1′-Acetoxychavicol Acetate from Alpinia galanga Represses Proliferation and Invasion, and Induces Apoptosis via HER2-signaling in Endocrine-Resistant Breast Cancer Cells

• Abstract
• Introduction
• Results
• Discussion
• Materials and Methods
• Extraction and purification of bioactive compound
• Molecular docking simulation
• Maintenance of breast cancer cells
• Cell viability assay
• Colony-forming assay
• Real-time qPCR
• Western blot analysis
• Invasion assay
• In vivo acute toxicity test
• In vivo growth and proliferation
• Statistical analysis
• Ethical Considerations
• Contributorsʼ Statement
• References

Abstract

Estrogen receptor-positive breast cancer patients have a good prognosis, but 30% of these patients will experience recurrence due to the development of resistance through various signaling pathways. This study aimed to evaluate the mode of anticancer effects of 1′-acetoxychavicol acetate, which is isolated from the rhizomes of Alpinia galanga in estrogen receptor positive (MCF7) human epidermal growth factor receptor 2-overexpressed (MCF7/HER2), and endocrine-resistant breast cancer cells (MCF7/LCC2 and MCF7/LCC9). 1′-Acetoxychavicol acetate showed antiproliferation in a concentration- and time-dependent fashion and had higher potency in human epidermal growth factor receptor 2-overexpressed cell lines. This was associated with down-regulation of human epidermal growth factor receptor 2, pERK1/2, pAKT, estrogen receptor coactivator, cyclin D1, and MYC proto-oncogene while in vivo and significant reduction in the tumor mass of 1′-acetoxychavicol acetate-treated zebrafish-engrafted breast cancer groups. The anti-invasive effects of 1′-acetoxychavicol acetate were confirmed in vitro by the matrigel invasion assay and with down-regulation of C – X-C chemokine receptor type 4, urokinase plasminogen activator, vascular endothelial growth factor, and basic fibroblast growth factor 2 genes. The down-regulation of urokinase plasminogen activator and fibroblast growth factor 2 proteins was also validated by molecular docking analysis. Moreover, 1′-acetoxychavicol acetate-treated cells exhibited lower expression levels of the anti-apoptotic Bcl-2 and Mcl-1 proteins in addition to enhanced stress-activated kinases/c-Jun N-terminal kinase 1/2 and poly-ADP ribose polymerase cleavage, indicating apoptotic cell induction by 1′-acetoxychavicol acetate. Moreover, 1′-acetoxychavicol acetate had higher potency in human epidermal growth factor receptor 2-overexpressed cell lines regarding its inhibition on human epidermal growth factor receptor 2, pAKT, pERK1/2, PSer¹¹⁸, and PSer¹⁶⁷-ERα proteins. Our findings suggest 1′-acetoxychavicol acetate mediates its anti-cancer effects via human epidermal growth factor receptor 2 signaling pathway.

Introduction

Estrogen receptor is expressed in 70% of breast cancer patients and is known to sensitize breast cancer to tamoxifen therapy [1]. However, approximately 30% of estrogen receptor-positive breast cancer patients will develop resistance to endocrine treatment [2]. Therefore, the attempt to find other promising medications for the treatment is crucially required to improve survival rate. The mechanisms of resistance include overexpression of HER2, hyperactivation of a PI3K and MAPK pathways, and NF-κB activation [2], [3], [4], [5]. Estrogen receptors can be phosphorylated at S118 and 167 at the AF-1 domain by the downstream molecules of HER2 signaling MAPK and AKT, respectively [6]. In addition, PI3K/AKT is a significant downstream molecule of the HER2 pathway that frequently mutates and can be the cause of hormonal-resistant breast cancer [6]. PI3K/AKT can also activate and stimulate the transcriptional activity of estrogen receptor-regulated genes in both estrogen-dependent and -independent pathways in estrogen receptor-positive breast cancers [6].

Natural compounds play a pivotal function in cancer therapy, and a great number of anticancer agents are derived from plants [7], [8]. ACA is found in the rhizomes of Alpinia galanga (L.) Willd (Zingiberaceae) (greater galangal). It has been found to inhibit the proliferation of the liver and esophagus and exhibits cytotoxic activity against myeloma cells and MCF7 and MDA-MB-231 breast cancer cell lines via the down-regulation of NF-κB-related functions [9], [10], [11], [12]. However, despite well-established ACA activity against several types of cancers, the ACA mode of action in estrogen receptor-positive breast cancer resistance following hormonal therapy has never been reported.

