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Plumbagin inhibited AKT signaling pathway in HER-2 overexpressed-endocrine resistant breast cancer cells
European Journal of Pharmacology 868 (2020) 172878
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Plumbagin inhibited AKT signaling pathway in HER-2 overexpressedendocrine resistant breast cancer cells
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Nithidol Sakunrangsit, Wannarasmi Ketchart∗ Overcoming Cancer Drug Resistance Research Unit, Department of Pharmacology, Faculty of Medicine, Chulalongkorn University, Bangkok, 10330, Thailand
ARTICLE INFO
ABSTRACT
Keywords: PLB HER-2 Endocrine-resistance AKT
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 HER2 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 endocrineresistant and HER-2 overexpressed cells. However, the inhibitory effect of PLB was more obvious when pretreated 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.
1. Introduction
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)
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
∗
Corresponding author. Department of Pharmacology, Chulalongkorn University, 1873 Rama IV Road, Pathumwan, Bangkok, 10330, Thailand. E-mail address: [email protected] (W. Ketchart).
https://doi.org/10.1016/j.ejphar.2019.172878 Received 5 July 2019; Received in revised form 11 December 2019; Accepted 17 December 2019 Available online 19 December 2019 0014-2999/ © 2019 Elsevier B.V. All rights reserved.
European Journal of Pharmacology 868 (2020) 172878
N. Sakunrangsit and W. Ketchart
Fig. 1. The expression of ER, NCOA3 and HER2 signaling in endocrine-resistant and HER-2 overexpressed breast cancer cells. (A) Morphology of wild-type MCF-7, endocrine-resistant MCF-7/LCC2, MCF-7/LCC9 and HER2-overexpressed SKBR3 cells. Cells were routinely cultured and then captured by using a light microscope with digital camera. Scale bar = 100 μm. (B) Real-time PCR results of ESR1, NCOA3 and ERBB2 mRNA levels for each cell line. Data were shown as mean ± S.E.M from three independent experiments (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. MCF-7 cells. (C) Western blots of HER2, NCOA3 and AKT/ ERK proteins. (D) The analysis of HER2, NCOA3 and AKT/ERK signaling protein expression in endocrine-resistant and HER2-overexpressed cells. Data were shown as mean ± S.E.M from three independent experiments (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. MCF-7 cells.
2. Materials and methods
Table 1 The IC50 values of PLB in endocrine resistant and HER-2 overexpressed breast cancer cells. Cell line MCF-7 MCF-7/LCC2 MCF-7/LCC9 SKBR3
IC50 (μM) 24 h 1.47 1.69 1.24 2.04
2.1. Cell lines and cultures
Reference
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 York, USA). SKBR3 cell line was maintained in McCoy's 5A Medium containing 5% FBS (ATCC; Virginia, USA). All cell lines were grown at 37 °C under 5% CO2. Endocrine resistant cell lines were routinely checked to ensure that they were resistant to tamoxifen using MTT assay.
48 h ± ± ± ±
0.05 0.01 0.05 0.04
1.22 1.58 1.17 1.60
± ± ± ±
0.01 0.06 0.02 0.01
Sakunrangsit et al. (2016) Sakunrangsit et al. (2016) Sakunrangsit et al. (2016) N/A
Results are expressed as mean ± S.E.M in triplicate from three-independent experiments (n = 3). IC50 represents the half maximal inhibitory concentration. PLB = Plumbagin, HER-2 = Human epidermal growth factor 2, N/A = Not assessed previously.
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.
2.2. Reagents Plumbagin (PLB) and 4-hydroxytamoxifen (4-OHT) were purchased from Sigma-Aldrich (Missouri, USA). Fulvestrant (ICI) and lapatinib (LAP) were obtained from Abcam (Cambridge, UK). MK2206 (AKT inhibitor) was obtained from PreproTech (New Jersey, USA). Human recombinant insulin (AKT activator) was purchased from Invitrogen (California, USA). PLB, LAP and MK2206 were dissolved in dimethyl sulfoxide (DMSO). 4-OHT and ICI were prepared in absolute ethanol as 10 mM stock solution. esiRNA human NCOA3 was purchased from Sigma-Aldrich (Missouri, USA). All antibodies include HER2, AKT, pAKT [Ser473], ERK1/2, pERK1/2 [Tyr702/Tyr704], NCOA3, Caspase3, Cleaved Caspase 3 and GAPDH were purchased from Cell Signaling Technology (Massachusetts, USA). 2
European Journal of Pharmacology 868 (2020) 172878
N. Sakunrangsit and W. Ketchart
Fig. 2. The inhibitory effect of PLB in HER2-overexpressed cells on cell proliferation and mRNA expressions of ERBB2, ER and NCOA3. (A, B) Relative cell viability and IC50 values of PLB in HER2-overexpressed (SKBR3) cells for 24 h and 48 h. 0.1% DMSO and lapatinib (LAP) were used as negative and positive controls, respectively. *P < 0.05, **P < 0.01, ***P < 0.001 vs. negative control. (C, D) mRNA expression of ERBB2, ESR1 and NCOA3 after 24-h treatment with PLB in MCF-7/LCC2 and MCF-7/LCC9 cells. Data were shown as mean ± S.E.M from three independent experiments (n = 3). *P < 0.05, **P < 0.01 vs. negative control.
2.3. MTT assay
from the cells. The amount and purity of RNA were determined using a NanoDrop-One (Thermo Scientific). One μg of total RNAs was converted to cDNA by amplifying the genes of interest. cDNA was obtained by using specific primers to estrogen receptor 1 (ESR1), epidermal growth factor 2 (ERBB2), NCOA3, B-cell lymphoma 2 (BCL2), and procaspase 3 (PROCASP3). The primers used in this study were designed by PrimerBLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) as shown in Supplementary Data. Amplification and detection was performed using SYBR Green qPCR supermix by StepOne™ Real-Time PCR System (Applied Biosystems, USA). GAPDH was used as an internal control. Relative expression was calculated with the comparative CT method.
2.3.1. Estrogen-induced proliferation MCF-7, MCF-7/LCC2 and MCF-7/LCC9 cells were maintained in phenol red-free IMEM supplemented with 5% charcoal dextran-treated FBS so that the cells were not exposed to hormones at least 4 days prior to the experiment. 5×103 cells/well of MCF-7 were seeded into 96-well plates with complete IMEM medium overnight and then treated with PLB at concentrations of 0.25, 0.5 and 0.75 μM with or without 10 nM estradiol (E2) for 7 days. 4-hydroxytamoxifen (4-OHT) and ICI at 0.1 μM were used as positive controls and 0.1% DMSO was used as a vehicle control. MTT assay was performed after incubation. Percentage of cell viability was calculated.
