Date Published: February 6, 2014
Publisher: Public Library of Science
Author(s): Cristina Federici, Francesco Petrucci, Stefano Caimi, Albino Cesolini, Mariantonia Logozzi, Martina Borghi, Sonia D’Ilio, Luana Lugini, Nicola Violante, Tommaso Azzarito, Costanza Majorani, Daria Brambilla, Stefano Fais, Irina V. Lebedeva.
Intrinsic resistance to cytotoxic drugs has been a main issue in cancer therapy for decades. Microenvironmental acidity is a simple while highly efficient mechanism of chemoresistance, exploited through impairment of drug delivery. The latter is achieved by extracellular protonation and/or sequestration into acidic vesicles. This study investigates the importance of extracellular acidosis and nanovesicle (exosome) release in the resistance of human tumour cell to cisplatin (CisPt); in parallel to proton pump inhibitors (PPI) ability of interfering with these tumour cell features. The results showed that CisPt uptake by human tumour cells was markedly impaired by low pH conditions. Moreover, exosomes purified from supernatants of these cell cultures contained various amounts of CisPt, which correlated to the pH conditions of the culture medium. HPLC-Q-ICP-MS analysis revealed that exosome purified from tumour cell culture supernatants contained CisPt in its native form. PPI pre-treatment increased cellular uptake of CisPt, as compared to untreated cells, in an acidic-depend manner. Furthermore, it induced a clear inhibition of exosome release by tumour cells. Human tumours obtained from xenografts pretreated with PPI contained more CisPt as compared to tumours from xenografts treated with CisPt alone. Further analysis showed that in vivo PPI treatment induced a clear reduction in the plasmatic levels of tumour-derived exosomes which also contained lower level of CisPt. Altogether, these findings point to the identification of a double mechanism that human malignant melanoma use in resisting to a dreadful cellular poison such as cisplatin. This framework of resistance includes both low pH-dependent extracellular sequestration and an exosome-mediated elimination. Both mechanisms are markedly impaired by proton pump inhibition, leading to an increased CisPt-dependent cytotoxicity.
Malignant melanoma is among the most chemoresistant tumours. Commonly used anticancer drugs do not substantially alter the prognosis of the progressive disease. Single agent or combined chemotherapies result in poor benefits for patients with malignant melanoma , . Standard treatment for metastatic melanoma based on the alkylating agent dacarbazine, frequently leads to poor outcomes , while combinations of chemotherapeutics have shown only marginally higher response rates, paying the price of systemic toxicity . Such unsatisfactory treatments highlight the urgency of implementing treatment strategies for malignant melanoma with novel, more effective and possibly less toxic approaches. Despite some mechanisms of tumour resistance to a variety of cytotoxic drugs have been proposed in pre-clinical studies  the former do not seem to have a clear role in tumour patients and this appears even more evident for tumours that are non-responsive rather than resistant to chemotherapy, such as melanoma. Cisplatin (CisPt) is an alkylating agent that binds to DNA bases causing crosslinks and breaks in DNA strands; interfering with DNA replication . An impaired uptake of CisPt appears to represent the most consistently identified feature of cells with resistance to this drug, both in vitro and in vivo, , as compared to other proposed mechanisms , . The mechanism by which CisPt enters into the cells is unknown, but earlier evidence suggested that CisPt enters relatively slowly as compared to most anticancer drugs, whereas in turn CisPt efflux occurs rapidly .
Melanoma is by far one of the most chemoresistant malignant tumours, showing an intrinsic resistance to Cisplatin as well. Between the mechanisms shown to have a role in resistance of cancer cells to Cisplatin there are increased efflux, or increased inactivation by sulfhydryl molecules, such as glutathione; altered expression of proteins in signal transduction pathways that control apoptosis; increased DNA repair . However, while without a clear molecular targetting, microenvironmental low pH appears to exert a major role in resistance to chemotherapy, proliferation and metastatic behavior of malignant tumours , , . In fact, the extracellular pH of normal tissue is neutral, the interstitial pH of tumour is acidic and the tumour cells have developed the capacity of surviving in hypoxic-acidic environment, condition not permissive to the normal cells. This selective advantage is exploited by the tumour cells to markedly impair the uptake of weakly basic chemotherapeutic drugs and as a consequence their effect on tumours. All in all, tumour acidity does not inhibit intracellular mechanism/s related to the effectiveness of the drugs, but it hampers their entry within a cell, thus getting under ways a simple, rough but extremely efficient mechanism that makes real poisons unable to operate. However, in this study we show that the same cells use an additional mechanism of resistance, that is the elimination of chemotherapeutics through extracellularly released nanovesicles, called exosomes. The two phenomena are linked in a complementary way, inasmuch as low pH increases the exosome release by tumour cells. In a previous study , we have extensively investigated the level of pH dependent resistance of various human tumour cell lines against different chemotherapeutics, including CisPt. In this study we wanted to obtain more mechanistic insights of CisPt tumour resistance in extreme experimental settings, such as the ability to grow in very acidic condition and high level of exosome release. In fact, this study has shown that in melanoma cells cultured at different low pH conditions (i.e. 6.0 and 5.0), there was an impaired CisPt uptake by tumour cells as compared to melanoma cells cultured at the physiological pH of 7.4. We further confirmed this result culturing melanoma cells in unbuffered condition, leading to a marked lowering of the medium pH (round 0.4 units, data not shown) . In this more “natural” acidic condition we had again a reduced CisPt uptake in melanoma cells, further supporting a clear role of the acidic microenvironment in chemoresistance.