Here, we extracted and purified ACA from the rhizomes of greater galangal and aimed to explore the mode of action of ACA though HER2 signaling and its downstream molecules related to resistance mechanisms of estrogen receptor-positive breast cancer cells.

Results

Fourteen protons in the ACA molecule have been displayed by the ¹H-NMR data ([Fig. 1 a]). Two symmetrical doublets appeared at 7.38 and 7.08 ppm and were 4 protons on a para-substitution of benzene ring. The resonance of protons at 5.99 (1H, m), 5.38 (1H, dd, J = 16.2, 1.2 Hz), and 6.23 (1H, d, J = 6.0 Hz) corresponded to protons on 2′ and 3′ olefinic carbons, respectively. The signal at 5.23 (1H, d, J = 10.2, 1.2 Hz) matched to proton 1′ carbon. Two methyl signals of 2 acetyl groups were evidently from the presence of 2 singlet signals at 2.27 (3H, s) and 2.08 (3H, s). The ¹³C-NMR ([Fig. 1 b]) displayed the ¹³C signals. Among these, the presence of chemical shift values of 20.8 and 20.9 ppm was clear due to the 2 methyl signals. Two carbons that showed the signals at 169.1 and 169.6 ppm were characteristically from carboxyl groups. An approval of a para-substituted benzene ring were provided by 2 parts of a carbon signal (chemical shifts at 128.2 and 135.8 ppm) and 2 quaternary carbon signals at 136.2 and 150.4. The downfield peak of sp2 carbons at δc 150.2 (C-1) specified the occurrence of an oxygen group closed to an aromatic carbon. The carbon signal with the chemical shift of 121.5 and 116.8 ppm should be assigned as sp2 carbons at 2′ and 3′, respectively. The downfield sp3 carbons at δc 75.3 (1′-C) indicated the presence of oxygen that attached to a carbon. Combination of the fragments mentioned above led to the assigned structure of compound ACA. Therefore, compound A was identified as ACA. Purity of ACA was > 97% based on analysis of ¹H-NMR and ¹³C-NMR spectra ([Fig. 1 a, b]). The high-resolution mass spectrum ([Fig. 1 c]) represented a molecular ion peak [M + Na] at m/z 257.0786 equivalent to a formula of a C₁₃H₁₄O₄+Na molecule. Therefore, the molecular ion peak at m/z 234.0889 was equivalent to a formula of an acetate ester ACA (C₁₃H₁₄O₄) ([Fig. 1 d]).

The molecular docking results ([Table 1]) demonstrated that ACA has similar binding affinity for HER2 kinase domain complexed with TAK-285 (3RCD), AKT1, ERK2, estrogen receptor, CDK6, uPA, and FGF2 ranging from − 21.61 to − 38.69 kcal/mol. However, among these 7 different targets, the binding affinity of ACA toward uPA (− 32.73 kcal/mol) and FGF2 (− 21.61 kcal/mol) was in the range of their known inhibitors 4-iodobenzo[b]thiophene-2-carboxamidine (4-IBTC) (− 37.43 kcal/mol) and Sm27 (− 29.96 kcal/mol), respectively. Due to hydrophobic structure of ACA, van der Waals (green sphere) were the main interactions underlying protein-ligand complexation for both uPA and FGF2 proteins ([Fig. 2]). On the other hand, some electrostatic contributions (e.g., salt bridge, pi-sulfur, and pi-cation) were involved in the binding of 4-IBTC and Sm27 due to the polar moieties in their chemical structures. Notably, the key binding amino acid residues of ACA toward both uPA and FGF2 as well as the hydrogen bond formation patterns (green dash) were relatively similar to those of the known inhibitors, implying that ACA could be an alternative potential inhibitor to uPA and FGF2.