2.6. NCOA3 siRNA transfection
2.3.2. AKT inhibitor and insulin pre-treatment MCF-7/LCC2, MCF-7/LCC9 and SKBR3 cells were cultured in serum-free media (SFM) for 2 h before being treated with 0.1 μM AKT inhibitor (MK2206) for 30 min or 0.1 μM insulin for 15 min. After that, different concentrations of PLB was used for MTT, apoptotic assay and Western blot. LAP was used as a positive control.
siRNA-NCOA3 (siNCOA3) or scramble control siRNA was transiently transfected into MCF7/LCC2 or MCF7/LCC9 cells using Lipofectamine. Cells were harvested after 24 h. NCOA3 expression was checked by Western blot. The expression of pAKT and AKT was determined in siNCOA3 transfected cells compared to wild-type cells by Western blot.
2.4. Apoptotic assay
2.7. Western blot analysis
MCF-7/LCC2, MCF-7/LCC9, and SKBR3 cells were seeded in 24-well plate with a density of 2x105 cells/ml. One groups of cells were pretreated with 0.1 μM MK2206 (AKT inhibitor). Then, all samples were treated with 0.5,1 and 2 μM of PLB. 1 μM of LAP was used as the positive control. Cells were harvested, washed with phosphate saline buffer (PBS), and stained with annexin-V and 7-AAD from Guava Nexin assay (Merck Millipore, USA) as per the manufacturer's instruction and then left at room temperature in the dark for 30 min. Cell apoptosis was determined using Guava EasyCyte HT flow cytometer (Merck Millipore, USA). Data were analyzed by Guava Suite software (Guava Technologies).
Cells were harvested in lysis buffer containing proteinase inhibitor cocktails (Merck Millipore, USA). Equal proteins were loaded and then transferred as previously described (Sakunrangsit et al., 2016). Blots were blocked in blocking buffer for 1 h and incubated with the following primary antibodies overnight at 4 °C: HER-2, AKT, pAKT [Ser473], ERK1/2, pERK1/2 [Tyr702/Tyr704], NCOA3, Caspase 3 and Cleaved Caspase3. After washing with TBST, the membranes were incubated with secondary antibody (Cell Signalling, USA) in TBST buffer for 1 h at ambient temperature. Protein bands were detected as previously described (Sakunrangsit et al., 2016). The band intensity of each protein relative to GAPDH was quantified by using Image Studio 5.2 software (LICOR, Lincoln, USA).
2.5. Real-time quantitative PCR (RT-qPCR)
2.8. Statistical analysis
TRIzol® reagent (Life technologies) was added to the cells according to the manufacturer's instructions so that the total RNA can be extracted
Data were expressed as mean ± SEM. from at least three 3
European Journal of Pharmacology 868 (2020) 172878
N. Sakunrangsit and W. Ketchart
Fig. 3. PLB was unable to inhibit E2-induced cell proliferation and expression of ER-targeted genes in endocrine-resistant cells. (A) Wild-type MCF-7 (B) MCF-7/LCC2 (C) MCF-7/LCC9 cells were treated with or without estrogen (10 nM E2) in the presence of non-toxic concentrations of PLB (0.25–0.75 μM), 0.1 μM 4hydroxytamoxifen (4-OHT) and 0.1 μM fulvestrant (ICI) for 7 days. 4-OHT and ICI were used as positive controls. 0.1% DMSO was used as the negative control. Data were shown as mean ± S.E.M from three independent experiments (n = 3). **P < 0.01 vs. negative control; #P < 0.05, ##P < 0.01 vs PLB alone. (D–F) Expression of CCND1 after the treatment with PLB, 4-OHT or ICI with or without E2 for 24 h *P < 0.05, **P < 0.01 vs. negative control; #P < 0.05 vs. PLB alone. (G–I) Expression of TFF1 (pS2) mRNA in MCF-7/LCC2, MCF-7/LCC9 and SKBR3 after being treated with PLB, 4-OHT and ICI with or without E2 for 24 h. Data were shown as mean ± S.E.M from three independent experiments (n = 3). *P < 0.05, **P < 0.01 vs. negative control; #P < 0.05 vs. PLB alone.
independent experiments and each experiment was performed in triplicates. A statistical hypothesis was determined by one-way ANOVA followed by the Tukey HSD post-hoc test. The statistical significance was accepted at *P < 0.05. The statistical analysis was done using SPSS 22.0 software (Chicago, IL, USA).
HER-2 and the phosphorylation of its downstream signaling proteins such as pAKT and pERK1/2 were upregulated in MCF-7/LCC2 and MCF7/LCC9 cells when compared to MCF-7 wild-type cells and the levels were almost similar to the levels of SKBR3 (HER-2 over-expressed) cell line (Fig. 1C–D).
3. Results
3.2. PLB was able to inhibit cell growth in HER-2 overexpressed cells and reversed the alteration of ESR1 and ERBB-2 mRNA expressions in endocrine resistant breast cancer cells
3.1. The alteration of ER, NCOA3 and HER2 signaling in endocrineresistant and HER-2 overexpressed breast cancer cell lines
Our previous study showed a potent cytotoxicity of PLB against ERpositive and endocrine-resistant breast cancer cells (Table 1) (Sakunrangsit et al., 2016). Since this study observed the increase of HER-2 mRMA and protein in endocrine-resistant cells, the inhibitory effect of PLB on cell growth of HER2-overexpressed cells was further studied to compare the effect in HER-2 overexpressed endocrine resistant cells. The results showed that PLB significantly inhibited cell viability of HER2-overexpressed (SKBR3) cells in a time- and concentration-dependent manner with the half-maximal inhibitory concentration (IC50) values of 2.04 μM and 1.60 μM after treatment for 24 and 48 h, respectively (Fig. 2A–B).
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 mRNA level in endocrine-resistant cells (Fig. 1B–C). However, NCOA3 protein did not express in HER-2 overexpressed (SKBR3) cells (Fig. 1C–D). In addition, 4
European Journal of Pharmacology 868 (2020) 172878
N. Sakunrangsit and W. Ketchart
Fig. 4. The inhibitory effect of PLB on the phosphorylation of AKT and ERK1/2 in endocrine-resistant and HER2-overexpressed breast cancer cells. (A–C) After treatment with PLB for 24 h, the phosphorylation of AKT was inhibited in MCF-7/LCC2, MCF-7/LCC9 and SKBR3 cells. 0.1% DMSO and LAP were used as negative and positive controls, respectively. Data were shown as mean ± S.E.M from three independent experiments (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. negative control. (D–F) After treatment with PLB for 24 h, the phosphorylation of ERK1/2 was inhibited in MCF-7/LCC2, MCF-7/LCC9 and SKBR3 cells. Data were shown as mean ± S.E.M from three independent experiments (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. negative control.
