Up-regulation of autophagy is a mechanism of resistance to chemotherapy and can be inhibited by pantoprazole to increase drug sensitivity

Abstract

Background

Autophagy is a survival mechanism that allows recycling of cellular breakdown products, particularly in stressed cells. Here we evaluate the hypotheses that up-regulation of autophagy is a common mechanism of resistance to chemotherapy, and that drug resistance can be reversed by inhibiting autophagy with a proton pump inhibitor.

Methods

We exposed human PC3, LNCaP and MCF7 cells to seven clinically-used chemotherapy drugs ± pantoprazole, examined the up-regulation of autophagy and the effect on cellular proliferation by Western Blots, MTS assay and colony-forming assay. The distribution of drug effects and of autophagy was quantified in LNCaP tumor sections in relation to blood vessels and hypoxia by immunohistochemistry using γH2AX, cleaved caspase-3 and p62.

Results

All anticancer drugs led to up-regulation of autophagy in cultured tumor cells. Pantoprazole inhibited the induction of autophagy in a time- and dose-dependent manner, and sensitized cancer cells to the seven anti-cancer drugs. Treatment of LNCaP xenografts with paclitaxel induced both DNA damage and autophagy; autophagy was inhibited and markers of toxicity were increased by pantoprazole.

Conclusions

Induction of autophagy is a general mechanism associated with resistance to anticancer drugs and that its inhibition is a promising therapeutic strategy to enhance the effects of chemotherapy and improve clinical outcomes.

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Acknowledgements

We thank all members of the Pathology Research Program (PRP), and the Advanced Optical Microscopy Facility (AOMF). Special thanks to Dr. Richard Hill for his guidance and helpful advice. Supported by grant KG100252 from the Komen Foundation, a grant from the Canadian Institutes of Health Research and a grant from Prostate Cancer Canada Rising Star award.

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Corresponding authors

Correspondence to Qian Tan or Ian F. Tannock.

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All authors declare that she/he has no conflict of interest.

Ethical approval

Animal experiments described in this paper were carried out using Animal Use Protocol (AUP1232.15, 09/05/14) approved by Princess Margaret Cancer Center, University Health Network (UHN) Animal Care Committee under the guidelines of the Canadian Council on Animal Care.

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A

. Quantitative analysis of relative LC3-II and p62 levels in MCF7, LNCaP and PC3 cells. B. Quantification of RFP+ and GFP+ punctae /cell in MCF7 and PC3 cell lines, ratio between pantoprazole and control, combination treatment and chemotherapy drug alone. Data are means ± SEM (N=3) (TIF 96 KB)

A

. Effect of pantoprazole on autophagy. LNCaP cells were treated with bafilomycin A1 (BAF:100nM 4hr), pantoprazole (PTP: 100µM for 26hr), doxorubicin (DOX: 50nM), docetaxel (DOCE: 1nM), gemcitabine (GEM: 50nM), melphalan (MEL: 1µM), methotrexate (MTX: 10nM), mitoxantrone (MIT: 50nM), paclitaxel (PAC: 1nM) alone (for 24hr) or pantoprazole 2 hours prior to chemotherapy drugs (pantoprazole 26 hr commencing 2h prior to chemotherapy drugs). The protein levels of LC3-II and p62 were assayed by western blots (N=3). B. Quantitative analysis of LC3-II and p62 levels in LNCaP cells. The relative density of the LC3-II band is indicated. C. The relative density of the p62 band is indicated. The relative levels of LC3-II and p62 were normalized to β-actin and the value of control was set as 1.0. * p<0.05 chemotherapy drugs treatment alone vs respective control; ** p<0.01 pre-treatment with PTP vs chemotherapy drug treatment alone. Error bars represent SEM for 3 independent experiments (TIF 112 KB)

B

A. Effect of pantoprazole on autophagy. PC3 cells were treated with bafilomycin A1 100nM (4hr), pantoprazole (100µM for 26hr), doxorubicin (DOX: 50nM), docetaxel (DOCE: 1nM), gemcitabine (GEM: 50nM), melphalan (MEL: 1µM), methotrexate (MTX: 10nM), mitoxantrone (MIT: 50nM), paclitaxel (PAC: 1nM) alone (for 24hr) or pantoprazole 2 hours prior to chemotherapy drugs (pantoprazole 26 hr commencing 2h prior to chemotherapy drugs). The protein levels of LC3-II and p62 were assayed by western blot (N=3). . Quantitative analysis of LC3-II and p62 levels in PC3 cells: The relative density of the LC3-II band is indicated. C. The relative density of the p62 band is indicated. The relative levels of LC3-II and p62 were normalized to β-actin and the value of control was set as 1.0. * p<0.05 chemotherapy drugs treatment alone vs respective control; ** p<0.01 pre-treatment with PTP vs chemotherapy drug treatment alone). Error bars represent SEM for 3 independent experiments (TIF 126 KB)

A

LNCaP tumor xenografts treated with paclitaxel, pantoprazole, pantoprazole 2 hours prior to paclitaxel, or untreated controls. Figures represent percent positive pixels of cleaved caspase-3 () and Ki67 (C) as a function of distance from the nearest blood vessel and of cleaved caspase-3 (B) and Ki67 (D) as a function of distance from the nearest hypoxic region. Error bars around curves represent SEM for 5–6 mice per group. Differences between combination group and paclitaxel group (p < 0.01), and these two groups compare to control curves in panels B-D are statistically significantly (p < 0.05) (TIF 78 KB)

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Tan, Q., Joshua, A.M., Wang, M. et al. Up-regulation of autophagy is a mechanism of resistance to chemotherapy and can be inhibited by pantoprazole to increase drug sensitivity. Cancer Chemother Pharmacol 79, 959–969 (2017). https://doi.org/10.1007/s00280-017-3298-5

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Keywords

  • Autophagy
  • Proton pump inhibitor
  • Drug distribution
  • Pharmacodynamic markers
  • Tumor microenvironment