The cytotoxic effect results of ACA were shown as the percentage viability in MCF7, MCF7/LCC2, MCF7/LCC9, and MCF7/HER2 cells at 24, 48, and 72 h ([Fig. 3 a – d]). ACA significantly inhibited cell viability in concentration- and time-dependent manners ([Table 2]). The IC₅₀ of ACA was higher in the resistant cell lines (MCF7/LCC2 and MCF7/LCC9) compared to the wide-type (MCF7), where treatment with vehicle control (0.1% ethanol [v/v]) did not affect cell viability. However, the IC₅₀ values (48 h) of ACA in HER2-overexpressed cell lines (MCF7/HER2, AU565, and BT474) were 2 – 4 times lower than the MCF7 cell line ([Table 2]). The results from this study suggested that ACA had higher potency in HER2-overexpressed cell lines compared to the MCF7 cell line. The positive controls for each cell lines were tested at 48 h, in which the result showed that the cell lines were not drastically dead ([Fig. 3 e]). We also determined the interaction between ACA and MTT tetrazolium in a cell-free condition by measuring the formazan formation (% of control). The result showed that ACA had no effect on MTT reduction in the concentration tested up to 160 µM (Fig. 1S, Supporting Information).

To further verify the antiproliferative effect of ACA, we examined the colony-forming capability (% of control) of the cells ([Fig. 4 a – d]) and the protein expression of HER2, p-ERK1/2, p-AKT, NCOA3, and c-Myc ([Fig. 5 a – c]). The results showed that the colony formation of the studyʼs cells were significantly inhibited by 20 – 80%. In addition to protein expression analysis, ACA treatment significantly down-regulated HER2 more profoundly in MCF7/LCC2 and MCF7/LCC9 than MCF7. ACA (15 – 40 µM) can significantly down-regulate p-ERK1/2 and p-AKT in MCF7/LCC2 and MCF7/LCC9. Only the highest concentration of ACA used for MCF7 treatment can repress p-AKT. However, no significant differences in the expression of p-ERK1/2 of 1′-acetoxychavicol treatment in MCF7. In addition, ACA repressed NCOA3 in both transcriptional and translational levels in MCF7/LCC2 and MCF7/LCC9 (Fig. 2Sg, Supporting Information and [Fig. 5 a – c]). Also, it can repress NCOA3 protein expression in MCF7 at the highest concentration. ACA can also repress c-Myc, which is the molecule responsible for cell proliferation. To investigate the target mechanism of ACA via HER2 signaling pathway, we started with examining the basal level of HER2 and its downstream molecules, including pAKT, pERK1/2, PSer¹¹⁸-ERα, and PSer¹⁶⁷-ERα ([Fig. 6 a]). Then, we investigated the mode of action of ACA through HER and its downstream molecules in MCF7/HER2 cell line. The results showed that ACA can significantly downregulate HER2 and its downstream molecules more efficiently in MCF7/HER2 compared to the MCF7 cell line. In addition, the results suggested that the preferable mechanism of ACA was through HER2 signaling pathway since its anticancer effect had a higher efficacy in MCF7/HER2 compared to the MCF7 cell line ([Fig. 6 b – d]).

The finding that ACA had the potential to bind both uPA and FGF2 proteins, in addition to its known activity as an NF-κB inhibitor, prompted us to examine the direct impact of ACA treatment on the expression levels of uPA and FGF2 as well as NF-κB regulated genes. ACA treatment reduced the expression levels of both uPA and FGF2 proteins ([Fig. 7 a – c]). Intriguingly, ACA-treated MCF7/LCC9 cells exhibited a more significant reduction of uPA and FGF2 proteins compared to wild-type MCF7. Additionally, NF-κB-targeted genes involved in breast cancer resistance including proliferative markers (CCND1 [13], [14], c-Myc [15]), invasive factors (CXCR4 [16] , uPA [17], [18]), and angiogenic factors (VEGF [19] , FGF2 [20], [21], [22]) were shown to be significantly inhibited in both resistant cells (Fig. 2SA-F, Supporting Information).

To investigate the anti-invasive effect of ACA, we examined the invasive ability of endocrine-resistant breast cancer cells using invasion chamber-coated Matrigel ([Fig. 8 a, b]). The use of ACA nontoxic concentrations was to avoid the result interference by cell death. Fixed concentration of ACA at 7.5 and 15 µM significantly inhibited invasive capability in MCF7/LCC2 by 12.57% and 22.23% (P ≤ 0.01 and 0.001), respectively. ACA at 10 and 20 µM significantly inhibited invasive capability in MCF7/LCC9 by 25% and 34.14% (P ≤ 0.01 and 0.001), respectively.

