Research Papers:

Altered pH gradient at the plasma membrane of osteosarcoma cells is a key mechanism of drug resistance

Sofia Avnet, Silvia Lemma, Margherita Cortini, Paola Pellegrini, Francesca Perut, Nicoletta Zini, Katsuyuki Kusuzaki, Tokuhiro Chano, Giulia Grisendi, Massimo Dominici, Angelo De Milito and Nicola Baldini _

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Oncotarget. 2016; 7:63408-63423. https://doi.org/10.18632/oncotarget.11503

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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

Correspondence to:

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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