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Combined N-terminal androgen receptor and autophagy inhibition increases the antitumor effect in enzalutamide sensitive and enzalutamide resistant prostate cancer cells


Kranzbühler, Benedikt; Salemi, Souzan; Mortezavi, Ashkan; Sulser, Tullio; Eberli, Daniel (2019). Combined N-terminal androgen receptor and autophagy inhibition increases the antitumor effect in enzalutamide sensitive and enzalutamide resistant prostate cancer cells. The Prostate, 79(2):206-214.

Abstract

INTRODUCTION AND OBJECTIVES Multiple androgen receptor (AR)-dependent and -independent resistance mechanisms limit the efficacy of current castration-resistant prostate cancer (CRPC) treatment. Novel N-terminal domain (NTD) binding AR-targeting compounds, including EPI-001 (EPI), have the promising ability to block constitutively active splice variants, which represent a major resistance mechanism in CRPC. Autophagy is a conserved lysosomal degradation pathway that acts as survival mechanism in cells exposed to anticancer treatments. We hypothesized, that promising NTD-AR treatment may upregulate autophagy and that a combination of NTD-AR and autophagy inhibition might therefore increase antitumor effects. METHODS AR-expressing prostate cancer cell lines (LNCaP, LNCaP-EnzR) were treated with different concentrations of EPI (10, 25, 50 μM) and in combination with the autophagy inhibitors chloroquine (CHQ, 20 μM) or 3-methyladenine (3-MA, 5 mM). Cell proliferation was assessed by WST-1-assays after 1 and 7 days. Ethidium bromide and Annexin V were used to measure viability and apoptosis on day 7 after treatment. Autophagosome formation was detected by AUTOdot staining. In addition, autophagic activity was monitored by immunocytochemistry and Western blot (WES) for the expression of ATG5, Beclin1, LC3-I/II and p62. RESULTS Treatment with EPI resulted in a dose-dependent reduction of cell growth and increased apoptosis in both cancer cell lines on day 7. In addition, EPI treatment demonstrated an upregulated autophagosome formation in LNCaP and LNCaP-EnzR cells. Assessment of autophagic activity by immunocytochemistry and WES revealed an increase of ATG5 and LC3-II expression and a decreased p62 expression in all EPI-treated cells. A combined treatment of EPI with autophagy inhibitors led to a further significant reduction of cell viability in both cell lines. CONCLUSIONS Our results demonstrate that NTD targeting AR inhibition using EPI leads to an increased autophagic activity in LNCaP and LNCaP-EnzR prostate cancer cells. A combination of NTD AR blockage with simultaneous autophagy inhibition increases the antitumor effect of EPI in prostate cancer cells. Double treatment may offer a promising strategy to overcome resistance mechanisms in advanced prostate cancer.

Abstract

INTRODUCTION AND OBJECTIVES Multiple androgen receptor (AR)-dependent and -independent resistance mechanisms limit the efficacy of current castration-resistant prostate cancer (CRPC) treatment. Novel N-terminal domain (NTD) binding AR-targeting compounds, including EPI-001 (EPI), have the promising ability to block constitutively active splice variants, which represent a major resistance mechanism in CRPC. Autophagy is a conserved lysosomal degradation pathway that acts as survival mechanism in cells exposed to anticancer treatments. We hypothesized, that promising NTD-AR treatment may upregulate autophagy and that a combination of NTD-AR and autophagy inhibition might therefore increase antitumor effects. METHODS AR-expressing prostate cancer cell lines (LNCaP, LNCaP-EnzR) were treated with different concentrations of EPI (10, 25, 50 μM) and in combination with the autophagy inhibitors chloroquine (CHQ, 20 μM) or 3-methyladenine (3-MA, 5 mM). Cell proliferation was assessed by WST-1-assays after 1 and 7 days. Ethidium bromide and Annexin V were used to measure viability and apoptosis on day 7 after treatment. Autophagosome formation was detected by AUTOdot staining. In addition, autophagic activity was monitored by immunocytochemistry and Western blot (WES) for the expression of ATG5, Beclin1, LC3-I/II and p62. RESULTS Treatment with EPI resulted in a dose-dependent reduction of cell growth and increased apoptosis in both cancer cell lines on day 7. In addition, EPI treatment demonstrated an upregulated autophagosome formation in LNCaP and LNCaP-EnzR cells. Assessment of autophagic activity by immunocytochemistry and WES revealed an increase of ATG5 and LC3-II expression and a decreased p62 expression in all EPI-treated cells. A combined treatment of EPI with autophagy inhibitors led to a further significant reduction of cell viability in both cell lines. CONCLUSIONS Our results demonstrate that NTD targeting AR inhibition using EPI leads to an increased autophagic activity in LNCaP and LNCaP-EnzR prostate cancer cells. A combination of NTD AR blockage with simultaneous autophagy inhibition increases the antitumor effect of EPI in prostate cancer cells. Double treatment may offer a promising strategy to overcome resistance mechanisms in advanced prostate cancer.

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Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Hospital Zurich > Urological Clinic
Dewey Decimal Classification:610 Medicine & health
Language:English
Date:1 February 2019
Deposited On:23 Nov 2018 07:49
Last Modified:18 Dec 2018 02:05
Publisher:Wiley-Blackwell Publishing, Inc.
ISSN:0270-4137
OA Status:Closed
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1002/pros.23725
PubMed ID:30345525

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