To investigate the induction of apoptosis, the protein expression of SAPKs/JNKs, Mcl-1 and Bcl-2 (anti-apoptotic proteins), and PARP and cleaved-PARP were examined. ACA-treated MCF7 showed no significant changes in the expression of phospho-SAPK/JNK, while ACA-treated MCF7/LCC2 and MCF7/LCC9 cell lines resulted in the up-regulation of phospho-SAPK/JNK expression up to tenfold compared to the control. ACA also down-regulated Mcl-1 and Bcl-2. Additionally, ACA exerted the apoptotic induction activity via cleavage of PARP and increased the expression of the cleaved form in all studied cells ([Fig. 9]).

In order to perform in vivo antiproliferation, the acute toxicity of ACA was studied first in zebrafish embryos. The 50% lethality concentration (LC₅₀) of ACA at 24, 48, 72, and 96 h post fertilization (hpf) was 41.20 µM ± 5.03, 40.47 µM ± 2.87, 35.45 µM ± 2.20, and 32.16 µM ± 1.54, respectively ([Fig. 10 a]). The concentration of ACA up to 20 µM did not cause acute toxicity to the zebrafish embryos in any time point. In in vivo anti-proliferation, CM-dil-labelled/MCF7/LCC9 zebrafish embryos were treated with ACA (5, 10, and 15 µM) at 1 day post injection (dpi), and the tumor-engrafted area was measured at 3 dpi ([Fig. 10 c]). ACA significantly inhibited the tumor-engrafted area compared to untreated zebrafish at 3 dpi ([Fig. 10 b, d]). The results proved that ACA had a significant antiproliferative effect on the animal model, which was concomitant with in vitro antiproliferative effect.

Discussion

ACA is a naturally derived plant-product that employs anticancer effects by inducing apoptosis and inhibiting angiogenesis and metastasis [23], [24]. In this study, the identification of ACA structure was confirmed by the NMR and mass spectra analysis. The ¹H NMR and ¹³H spectra from our study was similar to the study of Azuma et al. and Murakami et al. [25], [26]. Similarly, the mass spectrum showed the molecular ion peak m/z of ACA to equal 234.0889, which correlated with the work of Lin et al. [27].

Our results on cytotoxic activity showed that ACA treatment significantly inhibited the growth of MCF7 and the more aggressive MCF7/LCC2 and MCF7/LCC9 cells in dose- and time-dependent manners. Intriguingly, the cytotoxic effect of ACA was predominant in HER2-overexpressed cell lines as shown with the IC₅₀ values. To gain insight into the mechanism by which ACA inhibits the growth of MCF7, MCF7/LCC2, MCF7/LCC9, and MCF7/HER2 cells, HER2 signaling pathways and its downstream molecules were examined, since up-regulation of HER2 was clinically confirmed to affect estrogen receptor expression and the responsiveness to endocrine treatment of breast cancer patients [28], [29]. Also, HER2 downstream molecules ERK1/2 and AKT are found to be highly expressed and active in tamoxifen-resistant breast cancer and also mediate the activation of NF-κB [30], [31], [32]. Our results found that the effect of ACA on down-regulation of HER2, pAKT, pERK, and NCOA3 protein expression in the endocrine-resistant MCF7/LCC2 and MCF7/LCC9 was more profound compared to the parental MCF7. We particularly studied the anticancer mechanism of ACA via HER2 pathway through pAKT, pERK1/2, c-Myc, PSer¹¹⁸-ERα, and PSer¹⁶⁷-ERα, since in the HER2 signaling pathway, PSer¹¹⁸-ERα and PSer¹⁶⁷-ERα were phosphorylated by ERK1/2 and AKT, respectively [33]. From this study, we first reported that ACA can significantly downregulate PSer¹¹⁸-ERα and PSer¹⁶⁷-ERα in HER2-overexpressed breast cancer cell lines. Moreover, with an equal concentration (5 µM), ACA has higher potency in MCF7/HER2 regarding its repressive effects on HER2, pAKT, pERK1/2, and PSer¹¹⁸-ERα proteins. We also observed that ACA treatment resulted in down-regulation of the estrogen receptor coactivator NCOA3. Research shows that the overexpression of coactivator NCOA3 is related to tamoxifen-resistant breast cancer through the phosphorylation of the EGFR/HER2/MAPK-dependent pathway in HER2 overexpressing MCF-7 [2]. In breast cancer patients, NCOA3 overexpression was reported in 60% of the breast cancer tumors and correlated with responsiveness to tamoxifen treatment [34]. Furthermore, NCOA3 upstream regulators ERK1/2 and AKT pathways appeared to be mediating NCOA3 down-regulation as ACA-treated cells exhibited lower ERK1/2 and AKT, evidenced by less phosphorylated ERK1/2 and AKT compared to untreated control. This leads to the inhibition of NCOA3 activity since NCOA3 is phosphorylated by ERK1/2 and AKT [34]. Consequently, the transcription of estrogen receptor-targeted genes was terminated. We also evaluated the expression of NF-κB-regulated genes involved in endocrine-resistant MCF7/LCC2 and MCF7/LCC9 cells. We found that uPA, FGF2, CCND1, C-myc, CXCR4, and VEGF were downregulated in ACA-treated cells. Additionally, the results from invasion assay proved that ACA possessed anti-invasive effect in endocrine-resistant breast cancer cells. Furthermore, ACA could potentially target both FGF2 and VEGF simultaneously, therefore, possessing more effective anti-angiogenic activity on breast cancer via targeting both VEGF and FGF2. FGF2 and VEGF are known angiogenic factors, overexpressed in aggressive breast cancers and reported to be implicated in estrogen receptor nonclassical genomic pathway [35]. Blocking VEGF signaling by the antiVEGF-A monoclonal antibody, bevacizumab, neo-adjuvant for metastatic breast cancer did not improve the breast cancer patientʼs overall survival rate [36]. This was attributed to triggering breast tumor progression and angiogenesis mediated by the FGF2 pathway [37]. However, further in vitro and in vivo studies on antiangiogenesis of ACA in breast cancer are still required. The molecular docking results of the ACA also appear to support our observation that ACA treatment down-regulates uPA and FGF2 expression in MCF7/LCC2 and MCF7/LCC9 cells, as the binding affinity of both uPA and FGF2 to ACA were as competitive as their known uPA inhibitor (4-IBTC) and FGF2 ligand trap (Sm27). Therefore, ACA could be used as an alternative inhibitor to uPA and FGF2. However, ACA showed similar affinities for 3RCD (HER2), AKT, ERK, estrogen receptor, CDK6, highlighting that it can bind to multiple key molecules and affect signaling pathways involved in endocrine resistance.

