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. 2015 Mar 3;112(5):832-40.
doi: 10.1038/bjc.2015.17. Epub 2015 Feb 3.

Effect of pantoprazole to enhance activity of docetaxel against human tumour xenografts by inhibiting autophagy

Q Tan  1 , A M Joshua  2 , J K Saggar  1 , M Yu  1 , M Wang  1 , N Kanga  1 , J Y Zhang  1 , X Chen  3 , B G Wouters  1 , I F Tannock  4
Affiliations

Effect of pantoprazole to enhance activity of docetaxel against human tumour xenografts by inhibiting autophagy

Q Tan et al. Br J Cancer. .

Retraction in

Abstract

Background: Autophagy allows recycling of cellular components and may facilitate cell survival after chemotherapy. Pantoprazole inhibits proton pumps and is reported to inhibit autophagy. Here we evaluate the effects of pantoprazole to modify cytotoxicity of the anticancer drug docetaxel, and underlying mechanisms.

Methods: Effects of docetaxel±pantoprazole were studied against wild-type and autophagy-deficient PC3 cells and against four human xenografts. Effects of pantoprazole on autophagy were evaluated by quantifying LC3-I, LC3-II and p62 proteins in western blots, and by fluorescent microscopy of cells transfected with RFP-GFP-LC3. The distribution of drug effects and of autophagy was quantified in tumour sections in relation to blood vessels and hypoxia by immunohistochemistry using γH2AX, cleaved caspase-3, Ki67 and LC3/ p62.

Results: Pantoprazole increased the toxicity of docetaxel in vitro, increased docetaxel-induced expression of γH2AX and cleaved caspase-3, and decreased Ki67 in tumour sections. Pantoprazole increased growth delay of four human xenografts of low, moderate and high sensitivity to docetaxel, with minimal increase in toxicity. Docetaxel led to increased autophagy throughout tumour sections. Pantoprazole inhibited autophagy, and effects of pantoprazole were reduced against genetically modified cells with decreased ability to undergo autophagy.

Conclusions: Autophagy is a mechanism of resistance to docetaxel chemotherapy that may be modified by pantoprazole to improve therapeutic index.

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Figures

Figure 1
Figure 1
The effect of pantoprazole and docetaxel. (A) The effect of pantoprazole (PTP) on survival of MCF-7 cells alone (left panel) and with docetaxel (DOC: 50 nM) as determined by a clonogenic assay (note that surviving fraction is plotted on a logarithmic scale with condensed scale in right panel). Panel left, (control ●, pantoprazole alone at 10 μM ▪, 50 μM ▴,100 μM ♦, 200 μM , 500 μM ▾, 1 mM ◊); Panel right (docetaxel alone ●, plus pantoprazole at 10 μM ▪, 50 μM ▴, 100 μM ♦, 200 μM formula image,500 μM ▾and 1 mM ◊). Mean and s.e.m. are shown for three independent experiments. (B) Effect of pantoprazole and docetaxel on tumour growth in mice. Mice bearing MCF-7, A-431, PC3 and LNCaP xenografts were treated weekly for 3 weeks with saline, docetaxel (15 mg kg−1), pantoprazole (200 mg kg−1) or pantoprazole given 2 h before docetaxel. Tumour volume in mice was measured every 2–4 days (data represent mean±s.e.m.; n=12). Note that tumour volume is plotted on a logarithmic scale.
Figure 2
Figure 2
PC3 tumours treated with docetaxel (DOC: 15 mg kg−1 i.p.), pantoprazole (PTP: 200 mg kg−1 i.p.), pantoprazole 2 h before docetaxel or untreated controls. Figures represent per cent positive pixels for biomarkers as a function of distance from the nearest blood vessel in the section. (A) γH2aX at 10 min after docetaxel, (B) cleaved caspase-3 and (C) Ki67, both at 24 h after docetaxel. (D) Photomicrographs of Ki67 staining at 24 h in untreated (control), pantoprazole-treated alone and docetaxel-treated PC3 xenografts with or without pretreatment with pantoprazole.
Figure 3
Figure 3
Effect of pantoprazole on autophagy flux. Representative western blot (N=4) of PC3 cells treated with or without pantoprazole (PTP) 100 μM (2 h) in the presence or absence of bafilomycin A1 (Baf) 100 nM (4 h) to quantify LC3-I and LC3-II (A). The relative density of the LC3-II band is indicated. Pantoprazole alteration of the autophagic flux is confirmed by accumulation of p62 (B). The relative density of the p62 band is indicated. (C) Representative experiment (N=3) showing the effect of pantoprazole on accumulation of autophagosomes. PC3 cells were transfected with a RFP-GFP-LC3-II plasmid, with and without serum starvation (4 h), HCQ (50 μM for 24 h), pantoprazole (100 μM for 26 h), docetaxel (5 nM for 24 h) and combined treatment (pantoprazole 26 h commencing 2 h before docetaxel). The plasmid allows distinction of autophagosomes (GFP+ RFP+ yellow puncta) and autolysosomes (GFP− RFP+ red puncta) as GFP fluorescence is quenched in the acidic autolysosomes. Quantification was done by Image Pro software (Version premier 9).
Figure 4
Figure 4
The effect of pantoprazole on the toxicity of docetaxel, pantoprazole or both agents. (A–D) The effect of pantoprazole on the toxicity of docetaxel, pantoprazole or both agents for PC3 wild-type (WT) cells (A), autophagy-deficient knockdown cells lacking ATG7 (B), BECLIN1 (C) or both (D: double knockdown: DK) as determined by a clonogenic assay. Control (blue), docetaxel (50 nM, 24 h, purple), pantoprazole (100 μM, 24 h, green), pantoprazole followed by docetaxel (red). Cells were plated in triplicate and data are means±s.e.m. (N=3); Asterisk indicates a significant difference in cell survival as compared with control across indicated treatment groups. (E) Western blot assay confirmed knockdown of ATG7 and BECLIN1.
Figure 5
Figure 5
Treatment of PC3 and LNCaP tumours. PC-3 (A,B,D,E) and LNCaP (C,F) tumours treated with docetaxel (15 mg kg−1 i.p.), pantoprazole (200 mg kg i.p.), pantoprazole 2 h before docetaxel or untreated controls. Figures represent per cent positive pixels for LC3 (A,C), p62 (B) as a function of distance from the nearest blood vessel and LC3 (D,F) and p62 (E) as a function of distance from the nearest hypoxic region. (G) Photomicrographs of LC3 in untreated (control), pantoprazole-treated, docetaxel-treated PC3 tumour xenografts, with or without pretreatment with pantoprazole.
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