The alterations of the genes involved in tamoxifen resistance were ESR1, ERBB2 and NCoA3 which were observed in both endocrine-resistant cells (Fig. 1B) (Sakunrangsit et al., 2016). PLB was able to increase ESR1 and significantly decrease ERBB-2 and NCoA3 mRNA expressions in MCF-7/LCC2 and MCF-7/LCC9 cells (Fig. 2C–D). Therefore, PLB altered the expression of the genes involved in tamoxifen resistance in both ER and HER-2 signaling pathways.
compared to MCF-7 cells (Fig. 3D–I). These findings suggested that the anti-cancer mechanism of PLB in endocrine resistance was not mediated by ER-dependent pathway. 3.4. PLB inhibited cell growth and phosphorylation of downstream signaling of HER-2 in endocrine-resistant and HER-2 over-expressed cells The inhibitory effect of PLB on HER-2 and its downstream signaling proteins was further examined. PLB significantly decreased phosphorylation of AKT and ERK1/2 in a concentration-dependent manner in MCF-7/LCC2 and MCF-7/LCC9 cells (Fig. 4A–B, D-E). In contrast, the inhibitory effect of PLB on phosphorylation of AKT and ERK1/2 was not significantly different compared to the controls, but lapatinib significantly decreased the phosphorylation of AKT and ERK1/2 in SKBR3 cells (Fig. 4C, F). These results suggested that even though SKBR-3 cells also overexpressed HER-2 like MCF-7/LCC2 and MCF-7/LCC9 cells, however the mechanism of action of PLB through AKT and ERK1/2 pathways was different in endocrine-resistant cells. The effects of PLB on AKT signaling pathway were further investigated using AKT inhibitor (MK2206) or insulin (INS) pretreatment. The inhibitory effect of PLB on cell viability did not change among MCF-7/LCC2 and MCF-7/LCC9 cells pretreated with MK2206 (Fig. 5A–B) while cell viability decreased in the same manner as in
3.3. PLB has no effect on estrogen-induced cell proliferation and expression of ER-targeted genes in endocrine-resistant cells Since PLB treatment increased ESR1 mRNA expression in MCF-7/ LCC2 and MCF-7/LCC9 cells, experiment to investigate the mechanism of PLB in ER-signaling was further performed by inducing the cells with estrogen (estradiol or E2). Estrogen was able to induce cell proliferation only in MCF-7 wild-type cells while estrogen was unable to increase proliferation in MCF-7/LCC2 and MCF-7/LCC9 cells. PLB significantly suppressed estrogen-induced cell proliferation in MCF-7 cells (Fig. 3A). There were no differences in the suppression effect of PLB with or without estrogen between the two resistant MCF-7/LCC2 and MCF-7/ LCC9 cell lines (Fig. 3B–C). Moreover, the inhibitory effect of PLB on the mRNA expression of CCND1 and TFF1 (pS2) which are ER-targeted genes, were attenuated in MCF-7/LCC2 and MCF-7/LCC9 cells when 5
European Journal of Pharmacology 868 (2020) 172878
N. Sakunrangsit and W. Ketchart
Fig. 5. PLB inhibited growth and phosphorylation of AKT in endocrine-resistant and HER2-overexpressed cells. (A–C) MCF-7/LCC2, MCF-7/LCC9 and SKBR3 were pre-treated with AKT inhibitor (0.1 μM MK2206) for 30 min before incubating with PLB (0.5–2 μM) for 24 h. 0.1% DMSO and LAP were used as negative and positive controls, respectively. Data were shown as mean ± S.E.M from three independent experiments (n = 3). ***P < 0.001 vs. negative control; #P < 0.05, ## P < 0.01, ###P < 0.001 vs. PLB alone. (D–F) MCF-7/LCC2, MCF-7/LCC9 and SKBR3 were pre-treated with insulin (0.1 μM INS) for 15 min before incubating with PLB (0.5–2 μM) for 24 h. Data were shown as mean ± S.E.M from three independent experiments (n = 3). **P < 0.01, ***P < 0.001 vs. negative control; # P < 0.05, ##P < 0.01, ###P < 0.001 vs. PLB alone. (G–H) Phosphorylation of AKT was significantly inhibited by PLB in MK2206 or INS-pretreated SKBR3 cells. Data were shown as mean ± S.E.M from three independent experiments (n = 3). *P < 0.05 and **P < 0.01 vs. negative control.
SKBR3 cells (Fig. 5C). In addition, the PLB significantly decreased the phosphorylation of AKT when pretreated with AKT inhibitor when compared to PLB treatment alone in SKBR3 cells whereas the effect of PLB was not different to that of the MCF-7/LCC9 cells (Fig. 5G). Insulin was able to induce cell proliferation and phosphorylation of AKT in endocrine-resistant and HER-2 overexpressed cells (Fig. 5D–H). PLB significantly decreased insulin-induced cell proliferation and insulininduced AKT phosphorylation in both endocrine-resistant and HER-2 overexpressed cells (Fig. 5D–H). Thus, PLB and AKT inhibitor has synergistic effect in HER-2 overexpressed breast cancer cells.
of PLB on cell apoptosis increased when the cells were pre-treated with MK2206 in SKBR3 cells (Fig. 7C) but this effect was not seen in MCF-7/ LCC2 and MCF-7/LCC9 cells (Fig. 7A–B). Moreover, the effect of PLB on mRNA expression of anti-apoptotic genes also was similar to the apoptotic effect in all cell lines pretreated with MK2206 (Fig. 7D–F). Therefore, this finding also suggested the synergistic effect of PLB and AKT inhibitor in inducing cell apoptosis in only HER-2 overexpressed breast cancer cells. 3.6. The inhibitory effect of PLB on phosphorylation of AKT was attenuated when NCOA3 was down-regulated in endocrine resistant HER-2 overexpressed cells
3.5. PLB induced cell apoptosis in endocrine resistant cells and HER-2 overexpressed cells
The inhibitory mechanism of PLB on phosphorylation of AKT in endocrine resistant cells were further studied. Since the expression level of NCOA3 was higher in endocrine resistant HER-2 overexpressed cells when compared to HER-2 overexpressed cells, NCOA3siRNA was used to knockdown NCOA3 (Fig. S2) and determined the effect of PLB. The result showed that the inhibitory effect of PLB on phosphorylated AKT was attenuated in both MCF-7/LCC2 and MCF-7/LCC9 cells (Fig. 8A–B). Thus, NCOA3 mediated the inhibitory effect of PLB on phosphorylation of AKT in endocrine resistant HER-2 overexpressed cells.
PLB significantly induced cell apoptosis at very low concentrations. 0.5 μM PLB could induce apoptosis MCF-7/LCC9 and SKBR3 cells. 1.0 μM PLB could induce apoptosis in MCF-7/LCC2 cells (Fig. 6A–B). PLB was able to inhibit mRNA expression of anti-apoptotic genes such as BCL-2 and PROCASP3 (Fig. 6C–E). Moreover, PLB increased Cleaved Caspase 3 protein level in a concentration-dependent manner in all three cell lines (Fig. 6F–H). The inhibitory mechanism of PLB on cell apoptosis was further investigated using AKT inhibitor. The result demonstrated that the effect 6
European Journal of Pharmacology 868 (2020) 172878
N. Sakunrangsit and W. Ketchart
Fig. 6. PLB induced apoptosis in endocrine-resistant and HER2-overexpressed cells. (A) Dot plot distribution of cell apoptosis of MCF-7/LCC2, MCF-7/LCC9 and SKBR3 cells after PLB treatment for 48 h. 0.1% DMSO and LAP were used as negative and positive controls, respectively. (B) The proportion percentages of apoptotic (early and late apoptosis) cells were shown as percentage mean ± SEM from three independent experiments (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. negative control. (C–E) The mRNA expression of anti-apoptotic genes after treatment with PLB (0.5–2 μM) for 24 h. Data were shown as mean ± S.E.M from three independent experiments (n = 3). *P < 0.05, **P < 0.01 vs. negative control. (F–H) The protein expression of Cleaved Caspase 3after treatment with PLB (0.5–2 μM) for 24 h. LAP was used as a positive control. Data were shown as mean ± S.E.M from three independent experiments (n = 3). *P < 0.05, **P < 0.01 vs. negative control.