In contrast to the ACA anti-proliferative activity, ACA treatment resulted in up-regulating the basal level of the pro-apoptotic SAPK/JNK-affected hormonal resistant breast cancer cells. In line with the Liew et al. observation that ACA apoptotic induction is mediated by mitochondrial pathways [38], here we showed that ACA treatment resulted in down-regulation of the mitochondrial anti-apoptotic Mcl-1 and Bcl-2 proteins and enhancement of PARP cleavage. Therefore, we observe a shift from cancer cell survival and proliferation to apoptosis after ACA treatment.

A zebrafish xenograft model is considered more advantageous compared to the mice model particularly with its ability to real-time measure tumor mass. Utilizing this model, we were able to monitor cancer cell proliferation in a time-dependent manner. The finding that ACA treatment significantly reduced the tumor mass of MCF7/LCC9 implanted-zebrafish clearly confirmed the antiproliferative activity of ACA on breast cancer cells in in vivo models.

In summary, our study has explored for the first time the anti-tumor effect of ACA on a zebrafish xenograft model as well as ACAʼs mode of action on cell survival and invasion in endocrine-resistant breast cancer cells. Anticancer activities of ACA were through the HER2 signaling pathway and the estrogen receptor coactivator NCOA3 ([Fig. 11]). We also highlighted the shift from pro-survival to pro-apoptotic signals by targeting multiple genes involved in endocrine resistance. This finding proposed that the advantage of ACA might be a promised adjuvant therapy in endocrine-resistant breast cancer.

Materials and Methods

Extraction and purification of bioactive compound

The fresh A. galanga rhizomes were acquired locally in Bangkok, Thailand (November 2017). The plant was identified by Dr. Thanapat Songsak (Department of Pharmacognosy, College of Pharmacy, Rangsit University, Thailand), was archived at the College of Pharmacy, Rangsit University, Thailand, and given voucher specimen number CP-Ag-29.

