Plumbagin inhibited AKT signaling pathway in HER-2 overexpressed-endocrine resistant breast cancer cells

doi:10.1016/j.ejphar.2019.172878

European Journal of Pharmacology

Volume 868, 5 February 2020, 172878

https://doi.org/10.1016/j.ejphar.2019.172878 Get rights and content

Abstract

The important mechanism of endocrine resistance is the crosstalk between estrogen receptor (ER) and HER2 signaling pathways. Aside from ER downregulation, there was an increase in HER2 expression and increased activation of the downstream AKT/ERK pathways in endocrine-resistant cells (MCF-7/LCC2 and MCF-7/LCC9) which is similar to HER2-overexpressed (SKBR3) cells. However, nuclear receptor coactivator 3 (NCOA3), the important ER-coactivator, that upregulated in endocrine-resistant cells did not express in HER2-overexpressed (SKBR3) cells. NCOA3 was able to activate AKT/ERK signalling pathway. Our previous study reported that plumbagin (PLB), a naphthoquinone compound, had a potent cytotoxic activity against endocrine-resistant cells. This study aimed to further investigate the mechanism of anti-cancer effects of PLB on ER and HER-2 signaling. PLB can inhibit estradiol (E₂)-induced cell proliferation in MCF-7 wild-type cells but had no effect in the resistant cells. It also inhibited HER2 expression in both endocrine-resistant and HER-2 overexpressed cells. Therefore, the mechanism of PLB may be regulated through HER-2 signaling. PLB inhibited the phosphorylation of AKT (pAKT) and pERK1/2 and induced apoptosis and reduced the expression of anti-apoptotic genes Bcl-2 and pro-caspase 3 and Cleaved Caspase 3 protein in both endocrine-resistant and HER-2 overexpressed cells. However, the inhibitory effect of PLB was more obvious when pre-treated the cells with AKT inhibitor only in HER-2 overexpressed cells. In addition, the inhibitory effect of PLB on pAKT was attenuated when NCOA3 was downregulated. Our finding suggested that the inhibitory effect of PLB on AKT signaling pathways regulated through NCOA3 in HER2-overexpressed endocrine-resistant cells.

Graphical abstract

Introduction

Half of the ER-positive breast cancer patients in advanced stages do not respond to tamoxifen (Droog et al., 2013; Garcia-Becerra et al., 2012; Lewis and Jordan, 2005). The main mechanisms of the resistance are due to an alteration of ERα expression and over-expression of growth factor receptors especially human epidermal growth factor receptor 2 (HER-2) (Garcia-Becerra et al., 2012; Hosford and Miller, 2014). Aberrant activation of HER2 was able to stimulate PI3K/AKT and MAPK/ERK pathways in several types of cancer such as those in the lung, prostate and the breast (Gan et al., 2010; Hu et al., 2016; Zhu et al., 2018). Both of these two signaling pathways are involved in cell survival, inhibition of cancer cell apoptosis and metastasis (Atmaca et al., 2017; Kawiak and Lojkowska, 2016; Prasad et al., 2015). There are few drugs that can be used to treat ER-positive breast cancer patients due to drug resistance and adverse effects. Hence, novel therapeutic agents that target to decrease the endocrine resistance should be developed in order to prevent tumor recurrence and metastasis.

Plumbagin (PLB) is a naphthoquinone compound and derivative of Vitamin K3. PLB is isolated from the roots of Plumbago indica. PLB has apoptotic effects in prostate cancer cells (PC-3, LNCaP, C4-2 cell lines), HER-2 over-expressed breast cancer cells (SKBR3, BT474 cell lines) and colon cancer cells (HT-29 cell line) (Chen et al., 2013; Kawiak et al., 2012; Powolny and Singh, 2008). PLB had no effect in normal prostate epithelial RWPE-1 cells and non-cancerous breast epithelial MCF-10A cells (Ahmad et al., 2008; Aziz et al., 2008) which indicated that it was a safe product. In addition, an animal study demonstrated that there was no adverse effect when 25 mg/kg PLB was used for 4 days in mice (Sumsakul et al., 2014). Our previous study reported anti-cancer effect of PLB in endocrine-resistant cells in micromolar concentration (Sakunrangsit et al., 2016). IC50 values of MCF-7 wild-type cells and MCF-7/LCC2 and MCF-7/LCC9 resistant cells were in the same range. Our recent study reported NADPH: quinone oxidoreductase 1 (NQO1) enzyme was upregulated in endocrine-resistant cells. PLB was metabolized by this enzyme to an unstable form and generated more reactive oxygen species which induced apoptosis in cancer cells (Pradubyat et al., 2020). Thus, PLB is selective to NQO1 overexpressed cells (Pradubyat et al., 2020). Our study also demonstrated that PLB reduced nuclear receptor coactivator 3 (NCoA3) which is an ER co-activator that increased the expression in resistant cells (Sakunrangsit et al., 2016). The previous study also reported overexpression of NCOA3 was able to activate AKT signaling in breast cancer (Li et al., 2018). As previously mentioned, endocrine-resistant breast cancer cells have altered the expression of ERα and upregulated HER-2 (Garcia-Becerra et al., 2012). However, there are no data on the inhibitory mechanism of PLB on ERα expression and HER-2 signaling in endocrine-resistant cells. As a result of this, this study investigated the inhibitory mechanism of PLB on ER and downstream of HER-2 signaling in endocrine-resistant and HER-2 overexpressed breast cancer cells.