4. Discussion
estrogen-induced cell proliferation and ER-targeted genes, suggesting that the inhibitory effect of PLB was regulated through HER-2. The downstream signaling molecules of HER-2 were studied and the inhibitory effects of PLB on pAKT and pERK1/2 were observed. This mechanism of PLB is consistent with the effect of other nathoquinone compound, rementaceone, which can inhibit PI3K/AKT signaling pathway in HER-2 overexpressed breast cancer cells (Kawiak and Lojkowska, 2016). The result demonstrated that PLB significantly inhibited phosphorylation of AKT in HER-2 overexpressed endocrine-resistant cells. However, this inhibitory effect of PLB in HER-2 overexpressed (SKBR3) cells was different
The mechanisms of tamoxifen or endocrine resistance involve the alteration of ERα expression and the over-expression of HER-2 (GarciaBecerra 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 ERBB2 mRNA expression and HER-2 protein. However, PLB had no effect on 7
European Journal of Pharmacology 868 (2020) 172878
N. Sakunrangsit and W. Ketchart
Fig. 7. Inhibition of AKT by PLB affects cell apoptosis in endocrine-resistant cells and HER2-overexpressed cells. (A–C) The flow cytogram and the percentage of cell apoptosis after MCF-7/LCC2, MCF-7/LCC9 and SKBR3 cells were pre-treated with or without AKT inhibitor (0.1 μM MK2206) for 30 min and then treated with PLB (0.5–2 μM) for 24 h. 0.1% DMSO and LAP were used as negative and positive controls, respectively. Data were shown as mean ± S.E.M from three independent experiments (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. negative control; #P < 0.05, ##P < 0.01 vs. PLB alone. (D–F) The mRNA expression of antiapoptotic genes after pretreatment with AKT inhibitor for 30 min followed by treatment with PLB (0.5–2 μM) for 24 h. Data were shown as mean from three independent experiments (n = 3). *P < 0.05, **P < 0.01 vs. negative control.
from HER-2 over-expressed endocrine-resistant cells when pre-treated the cells with AKT inhibitor. The synergistic effect of PLB and AKT inhibitor was observed in the inhibition of cell proliferation, apoptosis and phosphorylation of AKT only in HER-2 overexpressed (SKBR3) cells. This finding suggested the direct mechanism of AKT inhibition of PLB in this cell line. Moreover, HER-2 overexpressed SKBR3 cells expressed very low levels of NCoA3. Overexpression of NCOA3 can activate AKT signaling pathway in breast cancer (Li et al., 2018). NCoA3 was also reported to regulate genes involved in insulin/AKT pathway in prostate cancer (Yan
et al., 2006). Our study demonstrated that the inhibitory effect of PLB on pAKT expression was attenuated when NCOA3 was downregulated. Therefore, the inhibitory effect of PLB in HER-2 overexpressed endocrineresistant cells regulated through NCoA3. In conclusion, PLB has better potency in endocrine-resistant breast cancer cells than HER-2 overexpressed cells since the better effects of HER-2 overexpressed cells were observed in lapatinib which is its standard treatment. PLB uses a different mechanism to inhibit the phosphorylation of AKT and apoptosis in endocrine-resistant cells when compared to overexpressed HER-2 breast 8
European Journal of Pharmacology 868 (2020) 172878
N. Sakunrangsit and W. Ketchart
Fig. 8. NCOA3 mediated the inhibitory effect of PLB on phosphorylation of AKT in endocrine resistant HER-2 overexpressed cells. (A) MCF7/LCC2 and (B) MCF7/LCC9 cells were transfected with control siRNA or siNCOA3 and treated with PLB (0.5–2 μM) for 24 h. Data were shown as mean from three independent experiments (n = 3). **P < 0.01 and ***P < 0.001 vs. control, and #P < 0.05 and ##P < 0.01 vs. PLB alone.
cancer cells. Thus, PLB is a good candidate in only HER-2 overexpressed endocrine resistant breast cancer cells.
Wannarasmi Ketchart: Conceptualization, Methodology, Investigation, Formal analysis, Writing- Original draft preparation, Figures, Review and Editing and Supervision.
Ethical consideration
References
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).
Ahmad, A., Banerjee, S., Wang, Z., Kong, D., Sarkar, F.H., 2008. Plumbagin-induced apoptosis of human breast cancer cells is mediated by inactivation of NF-kappaB and Bcl-2. J. Cell. Biochem. 105, 1461–1471. Atmaca, H., Ozkan, A.N., Zora, M., 2017. Novel ferrocenyl pyrazoles inhibit breast cancer cell viability via induction of apoptosis and inhibition of PI3K/Akt and ERK1/2 signaling. Chem. Biol. Interact. 263, 28–35. Aziz, M.H., Dreckschmidt, N.E., Verma, A.K., 2008. Plumbagin, a medicinal plant-derived naphthoquinone, is a novel inhibitor of the growth and invasion of hormone-refractory prostate cancer. Cancer Res. 68, 9024–9032. Chen, M.B., Zhang, Y., Wei, M.X., Shen, W., Wu, X.Y., Yao, C., Lu, P.H., 2013. Activation of AMP-activated protein kinase (AMPK) mediates plumbagin-induced apoptosis and growth inhibition in cultured human colon cancer cells. Cell. Signal. 25, 1993–2002. Droog, M., Beelen, K., Linn, S., Zwart, W., 2013. Tamoxifen resistance: from bench to bedside. Eur. J. Pharmacol. 717, 47–57. Gan, Y., Shi, C., Inge, L., Hibner, M., Balducci, J., Huang, Y., 2010. Differential roles of ERK and Akt pathways in regulation of EGFR-mediated signaling and motility in prostate cancer cells. Oncogene 29, 4947–4958. Garcia-Becerra, R., Santos, N., Diaz, L., Camacho, J., 2012. Mechanisms of resistance to endocrine therapy in breast cancer: focus on signaling pathways, miRNAs and genetically based resistance. Int. J. Mol. Sci. 14, 108–145. Hosford, S.R., Miller, T.W., 2014. Clinical potential of novel therapeutic targets in breast cancer: CDK4/6, Src, JAK/STAT, PARP, HDAC, and PI3K/AKT/mTOR pathways. Pharmgenomics Pers Med 7, 203–215. Hu, T., Zhou, R., Zhao, Y., Wu, G., 2016. Integrin alpha6/Akt/Erk signaling is essential for human breast cancer resistance to radiotherapy. Sci. Rep. 6, 33376. Kawiak, A., Lojkowska, E., 2016. Ramentaceone, a naphthoquinone derived from Drosera sp., induces apoptosis by suppressing PI3K/Akt signaling in breast cancer cells. PLoS One 11, e0147718. Kawiak, A., Zawacka-Pankau, J., Lojkowska, E., 2012. Plumbagin induces apoptosis in Her2-overexpressing breast cancer cells through the mitochondrial-mediated pathway. J. Nat. Prod. 75, 747–751. Lewis, J.S., Jordan, V.C., 2005. Selective estrogen receptor modulators (SERMs): mechanisms of anticarcinogenesis and drug resistance. Mutat. Res. 591, 247–263. Li, Z., Deng, X., Wu, G., Qiu, R., Ju, X., Wang, Y., Zhang, J., 2018. The PI3K and AIB1 interaction is involved in estrogen treated breast cancer cells. Cell. Mol. Biol. 64, 65–70. Powolny, A.A., Singh, S.V., 2008. Plumbagin-induced apoptosis in human prostate cancer cells is associated with modulation of cellular redox status and generation of reactive oxygen species. Pharm. Res. 25, 2171–2180. Pradubyat, N., Sakunrangsit, N., Mutirangura, A., Ketchart, W., 2020. NADPH: Quinone Oxidoreductase 1 (NQO1) Mediated Anti-cancer Effects of Plumbagin in Endocrine