Ten kilograms of A. galanga rhizomes were chopped before extraction with 25 L of hexane. The hexane extracts were combined and concentrated under vacuum at 40 °C and pooled together to obtain 7.2 g of a brown oily substance (crude hexane extract). Crude hexane extract was performed; chromatography on silica gel glass column and elution followed through a stepwise gradient of hexane-acetone. The yield of pure ACA after process through column chromatography was 6.5 g. The bioactive substance of the crude extract in the column chromatographic fraction (hexane 85: acetone 15) was spotted on TLC (hexane 8: ethyl acetate 2) and followed by UV detection using UV-Vis Spectrophotometer SHIMADZU, UV-1800 at the wavelength 254 nm. Proton nuclear magnetic resonance (¹H-NMR) and Carbon-13 nuclear magnetic resonance (¹³C-NMR) experimentations were implemented on a Bruker AV500 NMR spectrometer, running at 500 MHz for hydrogen (H) and carbon (C) and then documented in deuterochloroform (CDCl₃) with tetramethylsilane as an internal standard. ESI-TOF mass spectra were acquired from a Micromass LCT mass spectrometer.

Molecular docking simulation

The crystal structures of HER2 Kinase Domain Complexed with TAK-285 (PDB ID: 3RCD [39]), AKT1 (PDB ID: 4GV1 [40]), ERK2 (PDB ID: 5NHJ [41]), estrogen receptor (PDB ID: 3ERT [42]), uPA (PDB ID: 5YC7 [43]), CDK6 (PDB ID: 1XO2 [44]), and FGF2 (PDB ID: 1FQ9 [45]) were obtained from Protein Data Bank. The 3-dimensional structure of all the studied ligands were built and fully optimized by the HF/6 – 31(d) level of theory using Gaussian09 program [46]. The protein-ligand complexes were generated using CDOCKER module implemented in Accelrys Discovery Studio 2.5 [47] with 100 independent docking runs. The co-crystalized inhibitors were defined as docking center with a spherical radius of 15 Å.

Maintenance of breast cancer cells

ER-positive MCF7 and HER2-overexpressed AU565 and BT474 cell lines were purchased from the American Type Culture Collection (ATCC). MCF7/LCC2 (tamoxifen-resistant cell) and MCF7/LCC9 (tamoxifen/fulvestrant-resistant cell) were given from Dr. Robert Clarke (Lombardi Cancer Center, Georgetown University, Washington, DC, USA). The stable transfected HER2-overexpressed MCF7 cell line (MCF7/HER2) was obtained from Dr. Christopher Benz (Cancer Research Institute, University of California, San Francisco, USA). The cells were grown on 25-cm² flasks with DMEM (Gibco) supplemented with 100 U/mL penicillin/streptomycin and 5% FBS (Gibco) and incubated in 5% CO₂ at 37 °C in humidified air. The endocrine-resistant cells were regularly checked to confirm their resistance to tamoxifen.

Cell viability assay

Cell viability and the half maximal inhibitory concentration (IC₅₀) assessment were performed by MTT (Sigma) assay [48]. Briefly, 5000 cells in 100 µL/well were plated into a 96-well plate and incubated overnight. Following overnight incubation, the medium in each well was removed, and the cells were treated with 100 µL/well of ACA at increasing concentrations and incubated for 24, 38, and 72 h. As a negative control, 0.1% Ethanol in DMEM medium was used. 4-OHT and Pal were used as a positive control for hormonal-sensitive and resistant cells, respectively. MTT powder was weighted and dissolved in PBS at a 5 mg/mL concentration. After that, 10 µL of the 5 mg/mL MTT solution in PBS buffer was added into the cells (total volume of cell suspension and MTT solution in each well was 110 uL/well). MTT solution can be completely dissolved in cell culture medium, and the final concentration of MTT solution in the cells was 0.45 mg/mL and incubated for 4 h at 37 °C in the dark. Formazan crystals were solubilized with DMSO and optical density (O. D.) measured by a microplate reader at 570 nm. The same MTT method was also performed in a cell-free condition to check the potential interaction between ACA and MTT tetrazolium.

Colony-forming assay

The cells (MCF7, MCF7/LCC2, MCF7/LCC9, and MCF7/HER2) were seeded in 6-well plates at 1000 cells/well for 24 h. After that, the cells were treated with ACA (0 – 40 µM) for 24 h. After 24 h of the treatment, ACA-containing medium was removed, then the fresh medium was added, and the medium was changed every 48 h. The cells were allowed to grow for 7 days before the analysis. After 7 days of culture, the cells were washed with PBS and fixed with 1000 µL absolute methanol for 30 min and then stained with 700 µL of 0.5% crystal violet for 30 min at room temperature. The excess dye was removed and washed several times with deionized water. After allowing the colony to dry, the colonies were captured using a Nikon camera and counted (50 cells/colony). The colony formation was expressed as percentage colony-forming capability relative to untreated control. The colony-forming assay was performed in triplicate.