Section snippets

Cell lines and cultures

The MCF-7 (ER positive) and SKBR3 (HER-2 over-expressed) cell lines were purchased from the American Type Culture Collection (ATCC; Virginia, USA). MCF-7/LCC2 cell line is tamoxifen-resistant, and MCF-7/LCC9 cell line is resistant to both tamoxifen and fulvestrant. Both of these cell lines were obtained from Dr. Robert Clarke from the Lombardi Cancer Center, Georgetown University (Washington DC, USA). MCF-7, MCF-7/LCC2 and MCF-7/LCC9 cells were maintained in MEM containing 5% FBS (Gibco, New

The alteration of ER, NCOA3 and HER2 signaling in endocrine-resistant and HER-2 overexpressed breast cancer cell lines

The morphology of endocrine-resistant cells (MCF-7/LCC2, MCF-7/LCC9) and HER-2 overexpressed (SKBR3) cells are shown in Fig. 1A. The alteration of mRNA expression of estrogen receptor 1 (ESR1), NCOA3 and ERBB-2 were observed in MCF-7/LCC2 and MCF-7/LCC9 cells. ESR1 mRNA expression decreased while NCOA3 and ERBB-2 mRNA expression increased in both MCF-7/LCC2 and MCF-7/LCC9 cell lines when compared to MCF-7 wild-type cells (Fig. 1B). NCOA3 and ERBB-2 proteins were upregulated and correlated with

Discussion

The mechanisms of tamoxifen or endocrine resistance involve the alteration of ERα expression and the over-expression of HER-2 (Garcia-Becerra et al., 2012; Hosford and Miller, 2014). Both MCF-7/LCC2 and MCF-7/LCC9 endocrine-resistant cell lines had lower mRNA expression of ESR1 which is encoded for ERα and had higher expression of NCOA3, HER-2 and its downstream signaling proteins. This study observed the effect of PLB in inducing ESR1 mRNA expression and suppression of ERBB-2 mRNA expression

Ethical consideration

The experiments used human cell lines so were exempted from review by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University (IRB Number: 685/61).

Funding

This work was supported by Ratchadaphiseksomphot Endowment Fund, Faculty of Medicine, Chulalongkorn University, Thailand (RA62/034 to W.K.) and Special Task Force for Activating Research (STAR) Ratchadaphiseksomphot Endownment Fund to Overcoming Cancer Drug Resistance Research group (GSTAR 59-005-30-001 to W.K.).

Declaration of competing interest

The authors declare no conflict of interest.

Acknowledgements

We would like to thank the Research Affair Language Center for editing this paper and Prof. Robert Clarke M.D., The Lombardi Comprehensive Cancer Center (LCCC), Georgetown University School of Medicine, Washington DC, USA, for providing us with the LCC2 and LCC9 cell lines.

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Full length articlePlumbagin inhibited AKT signaling pathway in HER-2 overexpressed-endocrine resistant breast cancer cells

Abstract

Graphical abstract

Introduction

Section snippets

Cell lines and cultures

The alteration of ER, NCOA3 and HER2 signaling in endocrine-resistant and HER-2 overexpressed breast cancer cell lines

Discussion

Ethical consideration

Funding

Declaration of competing interest

Acknowledgements

Plumbagin-induced apoptosis of human breast cancer cells is mediated by inactivation of NF-kappaB and Bcl-2

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Chem. Biol. Interact.

Plumbagin, a medicinal plant-derived naphthoquinone, is a novel inhibitor of the growth and invasion of hormone-refractory prostate cancer

Cancer Res.

Activation of AMP-activated protein kinase (AMPK) mediates plumbagin-induced apoptosis and growth inhibition in cultured human colon cancer cells

Cell. Signal.

Tamoxifen resistance: from bench to bedside

Eur. J. Pharmacol.

Differential roles of ERK and Akt pathways in regulation of EGFR-mediated signaling and motility in prostate cancer cells

Oncogene

Mechanisms of resistance to endocrine therapy in breast cancer: focus on signaling pathways, miRNAs and genetically based resistance

Int. J. Mol. Sci.

Clinical potential of novel therapeutic targets in breast cancer: CDK4/6, Src, JAK/STAT, PARP, HDAC, and PI3K/AKT/mTOR pathways

Pharmgenomics Pers Med

Integrin alpha6/Akt/Erk signaling is essential for human breast cancer resistance to radiotherapy

Sci. Rep.

Ramentaceone, a naphthoquinone derived from Drosera sp., induces apoptosis by suppressing PI3K/Akt signaling in breast cancer cells

PLoS One

Full length article
Plumbagin inhibited AKT signaling pathway in HER-2 overexpressed-endocrine resistant breast cancer cells