Elsevier

Digestive and Liver Disease

Volume 44, Issue 2, February 2012, Pages 134-142
Digestive and Liver Disease

Liver, Pancreas and Biliary Tract
An oestrogen receptor β-selective agonist exerts anti-neoplastic effects in experimental intrahepatic cholangiocarcinoma

https://doi.org/10.1016/j.dld.2011.06.014Get rights and content

Abstract

Background

Cholangiocarcinoma cells over-express oestrogen receptor-β, which displays anti-proliferative and pro-apoptotic effects.

Aim

To evaluate the effects of a newly developed and highly selective oestrogen receptor-β agonist (KB9520) on experimental intrahepatic cholangiocarcinoma.

Methods

In vitro, the effects of KB9520 on apoptosis and proliferation of HuH-28 cells, HuH-28 cells with selective oestrogen receptor-β silencing (by small interfering RNA), HepG2 cells (oestrogen receptor-α and oestrogen receptor-β negative) and HepER3 cells (HepG2 cells transformed to stably express oestrogen receptor-α) were evaluated. In vivo, the effects of KB9520 on experimental intrahepatic cholangiocarcinoma, induced by thioacetamide administration were tested.

Results

In vitro, KB9520 induced apoptosis and inhibited proliferation of HuH-28 cells. KB9520 effects were absent in cells lacking oestrogen receptor-α and β (HepG2) and in cells expressing only oestrogen receptor-α (HepER3); its pro-apoptotic effect was impaired in cells where oestrogen receptor-β expression was decreased by specific small interfering RNA. In vivo, KB9520 inhibited experimental intrahepatic cholangiocarcinoma development in thioacetamide-treated rats and promoted tumour regression in rats where tumour was already established. In treated animals, tumour areas showed reduced proliferation but increased apoptosis.

Conclusions

KB9520 induced apoptosis in cholangiocarcinoma by selectively acting on oestrogen receptor-β, suggesting that oestrogen receptor-β selective agonists may be a novel and effective therapeutic option for the medical treatment of intrahepatic cholangiocarcinoma.

Introduction

Intrahepatic cholangiocarcinoma (CCA) is a devastating and highly aggressive cancer with an overall 5 year survival rate of less than 10% after diagnosis [1], [2], [3], [4]. There is no effective pharmacological treatment since CCA cells show high resistance to chemotherapeutic agents [1]; thus, there is an urgent need to develop novel therapeutic approaches.

Liver biopsies from patients with intrahepatic CCA were strongly positive for both oestrogen receptor (ER)-α and ER-β subtypes in 100% of examined patients, whilst cholangiocytes from normal liver were negative [5], [6]. Compared to benign cholangiocyte proliferation, intrahepatic CCA cells showed a higher expression of ER-α and ER-β [5], [6], [7] with an enhanced ER-α/ER-β ratio. This observation is in agreement with many different reports showing an increased ER-α/ER-β ratio in cancerous versus normal tissues, including ovary, prostate, colon and breast cancers [8], [9], [10], [11]. In these tissues, primary events in neoplastic transformation and progression have been correlated with up-regulation of ER-α and down-regulation of ER-β, which mainly occurs during adenoma–carcinoma transition, thus, associating the function of ER-α subtype with carcinogenesis rather than cancer cell proliferation [8], [9], [10], [11]. Recently, we have shown that ER-α acts synergistically with the IGF1 axis in promoting CCA cell proliferation and also that this receptor subtype stimulates VEGF synthesis and neoangiogenesis [5], [6]. The opposite was suggested for ER-β, which has been reported to have a protective effect against aberrant cell proliferation and carcinogenesis [12]. Genetic or pharmacological strategies aiming to induce expression or activation of ER-β are currently under investigation for cancer prevention or treatment [12]. Since, in contrast to other cancers, high ER-β expression is maintained in intrahepatic CCA even at late stages [5], it can be hypothesized that selective activation of ER-β may be beneficial to limit tumour growth.

Aim of this study was to evaluate in vitro and in vivo the effects of a newly developed and highly selective ER-β agonist (KB9520) on experimental intrahepatic CCA.

Section snippets

Materials

Reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated. Media and serum for cell culturing were obtained from Life Technologies, Inc. (Gaithersburg, MD). KB9520 [13] was synthesized and provided by Karo Bio (Rhönnstad P, Kallin E, Apelqvist T et al. Novel estrogen receptor ligands. Patent application # WO2009/127686, 22 October 2009). ICI 182,780 was purchased from Tocris Bioscience. ER-β antibodies were purchased from Santa Cruz Biotechnologies Inc. (Santa

Effect of KB9520 on apoptosis of HuH-28 cells

The expression of ER-α and -β in HuH-28 cell line derived from human intrahepatic CCA has been demonstrated in our previous studies by both RT-PCR and western-blot [5], [6]. The effect of increasing concentrations of KB9520 on apoptosis of HuH-28 cells is shown in Fig. 1. HuH-28 cells cultivated in growth medium containing 10% foetal serum were exposed for 72 h to KB9520 or DMSO (carrier) and the effect on apoptosis was evaluated by measuring caspase-3 activity or by counting TUNEL positive

Discussion

Estrogens via ER-α elicit proliferation of breast, uterus and developing prostate [23], [24], [25], [26], whereas estrogens via ER-β inhibit proliferation and promote differentiation in the prostate, mammary gland, colon, lung, and in stem cells in the bone marrow [8], [9], [10], [11], [27]. The ER-β subtype has an anti-proliferative and pro-apoptotic role in cancer cells and its down regulation has been linked to a worse prognosis [12]. The promoter of ER-β is frequently methylated in cancer

Disclosure

MM and DA received research grants from Karo Bio AB to support and develop the study.

Conflict of interest statement

Marco Marzioni and Domenico Alvaro received research grants from Karo Bio AB to support and develop the study. Patrik Rhönnstad, Theresa Apelqvist, Elisabet Kallin and Stefan Nilsson are Karo Bio employers. The other authors have nothing to declare.

List of abbreviations

    CCA

    cholangiocarcinoma

    CK

    cytokeratin

    DMSO

    dimethyl sulfoxide

    ER

    oestrogen receptor

    ODN

    oligonucleotides

    PCNA

    proliferating cell nuclear antigen

    SiRNA

    small interfering RNA

    TAA

    thioacetamide

    TUNEL

    terminal deoxynucleotidyl transferase-mediated triphosphate

Acknowledgements

Financial support: This work was supported by a MIUR grant PRIN 2007-prot. 2007HPT7BA_002 to Dr. Marzioni and by Intramural Grants by the Università Politecnica delle Marche to Dr. Marzioni and to Dr. Benedetti; D. Alvaro is supported by MIUR grant PRIN 2007-prot. 2007HPT7BA-003 and by Federate Atheneaum funds from the University “Sapienza” of Rome. Dr. Gaudio was supported by MIUR grants PRIN 2007-prot. 2007HPT7BA_001 and Federate Atheneaum funds from the Univ. “Sapienza” of Rome.

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