - National Research Council of Italy, Institute of Molecular Genetics, Bologna, Italy
4Laboratory of Musculoskeletal Cell Biology, Istituto Ortopedico Rizzoli, Bologna, Italy
5Musculoskeletal Oncology Unit, Takai Hospital, Nara, Japan
6Department of Clinical Laboratory Medicine, Shiga University of Medical Science, Otsu, Shiga, Japan
7Department of Medical and Surgical Sciences for Children and Adults, University-hospital of Modena e Reggio Emilia, Modena, Italy
8Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy
Nicola Baldini, email: [email protected]
Keywords: osteosarcoma, doxorubicin, drug resistance, plasma membrane pH gradient, tumor microenvironment
Received: April 19, 2016 Accepted: August 10, 2016 Published: August 22, 2016
Current therapy of osteosarcoma (OS), the most common primary bone malignancy, is based on a combination of surgery and chemotherapy. Multidrug resistance mediated by P-glycoprotein (P-gp) overexpression has been previously associated with treatment failure and progression of OS, although other mechanisms may also play a role. We considered the typical acidic extracellular pH (pHe) of sarcomas, and found that doxorubicin (DXR) cytotoxicity is reduced in P-gp negative OS cells cultured at pHe 6.5 compared to standard 7.4. Short-time (24–48 hours) exposure to low pHe significantly increased the number and acidity of lysosomes, and the combination of DXR with omeprazole, a proton pump inhibitor targeting lysosomal acidity, significantly enhanced DXR cytotoxicity. In OS xenografts, the combination treatment of DXR and omeprazole significantly reduced tumor volume and body weight loss. The impaired toxicity of DXR at low pHe was not associated with increased autophagy or lysosomal acidification, but rather, as shown by SNARF staining, with a reversal of the pH gradient at the plasma membrane (ΔpHcm), eventually leading to a reduced DXR intracellular accumulation. Finally, the reversal of ΔpHcm in OS cells promoted resistance not only to DXR, but also to cisplatin and methotrexate, and, to a lesser extent, to vincristine. Altogether, our findings show that, in OS cells, short-term acidosis induces resistance to different chemotherapeutic drugs by a reversal of ΔpHcm, suggesting that buffer therapies or regimens including proton pump inhibitors in combination to low concentrations of conventional anticancer agents may offer novel solutions to overcome drug resistance.
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