Real-time qPCR

The cells were treated with ACA (0 – 40 µM) for 24 h. Total RNA was extracted using Trizol reagent. ImProm-II Reverse Transcription System (Promega) was used for synthesizing complementary DNA (cDNA). The real-time qPCR was performed using cDNA as a template with primers specific for CCND1, NCOA3, c-Myc, CXCR4, uPA, VEGF, and FGF2 under the modified conditions stated in Table 1S (Supporting Information).

Western blot analysis

The cells were treated with ACA (0 – 40 µM) for 24 h. Total protein was extracted and analyzed by western blotting, utilizing specific antibodies (1 : 1000) against NCOA3, p-ERK1/2, total-ERK1/2, p-AKT, pan-AKT, SAPK/JNK, pSAPK/JNK, c-Myc, Bcl-2, Mcl-1, PARP and cleaved-PARP (Cell Signaling Technology), uPA, and FGF2 (Merck), and detected with an appropriate HRP-conjugated antimouse or antirabbit IgG secondary antibodies (1 : 2000) (Cell Signaling Technology). Protein density was evaluated using Immobilon Forte Western HRP substrate (Merck). GAPDH (Cell Signaling Technology) was used as a loading control.

Invasion assay

MCF7/LCC2 and MCF7/LCC9 cells were cultured in DMEM treated with nontoxic concentrations of ACA (0 – 20 µM) or 0.1% ethanol as a negative control and were plated into matrigel-coated transwell invasion inserts (24-well plates of 8 µm pore size) (Corning) for 24 h at a density of 1 × 10⁵ cells/mL. Noninvaded cells were scrapped off from the upper chamber with a cotton swab. Invasive cells were fixed with 4% formaldehyde, stained with crystal violet dye, and counted (25 random fields) under an inverted microscope.

In vivo acute toxicity test

The zebrafish embryo toxicity test was performed according to the published Organisation for Economic Co-operation and Development (OECD) Test Guidelines (TG236) for a Fish Embryo Toxicity test [49]. For each experiment, 20 fertilized eggs at the beginning of the epiboly stage (0.50 h) were used. The selected eggs were transferred into 1000 µL of 10, 20, 40, 80, and 160 µM ACA in 0.1% ethanol in E3/phenylthiourea (PTU) medium. The samples were incubated at 28 °C for 24, 48, 72, and 96 h. Four apical endpoints–which were coagulation of fertilized eggs, lack of somite formation, nondetachment of the tail-bud from the yolk sac, and lack of heartbeat–were observed as the indicator of lethality under Leica microscope every 24 h, and the LC₅₀ values of 24, 48, 72, and 96 h of ACA-treated zebrafish were calculated.

In vivo growth and proliferation

An early-stage of wild type zebrafish embryos (Danio Rerio, AB/Tuebingen) were used to determine the growth changes of the CM-Dil-labeled MCF7/LCC9 cell transplantation. CM-Dil-labeled MCF7/LCC9 cells were injected into the yolk of zebrafish at 2 days post fertilization (dpf). One day post injection (1 dpi), xenografted zebrafish embryos were separated into control and treatment groups (n = 23/group; the sample size was determined using G*power analysis); the treatment group were treated with ACA (5, 10, and 15 µM) for 2 days. Fluorescent images on xenografted embryos at 1 dpi and 3 dpi were acquired using Nikon (model DS-Ri2). Images were analyzed and tumor areas were estimated using imaging software NIS elements.

Statistical analysis

Results were defined as the mean and standard error of the mean. Graphical and analyzed graphed data obtained were done using GraphPad Prism V8. Significant differences between the mean values of intra-group were analyzed using 1-way analysis of variance (ANOVA) and a Tukeyʼs test for multiple comparisons. Specifically, p ≤ 0.05 indicates the significance level.

Ethical Considerations

The human cell lines used in this study were reviewed and exempted by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University, Thailand (IRB Number: 616/60). The animal ethic for zebrafish study was reviewed by Siriraj Animal Care and Use Committee (SiACUC) (date of approval August 8, 2018), Faculty of Medicine, Siriraj Hospital, Thailand (SI-ACUP 006/2559).