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. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ejphar.2019.172878. Author contribution Nithidol Sakunrangsit: Methodology, Investigation, Formal analysis, Writing- Figures. 9
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Bangchang, K., 2014. Antimalarial activity of plumbagin in vitro and in animal models. BMC Complement Altern. Med. 14, 15. Yan, J., Yu, C.T., Ozen, M., Ittmann, M., Tsai, S.Y., Tsai, M.J., 2006. Steroid receptor coactivator-3 and activator protein-1 coordinately regulate the transcription of components of the insulin-like growth factor/AKT signaling pathway. Cancer Res. 66, 11039–11046. Zhu, J., Yao, J., Huang, R., Wang, Y., Jia, M., Huang, Y., 2018. Ghrelin promotes human non-small cell lung cancer A549 cell proliferation through PI3K/Akt/mTOR/P70S6K and ERK signaling pathways. Biochem. Biophys. Res. Commun. 498, 616–620.
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Full length article
Plumbagin inhibited AKT signaling pathway in HER-2 overexpressedendocrine resistant breast cancer cells
T
Nithidol Sakunrangsit, Wannarasmi Ketchart∗ Overcoming Cancer Drug Resistance Research Unit, Department of Pharmacology, Faculty of Medicine, Chulalongkorn University, Bangkok, 10330, Thailand
ARTICLE INFO
ABSTRACT
Keywords: PLB HER-2 Endocrine-resistance AKT
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 HER2 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 endocrineresistant and HER-2 overexpressed cells. However, the inhibitory effect of PLB was more obvious when pretreated 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.
1. Introduction
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)
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
∗
Corresponding author. Department of Pharmacology, Chulalongkorn University, 1873 Rama IV Road, Pathumwan, Bangkok, 10330, Thailand. E-mail address: [email protected] (W. Ketchart).
https://doi.org/10.1016/j.ejphar.2019.172878 Received 5 July 2019; Received in revised form 11 December 2019; Accepted 17 December 2019 Available online 19 December 2019 0014-2999/ © 2019 Elsevier B.V. All rights reserved.
European Journal of Pharmacology 868 (2020) 172878
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Fig. 1. The expression of ER, NCOA3 and HER2 signaling in endocrine-resistant and HER-2 overexpressed breast cancer cells. (A) Morphology of wild-type MCF-7, endocrine-resistant MCF-7/LCC2, MCF-7/LCC9 and HER2-overexpressed SKBR3 cells. Cells were routinely cultured and then captured by using a light microscope with digital camera. Scale bar = 100 μm. (B) Real-time PCR results of ESR1, NCOA3 and ERBB2 mRNA levels for each cell line. Data were shown as mean ± S.E.M from three independent experiments (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. MCF-7 cells. (C) Western blots of HER2, NCOA3 and AKT/ ERK proteins. (D) The analysis of HER2, NCOA3 and AKT/ERK signaling protein expression in endocrine-resistant and HER2-overexpressed cells. Data were shown as mean ± S.E.M from three independent experiments (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. MCF-7 cells.
2. Materials and methods
Table 1 The IC50 values of PLB in endocrine resistant and HER-2 overexpressed breast cancer cells. Cell line MCF-7 MCF-7/LCC2 MCF-7/LCC9 SKBR3
IC50 (μM) 24 h 1.47 1.69 1.24 2.04
2.1. Cell lines and cultures
Reference
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 York, USA). SKBR3 cell line was maintained in McCoy's 5A Medium containing 5% FBS (ATCC; Virginia, USA). All cell lines were grown at 37 °C under 5% CO2. Endocrine resistant cell lines were routinely checked to ensure that they were resistant to tamoxifen using MTT assay.
48 h ± ± ± ±
0.05 0.01 0.05 0.04
1.22 1.58 1.17 1.60
± ± ± ±
0.01 0.06 0.02 0.01
Sakunrangsit et al. (2016) Sakunrangsit et al. (2016) Sakunrangsit et al. (2016) N/A
Results are expressed as mean ± S.E.M in triplicate from three-independent experiments (n = 3). IC50 represents the half maximal inhibitory concentration. PLB = Plumbagin, HER-2 = Human epidermal growth factor 2, N/A = Not assessed previously.
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.
2.2. Reagents Plumbagin (PLB) and 4-hydroxytamoxifen (4-OHT) were purchased from Sigma-Aldrich (Missouri, USA). Fulvestrant (ICI) and lapatinib (LAP) were obtained from Abcam (Cambridge, UK). MK2206 (AKT inhibitor) was obtained from PreproTech (New Jersey, USA). Human recombinant insulin (AKT activator) was purchased from Invitrogen (California, USA). PLB, LAP and MK2206 were dissolved in dimethyl sulfoxide (DMSO). 4-OHT and ICI were prepared in absolute ethanol as 10 mM stock solution. esiRNA human NCOA3 was purchased from Sigma-Aldrich (Missouri, USA). All antibodies include HER2, AKT, pAKT [Ser473], ERK1/2, pERK1/2 [Tyr702/Tyr704], NCOA3, Caspase3, Cleaved Caspase 3 and GAPDH were purchased from Cell Signaling Technology (Massachusetts, USA). 2
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Fig. 2. The inhibitory effect of PLB in HER2-overexpressed cells on cell proliferation and mRNA expressions of ERBB2, ER and NCOA3. (A, B) Relative cell viability and IC50 values of PLB in HER2-overexpressed (SKBR3) cells for 24 h and 48 h. 0.1% DMSO and lapatinib (LAP) were used as negative and positive controls, respectively. *P < 0.05, **P < 0.01, ***P < 0.001 vs. negative control. (C, D) mRNA expression of ERBB2, ESR1 and NCOA3 after 24-h treatment with PLB in MCF-7/LCC2 and MCF-7/LCC9 cells. Data were shown as mean ± S.E.M from three independent experiments (n = 3). *P < 0.05, **P < 0.01 vs. negative control.
