Inhibition of LSD1 by Pargyline inhibited process of EMT and delayed progression of prostate cancer in vivo

https://doi.org/10.1016/j.bbrc.2015.09.164Get rights and content

Highlights

  • We mimic progression of PCa in an animal model, being more representative clinic.

  • LSD1 is up-regulated in CRPC consistent with our previous study.

  • Inhibition of LSD1 by Pargyline inhibits process of EMT in vitro and in vivo.

  • Inhibition of LSD1 is a promise adjunctive therapy with ADT.

Abstract

Recently, lysine-specific demethylase 1 (LSD1) was identified as the first histone demethylase. LSD1 interacted with androgen receptor (AR) and promoted androgen-dependent transcription of target genes, such as PSA, by ligand-induced demethylation of mono- and dimethylated histone H3 at Lys 9 (H3K9). Meanwhile, the phenomenon of epithelial–mesenchymal transition (EMT) had received considerable attention in tumor recurrence and metastasis. This study examined the effect of Pargyline (an inhibitor of LSD1) on the process of EMT in vitro and in vivo. SCID mice were injected subcutaneously with LNCap cells. Pargyline was given intraperitoneally or not after castration (implemented with Bilateral orchidectomy), then PSA levels in serum and tumor were determined to assess time to androgen-independent progression. The results showed that LSD1 expression was up-regulated when PCa progressed to Castration Resistant Prostate Cancer (CRPC). Pargyline reduced LNCap cells migration and invasion ability, and inhibited the process of EMT by up-regulating expression of E-cadherin, and down-regulating expressions of N-cadherin and Vimentin in vitro and in vivo. Although, Pargyline did not change the level of AR, it reduced PSA expression both in vitro and in vivo. Furthermore, Pargyline delayed prostate cancer transition from androgen-dependent to androgen-independent state (CRPC). These findings indicated that inhibition of LSD1 might be a promise adjunctive therapy with androgen deprivation therapy (ADT) for locally advanced or metastatic prostate cancer.

Introduction

Prostate cancer (PCa) was considered as one of the most commonly diagnosed cancers in men of developed Western countries. Worldwide, it was the 2nd most commonly diagnosed and 6th leading cause of cancer death in men [1]. Androgen deprivation therapy (ADT) was still the standard systemic therapy for locally advanced or metastatic PCa. Initially, PCa was dependent on androgen for growth and could be successfully suppressed by ADT. However, almost all advanced PCa eventually progressed toward a more aggressive PCa form defined as castration resistant prostate cancer (CRPC), for which there was no available curative therapy [2].

The amino-terminal tails of histones were subject to a series of posttranslational modifications (including acetylation, phosphorylation, ubiquitination and methylation), resulting in a combination of histone marks which were known as the histone code [3]. It was believed that lysine methylation was irreversible until lysine-specific demethylase 1 (LSD1) was discovered recently [4]. Our previous study [5] and several studies [6], [7], [8] had demonstrated that over-expression of LSD1 was implicated in prostate cancer. LSD1 was strongly expressed in prostate cancers with high Gleason score. As well as, it might be regarded as a marker for prostate cancer with aggressive biology. It had been demonstrated that LSD1 co-localized with AR in nucleus of normal human prostate and PCa [6]. It was verified in vitro study [9] that LSD1 formed chromatin-associated complexes with AR in a ligand-dependent manner to demethylate the repressing histone marks mono- and dimethyl H3-K9, then led to de-repression of AR target genes, including PSA, which was considered as a key marker for PCa recurrence and progression.

The process of epithelial cells acquire characteristics of mesenchymal cells during embryogenesis, wound healing and malignant progression was now extensively accepted as a core biological process termed epithelial–mesenchymal transition (EMT). EMT was regarded as an important step in PCa metastasis [10], and was involved in deregulation of the androgen axis [11], which might be correlated with CRPC. Loss of E-cadherin and gain of N-cadherin and Vimentin were considered as a hallmark of EMT. Previous studies [12], [13] had demonstrated that LSD1 interacted with Snail1 and CoREST to form a ternary complex, keeping the stability and function of these proteins, and resulting in demethylation of H3K4me2 at the E-cadherin promoter to suppress its expression.

So, we speculated that inhibition of LSD1 by Pargyline might reduce the malignant potential of PCa, and delay the recurrence and progression of PCa to CRPC. In this study, firstly, we examined the effect of inhibition of LSD1 by Pargyline on the process of EMT, invasion ability and clonality of LNCap cells. Next, subcutaneous transplantation tumors were set up in SCID mice using LNCap cells. ADT (implemented with Bilateral orchidectomy) associated with Pargyline or not were given to the mice, then PSA levels in serum and tumor were determined to assess time to androgen-independent progression.

Section snippets

Cell culture

LNCaP cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Beijing, China). LNCaP cells were cultured in RPMI1640 medium supplemented with 10% FBS (Gibco/Invitrogen, Australia), and 1% penicillin/streptomycin (Invitrogen) at 37 °C in a humidified incubator in the presence of 5% CO2 and 1% penicillin/streptomycin (Invitrogen) at 37 °C in a humidified incubator. Cells were treated with 0 or 3 mM Pargiline (Sigma, USA) for 48 h, respectively.

Migration and invasion activity assay

Transwell migration and invasion

Pargyline inhibited process of EMT in vitro and in vivo

Firstly, exposure of LNCap prostate cancer cells to Pargyline resulted in induction expression of the epithelial marker, E-cadherin, and reduction of the mesenchymal markers, N-cadherin and Vimentin expression (Fig. 1A), changes characteristic of EMT. Furthermore, as shown by immunofluorescence (Fig. 1C), E-cadherin mainly expressed in cell membrane, expression of which was up-regulated after being treated with Pargyline.

Subsequently, xenograft tumors were established using LNCap cells in

Discussion

The present study demonstrated that Pargyline could effectively inhibit process of EMT, migration ability, and invasion activity of LNCap cells. Meanwhile, LSD1 expression was up-regulated when PCa progressed to CRPC. Moreover, Pargyline inhibited process of EMT as well, reduced PSA expression, and delayed prostate cancer progression from androgen-dependent to androgen-independent state in vivo.

It was well known that organogenesis, morphology and normal functioning of prostate were regulated

Conflict of interest

None.

Acknowledgments

This study is supported by the grants from the National Natural Science Foundation of China (No.2013RMFH012), the Province Natural Science Foundation of Hubei (No.2012FFA096), and supported by the Fundamental Research Funds for the Central Universities (NO. 2042014kf0115). We thank for Key laboratory of Hubei Province for Digestive System Disease and for all the authors whose work is included in this study.

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