Abstract A54: Pantoprazole enhances the activity of docetaxel chemotherapy for solid tumors by inhibition of autophagy

Purpose: Resistance to anti-tumour agents is a major cause of treatment failure in patients with cancer. Luciani and colleagues (JNCI 2004; 96:1702-13) have shown that PPIs can augment the effects of chemotherapy in vitro and in vivo, most likely by modifying the acid-base balance across the cell membrane and/or of intracellular organelles such as lysosomes and endososmes. Endosomes are important intermediaries of the process of autophagy, a survival mechanism for stressed cells and a possible target for anticancer therapy. Here we evaluated the effect of pantoprazole (PTP) (i) to modify cytotoxic effects of the commonly-used anticancer drug docetaxel (DOC) for cultured cells and for human tumour xenografts. (ii) to modify the distribution of biomarkers of activity of docetaxel in relation to tumour blood vessels. (iii) to inhibit autophagy. Methods: Endosomal pH of cells was measured by fluorescence spectroscopy following exposure to the endosomal probe Lysosensor. Levels of autophagy were evaluated by quantifying the level of the LC3B protein in Western blots. Human prostate cancer PC3 cells were transfected with a construct where LC3B was linked to red (RFP) and green (GFP) fluorescent protein (mRFP-GFP-LC3) such that cells with high levels of autophagy stained red while those with low levels stained green. Cytotoxicity of DOC +/-PTP was evaluated in vitro by a colony-forming assay for PC3 parental cells, and for cells transfected with shRNAs to inhibit autophagy. Growth delay of three human xenografts in nude mice was evaluated following treatment with single or multiple doses of DOC+/- PTP 3. The distribution of drug effects in tumour sections in relation to functional blood vessels (recognized by Dioc7) was quantified (at early intervals of ~10 minutes) by γH2AX, a marker of DNA damage, (at 24 hours) by cleaved caspase 3, a marker of apoptosis, Ki67, a marker of cell proliferation and LC3, a marker of autophagy. Results: PTP increased endosomal pH and inhibited autophagy in a dose and time dependent manner. PTP was not cytotoxic when used alone but increased the cytotoxicity of DOC for cultured PC3 cells; the effects of PTP were reduced against genetically-modified cells that have reduced ability to undergo autophagy. Pre-treatment with PTP increased markedly the growth delay due to DOC of three different xengografts, especially after three treatments at weekly intervals, with minimal effects on DOC toxicity. Cellular effects of DOC evaluated by γH2A