2.3. MTT assay
from the cells. The amount and purity of RNA were determined using a NanoDrop-One (Thermo Scientific). One μg of total RNAs was converted to cDNA by amplifying the genes of interest. cDNA was obtained by using specific primers to estrogen receptor 1 (ESR1), epidermal growth factor 2 (ERBB2), NCOA3, B-cell lymphoma 2 (BCL2), and procaspase 3 (PROCASP3). The primers used in this study were designed by PrimerBLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) as shown in Supplementary Data. Amplification and detection was performed using SYBR Green qPCR supermix by StepOne™ Real-Time PCR System (Applied Biosystems, USA). GAPDH was used as an internal control. Relative expression was calculated with the comparative CT method.
2.3.1. Estrogen-induced proliferation MCF-7, MCF-7/LCC2 and MCF-7/LCC9 cells were maintained in phenol red-free IMEM supplemented with 5% charcoal dextran-treated FBS so that the cells were not exposed to hormones at least 4 days prior to the experiment. 5×103 cells/well of MCF-7 were seeded into 96-well plates with complete IMEM medium overnight and then treated with PLB at concentrations of 0.25, 0.5 and 0.75 μM with or without 10 nM estradiol (E2) for 7 days. 4-hydroxytamoxifen (4-OHT) and ICI at 0.1 μM were used as positive controls and 0.1% DMSO was used as a vehicle control. MTT assay was performed after incubation. Percentage of cell viability was calculated.
2.6. NCOA3 siRNA transfection
2.3.2. AKT inhibitor and insulin pre-treatment MCF-7/LCC2, MCF-7/LCC9 and SKBR3 cells were cultured in serum-free media (SFM) for 2 h before being treated with 0.1 μM AKT inhibitor (MK2206) for 30 min or 0.1 μM insulin for 15 min. After that, different concentrations of PLB was used for MTT, apoptotic assay and Western blot. LAP was used as a positive control.
siRNA-NCOA3 (siNCOA3) or scramble control siRNA was transiently transfected into MCF7/LCC2 or MCF7/LCC9 cells using Lipofectamine. Cells were harvested after 24 h. NCOA3 expression was checked by Western blot. The expression of pAKT and AKT was determined in siNCOA3 transfected cells compared to wild-type cells by Western blot.
2.4. Apoptotic assay
2.7. Western blot analysis
MCF-7/LCC2, MCF-7/LCC9, and SKBR3 cells were seeded in 24-well plate with a density of 2x105 cells/ml. One groups of cells were pretreated with 0.1 μM MK2206 (AKT inhibitor). Then, all samples were treated with 0.5,1 and 2 μM of PLB. 1 μM of LAP was used as the positive control. Cells were harvested, washed with phosphate saline buffer (PBS), and stained with annexin-V and 7-AAD from Guava Nexin assay (Merck Millipore, USA) as per the manufacturer's instruction and then left at room temperature in the dark for 30 min. Cell apoptosis was determined using Guava EasyCyte HT flow cytometer (Merck Millipore, USA). Data were analyzed by Guava Suite software (Guava Technologies).
Cells were harvested in lysis buffer containing proteinase inhibitor cocktails (Merck Millipore, USA). Equal proteins were loaded and then transferred as previously described (Sakunrangsit et al., 2016). Blots were blocked in blocking buffer for 1 h and incubated with the following primary antibodies overnight at 4 °C: HER-2, AKT, pAKT [Ser473], ERK1/2, pERK1/2 [Tyr702/Tyr704], NCOA3, Caspase 3 and Cleaved Caspase3. After washing with TBST, the membranes were incubated with secondary antibody (Cell Signalling, USA) in TBST buffer for 1 h at ambient temperature. Protein bands were detected as previously described (Sakunrangsit et al., 2016). The band intensity of each protein relative to GAPDH was quantified by using Image Studio 5.2 software (LICOR, Lincoln, USA).
2.5. Real-time quantitative PCR (RT-qPCR)
2.8. Statistical analysis
TRIzol® reagent (Life technologies) was added to the cells according to the manufacturer's instructions so that the total RNA can be extracted
Data were expressed as mean ± SEM. from at least three 3
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Fig. 3. PLB was unable to inhibit E2-induced cell proliferation and expression of ER-targeted genes in endocrine-resistant cells. (A) Wild-type MCF-7 (B) MCF-7/LCC2 (C) MCF-7/LCC9 cells were treated with or without estrogen (10 nM E2) in the presence of non-toxic concentrations of PLB (0.25–0.75 μM), 0.1 μM 4hydroxytamoxifen (4-OHT) and 0.1 μM fulvestrant (ICI) for 7 days. 4-OHT and ICI were used as positive controls. 0.1% DMSO was used as the negative control. Data were shown as mean ± S.E.M from three independent experiments (n = 3). **P < 0.01 vs. negative control; #P < 0.05, ##P < 0.01 vs PLB alone. (D–F) Expression of CCND1 after the treatment with PLB, 4-OHT or ICI with or without E2 for 24 h *P < 0.05, **P < 0.01 vs. negative control; #P < 0.05 vs. PLB alone. (G–I) Expression of TFF1 (pS2) mRNA in MCF-7/LCC2, MCF-7/LCC9 and SKBR3 after being treated with PLB, 4-OHT and ICI with or without E2 for 24 h. Data were shown as mean ± S.E.M from three independent experiments (n = 3). *P < 0.05, **P < 0.01 vs. negative control; #P < 0.05 vs. PLB alone.
independent experiments and each experiment was performed in triplicates. A statistical hypothesis was determined by one-way ANOVA followed by the Tukey HSD post-hoc test. The statistical significance was accepted at *P < 0.05. The statistical analysis was done using SPSS 22.0 software (Chicago, IL, USA).
HER-2 and the phosphorylation of its downstream signaling proteins such as pAKT and pERK1/2 were upregulated in MCF-7/LCC2 and MCF7/LCC9 cells when compared to MCF-7 wild-type cells and the levels were almost similar to the levels of SKBR3 (HER-2 over-expressed) cell line (Fig. 1C–D).
