Zoledronic Acid Restores Doxorubicin Chemosensitivity and Immunogenic Cell Death in Multidrug-Resistant Human Cancer Cells

I Campia1, J Kopecka1, B Castella2, M Rigoni2, M Coscia2,3, A Bosia1,2, D Ghigo1,2, M Massaia2,3, C Riganti1,2. 1Department of Genetics, Biology and Biochemistry, University of Torino, 10126 Torino, Italy, 2Research Center on Experimental Medicine (Ce.R.M.S.), University of Torino, 10126 Torino, Italy, 3Division of Hematology, University of Torino, 10126 Torino, Italy

The mevalonate (Mev) pathway is a conserved metabolic cascade which mediates the synthesis of sterols, such as cholesterol, and non-sterol isoprenoids, such as farnesyl pyrophosphate (FPP). The over-expression of Mev pathway enzymes correlates to poor clinical outcome in several human cancers [1]. High intracellular cholesterol levels and activity of isoprenylated G-proteins promote resistance to chemotherapy [2, 3]. These data suggest Mev pathway as an attractive target to counteract chemoresistance in cancer cells. The aminobisphosphonate zoledronic acid (ZA) specifically inhibits the Mev pathway enzyme FPP synthase. The potential role of ZA as chemoresistance revertant has not been investigated.

To address this issue, we used a panel of human drug-sensitive (HT29, A549, MCF-7) and drug-resistant cancer cells, with acquired resistant phenotype (HT29-dx, A549-dx, MCF-7-DX) or constitutive resistant phenotype (HMM, HepG2, HP06), and samples of patients with chemosensitive (28) and chemoresistant (20) chronic lymphocytic leukemia.

Compared with drug-sensitive cells, all the drug-resistant cells had higher expression of mdr1 gene (p < 0.002), which encodes for P-glycoprotein (Pgp), higher activity of Mev pathway (p<0.002), increased levels of Ras/Rho isoprenylated proteins, enhanced activation of Ras/ERKs- and RhoA/RhoA kinase pathways. These events culminated in the Hypoxia-Inducible-Factor-1alpha (HIF-1alpha)-driven expression of Pgp, in the lower intracellular accumulation of drugs substrates of Pgp, like doxorubicin, in the reduced drug cytotoxicity.

In drug-resistant cells, the treatment with ZA (1 µM for 48h) lowered the rate of Mev pathway (p<0.005), disrupted Ras/RhoA-dependent downstream pathways, reduced the HIF-1alpha-dependent expression of Pgp, increased the doxorubicin intracellular retention (p < 0.005) and cytotoxicity (p < 0.05). Of note, doxorubicin also induces an immunogenic cell death in tumour cells, by increasing the surface exposure of the protein calreticulin, that allows the subsequent phagocytosis by dendritic cells [4]. This response was present in drug-sensitive cells, absent in all the drug-resistant cells analyzed. ZA restored the doxorubicin-induced translocation of calreticulin, the tumour cells phagocytosis by dendritic cells and the subsequent recruitment of specific anti-tumor cytotoxic T cells against resistant cells.

We suggest that Mev pathway is a master regulator of chemoresistance; its targeting with ZA may represent a potential therapeutic tool to restore the cytotoxic and pro-immunogenic effects of doxorubicin in resistant cancer cells.

References:

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