Abstract 537: Mechanisms by which pantoprazole, a proton pump inhibitor, enhances the activity of doxorubicin in solid tumors

Purpose: Mechanisms of tumour resistance to cytotoxic drugs in solid tumours include limited drug distribution from tumour blood vessels and other effects of the tumour microenvironment. Proton pump inhibitors, such as pantoprazole (PTP), raise endosomal pH and thereby decrease sequestration of basic drugs in acidic compartments. This may allow more drugs to interact with DNA, while decreased net cellular uptake of drug, may allow more drugs to penetrate tissue to distal cells. PTP might also inhibit autophagy, a possible target for anticancer therapy, since endosomes are important intermediaries of this process. Here we describe the effects of PTP on doxorubicin uptake and distribution in cell cultures and solid tumours, and effects to cause growth delay. We have also evaluated the effect of PTP on autophagy of tumour cells. Methods: Endosomal pH of cells was measured by using Lysosensor and fluorescence spectroscopy. Cytotoxicity was evaluated by a colony-forming assay. Doxorubicin distribution was quantified in MCF-7 xenografts by immunohistochemistry, in relation to blood vessels (recognized by an antibody to CD31), and regions of hypoxia (recognized by an antibody to EF5), with and without pre-treatment with PTP. Anti-tumour effects were evaluated by growth delay following treatment with single or multiple doses of doxorubicin with and without PTP. Effects of PTP on autophagy were evaluated by (i) expression of the LC3II protein by Western blotting and (ii) effects of PTP on prostate cancer PC3 cells transfected with green fluorescent protein) and with LC3 protein (a marker of autophagy) tagged with red fluorescent protein (mRFP-GFP-LC3 cells) Results: PTP increased endosomal pH and decreased endosomal sequestration of doxorubicin in cultured cells. Pre-treatment with PTP increased the distribution of doxorubicin in solid tumours. PTP alone had no effect on tumour growth but increased the effect of doxorubicin to cause growth delay of human tumour xenografts with no apparent increase in toxicity. PTP showed marked effects to inhibit autophagy in cultured tumour cells in a dose and time dependent manner. Conclusions/Discussion: PTP increases the effectiveness of doxorubicin against solid tumours and this combination is being investigated in a phase I clinical trial. Potential mechanisms underlying this effect include inhibition of sequestration of doxorubicin in endosomes permitting better drug distribution, and inhibition of autophagy. Supported by a