3. Results
3.2. PLB was able to inhibit cell growth in HER-2 overexpressed cells and reversed the alteration of ESR1 and ERBB-2 mRNA expressions in endocrine resistant breast cancer cells
3.1. The alteration of ER, NCOA3 and HER2 signaling in endocrineresistant and HER-2 overexpressed breast cancer cell lines
Our previous study showed a potent cytotoxicity of PLB against ERpositive and endocrine-resistant breast cancer cells (Table 1) (Sakunrangsit et al., 2016). Since this study observed the increase of HER-2 mRMA and protein in endocrine-resistant cells, the inhibitory effect of PLB on cell growth of HER2-overexpressed cells was further studied to compare the effect in HER-2 overexpressed endocrine resistant cells. The results showed that PLB significantly inhibited cell viability of HER2-overexpressed (SKBR3) cells in a time- and concentration-dependent manner with the half-maximal inhibitory concentration (IC50) values of 2.04 μM and 1.60 μM after treatment for 24 and 48 h, respectively (Fig. 2A–B).
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 mRNA level in endocrine-resistant cells (Fig. 1B–C). However, NCOA3 protein did not express in HER-2 overexpressed (SKBR3) cells (Fig. 1C–D). In addition, 4
European Journal of Pharmacology 868 (2020) 172878
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Fig. 4. The inhibitory effect of PLB on the phosphorylation of AKT and ERK1/2 in endocrine-resistant and HER2-overexpressed breast cancer cells. (A–C) After treatment with PLB for 24 h, the phosphorylation of AKT was inhibited in MCF-7/LCC2, MCF-7/LCC9 and SKBR3 cells. 0.1% DMSO and LAP were used as negative and positive controls, respectively. Data were shown as mean ± S.E.M from three independent experiments (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. negative control. (D–F) After treatment with PLB for 24 h, the phosphorylation of ERK1/2 was inhibited in MCF-7/LCC2, MCF-7/LCC9 and SKBR3 cells. Data were shown as mean ± S.E.M from three independent experiments (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. negative control.
The alterations of the genes involved in tamoxifen resistance were ESR1, ERBB2 and NCoA3 which were observed in both endocrine-resistant cells (Fig. 1B) (Sakunrangsit et al., 2016). PLB was able to increase ESR1 and significantly decrease ERBB-2 and NCoA3 mRNA expressions in MCF-7/LCC2 and MCF-7/LCC9 cells (Fig. 2C–D). Therefore, PLB altered the expression of the genes involved in tamoxifen resistance in both ER and HER-2 signaling pathways.
compared to MCF-7 cells (Fig. 3D–I). These findings suggested that the anti-cancer mechanism of PLB in endocrine resistance was not mediated by ER-dependent pathway. 3.4. PLB inhibited cell growth and phosphorylation of downstream signaling of HER-2 in endocrine-resistant and HER-2 over-expressed cells The inhibitory effect of PLB on HER-2 and its downstream signaling proteins was further examined. PLB significantly decreased phosphorylation of AKT and ERK1/2 in a concentration-dependent manner in MCF-7/LCC2 and MCF-7/LCC9 cells (Fig. 4A–B, D-E). In contrast, the inhibitory effect of PLB on phosphorylation of AKT and ERK1/2 was not significantly different compared to the controls, but lapatinib significantly decreased the phosphorylation of AKT and ERK1/2 in SKBR3 cells (Fig. 4C, F). These results suggested that even though SKBR-3 cells also overexpressed HER-2 like MCF-7/LCC2 and MCF-7/LCC9 cells, however the mechanism of action of PLB through AKT and ERK1/2 pathways was different in endocrine-resistant cells. The effects of PLB on AKT signaling pathway were further investigated using AKT inhibitor (MK2206) or insulin (INS) pretreatment. The inhibitory effect of PLB on cell viability did not change among MCF-7/LCC2 and MCF-7/LCC9 cells pretreated with MK2206 (Fig. 5A–B) while cell viability decreased in the same manner as in
3.3. PLB has no effect on estrogen-induced cell proliferation and expression of ER-targeted genes in endocrine-resistant cells Since PLB treatment increased ESR1 mRNA expression in MCF-7/ LCC2 and MCF-7/LCC9 cells, experiment to investigate the mechanism of PLB in ER-signaling was further performed by inducing the cells with estrogen (estradiol or E2). Estrogen was able to induce cell proliferation only in MCF-7 wild-type cells while estrogen was unable to increase proliferation in MCF-7/LCC2 and MCF-7/LCC9 cells. PLB significantly suppressed estrogen-induced cell proliferation in MCF-7 cells (Fig. 3A). There were no differences in the suppression effect of PLB with or without estrogen between the two resistant MCF-7/LCC2 and MCF-7/ LCC9 cell lines (Fig. 3B–C). Moreover, the inhibitory effect of PLB on the mRNA expression of CCND1 and TFF1 (pS2) which are ER-targeted genes, were attenuated in MCF-7/LCC2 and MCF-7/LCC9 cells when 5
European Journal of Pharmacology 868 (2020) 172878
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Fig. 5. PLB inhibited growth and phosphorylation of AKT in endocrine-resistant and HER2-overexpressed cells. (A–C) MCF-7/LCC2, MCF-7/LCC9 and SKBR3 were pre-treated with AKT inhibitor (0.1 μM MK2206) for 30 min before incubating with PLB (0.5–2 μM) for 24 h. 0.1% DMSO and LAP were used as negative and positive controls, respectively. Data were shown as mean ± S.E.M from three independent experiments (n = 3). ***P < 0.001 vs. negative control; #P < 0.05, ## P < 0.01, ###P < 0.001 vs. PLB alone. (D–F) MCF-7/LCC2, MCF-7/LCC9 and SKBR3 were pre-treated with insulin (0.1 μM INS) for 15 min before incubating with PLB (0.5–2 μM) for 24 h. Data were shown as mean ± S.E.M from three independent experiments (n = 3). **P < 0.01, ***P < 0.001 vs. negative control; # P < 0.05, ##P < 0.01, ###P < 0.001 vs. PLB alone. (G–H) Phosphorylation of AKT was significantly inhibited by PLB in MK2206 or INS-pretreated SKBR3 cells. Data were shown as mean ± S.E.M from three independent experiments (n = 3). *P < 0.05 and **P < 0.01 vs. negative control.
SKBR3 cells (Fig. 5C). In addition, the PLB significantly decreased the phosphorylation of AKT when pretreated with AKT inhibitor when compared to PLB treatment alone in SKBR3 cells whereas the effect of PLB was not different to that of the MCF-7/LCC9 cells (Fig. 5G). Insulin was able to induce cell proliferation and phosphorylation of AKT in endocrine-resistant and HER-2 overexpressed cells (Fig. 5D–H). PLB significantly decreased insulin-induced cell proliferation and insulininduced AKT phosphorylation in both endocrine-resistant and HER-2 overexpressed cells (Fig. 5D–H). Thus, PLB and AKT inhibitor has synergistic effect in HER-2 overexpressed breast cancer cells.
of PLB on cell apoptosis increased when the cells were pre-treated with MK2206 in SKBR3 cells (Fig. 7C) but this effect was not seen in MCF-7/ LCC2 and MCF-7/LCC9 cells (Fig. 7A–B). Moreover, the effect of PLB on mRNA expression of anti-apoptotic genes also was similar to the apoptotic effect in all cell lines pretreated with MK2206 (Fig. 7D–F). Therefore, this finding also suggested the synergistic effect of PLB and AKT inhibitor in inducing cell apoptosis in only HER-2 overexpressed breast cancer cells. 3.6. The inhibitory effect of PLB on phosphorylation of AKT was attenuated when NCOA3 was down-regulated in endocrine resistant HER-2 overexpressed cells
3.5. PLB induced cell apoptosis in endocrine resistant cells and HER-2 overexpressed cells
The inhibitory mechanism of PLB on phosphorylation of AKT in endocrine resistant cells were further studied. Since the expression level of NCOA3 was higher in endocrine resistant HER-2 overexpressed cells when compared to HER-2 overexpressed cells, NCOA3siRNA was used to knockdown NCOA3 (Fig. S2) and determined the effect of PLB. The result showed that the inhibitory effect of PLB on phosphorylated AKT was attenuated in both MCF-7/LCC2 and MCF-7/LCC9 cells (Fig. 8A–B). Thus, NCOA3 mediated the inhibitory effect of PLB on phosphorylation of AKT in endocrine resistant HER-2 overexpressed cells.
PLB significantly induced cell apoptosis at very low concentrations. 0.5 μM PLB could induce apoptosis MCF-7/LCC9 and SKBR3 cells. 1.0 μM PLB could induce apoptosis in MCF-7/LCC2 cells (Fig. 6A–B). PLB was able to inhibit mRNA expression of anti-apoptotic genes such as BCL-2 and PROCASP3 (Fig. 6C–E). Moreover, PLB increased Cleaved Caspase 3 protein level in a concentration-dependent manner in all three cell lines (Fig. 6F–H). The inhibitory mechanism of PLB on cell apoptosis was further investigated using AKT inhibitor. The result demonstrated that the effect 6
European Journal of Pharmacology 868 (2020) 172878
N. Sakunrangsit and W. Ketchart
Fig. 6. PLB induced apoptosis in endocrine-resistant and HER2-overexpressed cells. (A) Dot plot distribution of cell apoptosis of MCF-7/LCC2, MCF-7/LCC9 and SKBR3 cells after PLB treatment for 48 h. 0.1% DMSO and LAP were used as negative and positive controls, respectively. (B) The proportion percentages of apoptotic (early and late apoptosis) cells were shown as percentage mean ± SEM from three independent experiments (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. negative control. (C–E) The mRNA expression of anti-apoptotic genes after treatment with PLB (0.5–2 μM) for 24 h. Data were shown as mean ± S.E.M from three independent experiments (n = 3). *P < 0.05, **P < 0.01 vs. negative control. (F–H) The protein expression of Cleaved Caspase 3after treatment with PLB (0.5–2 μM) for 24 h. LAP was used as a positive control. Data were shown as mean ± S.E.M from three independent experiments (n = 3). *P < 0.05, **P < 0.01 vs. negative control.
4. Discussion
estrogen-induced cell proliferation and ER-targeted genes, suggesting that the inhibitory effect of PLB was regulated through HER-2. The downstream signaling molecules of HER-2 were studied and the inhibitory effects of PLB on pAKT and pERK1/2 were observed. This mechanism of PLB is consistent with the effect of other nathoquinone compound, rementaceone, which can inhibit PI3K/AKT signaling pathway in HER-2 overexpressed breast cancer cells (Kawiak and Lojkowska, 2016). The result demonstrated that PLB significantly inhibited phosphorylation of AKT in HER-2 overexpressed endocrine-resistant cells. However, this inhibitory effect of PLB in HER-2 overexpressed (SKBR3) cells was different
The mechanisms of tamoxifen or endocrine resistance involve the alteration of ERα expression and the over-expression of HER-2 (GarciaBecerra 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 ERBB2 mRNA expression and HER-2 protein. However, PLB had no effect on 7
European Journal of Pharmacology 868 (2020) 172878
N. Sakunrangsit and W. Ketchart
Fig. 7. Inhibition of AKT by PLB affects cell apoptosis in endocrine-resistant cells and HER2-overexpressed cells. (A–C) The flow cytogram and the percentage of cell apoptosis after MCF-7/LCC2, MCF-7/LCC9 and SKBR3 cells were pre-treated with or without AKT inhibitor (0.1 μM MK2206) for 30 min and then treated with PLB (0.5–2 μM) for 24 h. 0.1% DMSO and LAP were used as negative and positive controls, respectively. Data were shown as mean ± S.E.M from three independent experiments (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. negative control; #P < 0.05, ##P < 0.01 vs. PLB alone. (D–F) The mRNA expression of antiapoptotic genes after pretreatment with AKT inhibitor for 30 min followed by treatment with PLB (0.5–2 μM) for 24 h. Data were shown as mean from three independent experiments (n = 3). *P < 0.05, **P < 0.01 vs. negative control.
from HER-2 over-expressed endocrine-resistant cells when pre-treated the cells with AKT inhibitor. The synergistic effect of PLB and AKT inhibitor was observed in the inhibition of cell proliferation, apoptosis and phosphorylation of AKT only in HER-2 overexpressed (SKBR3) cells. This finding suggested the direct mechanism of AKT inhibition of PLB in this cell line. Moreover, HER-2 overexpressed SKBR3 cells expressed very low levels of NCoA3. Overexpression of NCOA3 can activate AKT signaling pathway in breast cancer (Li et al., 2018). NCoA3 was also reported to regulate genes involved in insulin/AKT pathway in prostate cancer (Yan
et al., 2006). Our study demonstrated that the inhibitory effect of PLB on pAKT expression was attenuated when NCOA3 was downregulated. Therefore, the inhibitory effect of PLB in HER-2 overexpressed endocrineresistant cells regulated through NCoA3. In conclusion, PLB has better potency in endocrine-resistant breast cancer cells than HER-2 overexpressed cells since the better effects of HER-2 overexpressed cells were observed in lapatinib which is its standard treatment. PLB uses a different mechanism to inhibit the phosphorylation of AKT and apoptosis in endocrine-resistant cells when compared to overexpressed HER-2 breast 8
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Fig. 8. NCOA3 mediated the inhibitory effect of PLB on phosphorylation of AKT in endocrine resistant HER-2 overexpressed cells. (A) MCF7/LCC2 and (B) MCF7/LCC9 cells were transfected with control siRNA or siNCOA3 and treated with PLB (0.5–2 μM) for 24 h. Data were shown as mean from three independent experiments (n = 3). **P < 0.01 and ***P < 0.001 vs. control, and #P < 0.05 and ##P < 0.01 vs. PLB alone.
cancer cells. Thus, PLB is a good candidate in only HER-2 overexpressed endocrine resistant breast cancer cells.
Wannarasmi Ketchart: Conceptualization, Methodology, Investigation, Formal analysis, Writing- Original draft preparation, Figures, Review and Editing and Supervision.
Ethical consideration
References
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).
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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. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ejphar.2019.172878. Author contribution Nithidol Sakunrangsit: Methodology, Investigation, Formal analysis, Writing- Figures. 9
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