Contributorsʼ Statement

Conception and design of the work: W. Ketchart, N. Pradubyat, C. Mitrpant, C. Palmieri; data collection: N. Pradubyat, P. Mahalapbutr, T. Elmitwali, A. Giannoudis; analysis and interpretation of the data: N. Pradubyat, W. Ketchart; statistical analysis: N. Pradubyat; drafting the manuscript: N. Pradubyat; critical revision of the manuscript: W. Ketchart.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgements

We gratefully acknowledged Dr. Robert Clark (Georgetown University, US) for endocrine-resistant breast cancer cell lines, Assoc. Prof. Dr. Prasat Kittakoop (Chulabhorn Research Institute, Thailand) for chemical isolation protocol. We would like to acknowledge Asst. Prof. Dr. Thanyada Rungrotmongkol (Chulalongkorn University, Thailand) for computing sources. This study has been funded by Ratchadaphiseksomphot fund (RA 61/093 to WK), the 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund) (GCUGR1125611027D to NP), and Capacity Building Program for New Researchers 2018 from National Research Council of Thailand (NRCT) (NP). This work was partly supported by National Research Council Thailand (Thailand Grand Challenge: Precision Medicine) (CM).

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• 11 Campbell CT, Prince M, Landry GM, Kha V, Kleiner HE. Pro-apoptotic effects of 1′-acetoxychavicol acetate in human breast carcinoma cells. Toxicol Lett 2007; 173: 151-160
  CrossrefPubMedSearch in Google Scholar
• 12 Sagawa M, Tabayashi T, Kimura Y, Tomikawa T, Nemoto-Anan T, Watanabe R, Tokuhira M, Ri M, Hashimoto Y, Iida S, Kizaki M. TM-233, a novel analog of 1′-acetoxychavicol acetate, induces cell death in myeloma cells by inhibiting both JAK/STAT and proteasome activities. Cancer Sci 2015; 106: 438-446
  CrossrefPubMedSearch in Google Scholar
• 13 Guttridge DC, Albanese C, Reuther JY, Pestell RG, Baldwin AS. NF-κB controls cell growth and differentiation through transcriptional regulation of cyclin D1. Mol Cell Biol 1999; 19: 5785-5799
  CrossrefPubMedSearch in Google Scholar
• 14 Hinz M, Krappmann D, Eichten A, Heder A, Scheidereit C, Strauss M. NF-κB function in growth control: regulation of cyclin D1 expression and G0/G1-to-S-phase transition. Mol Cell Biol 1999; 19: 2690-2698
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• 15 Duyao MP, Buckler AJ, Sonenshein GE. Interaction of an NF-kappa B-like factor with a site upstream of the c-myc promoter. Proc Natl Acad Sci U S A 1990; 87: 4727-4731
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Correspondence

Publication History

Received: 03 July 2020

Accepted after revision: 10 November 2020

Article published online:
14 January 2021

© 2021. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

• References

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• 20 Kim HR, Heo YM, Jeong KI, Kim YM, Jang HL, Lee KY, Yeo CY, Kim SH, Lee HK, Kim SR. FGF-2 inhibits TNF-α mediated apoptosis through up-regulation of Bcl2-A1 and Bcl-xL in ATDC5 cells. BMB Rep 2012; 45: 287-292
  CrossrefPubMedSearch in Google Scholar
• 21 Aukes K, Forsman C, Brady NJ, Astleford K, Blixt N, Sachdev D, Jensen ED, Mansky KC, Schwertfeger KL. Breast cancer cell-derived fibroblast growth factors enhance osteoclast activity and contribute to the formation of metastatic lesions. PLoS One 2017; 12: e0185736
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• 23 Pang X, Zhang L, Lai L, Chen J, Wu Y, Yi Z, Zhang J, Qu W, Aggarwal BB, Liu M. 1′-acetoxychavicol acetate suppresses angiogenesis-mediated human prostate tumor growth by targeting VEGF-mediated Src-FAK-Rho GTPase-signaling pathway. Carcinogenesis 2011; 32: 904-912
  CrossrefPubMedSearch in Google Scholar
• 24 Wang J, Zhang L, Chen G, Zhang J, Li Z, Lu W, Liu M, Pang X. Small molecule 1′-acetoxychavicol acetate suppresses breast tumor metastasis by regulating the SHP-1/STAT3/MMPs signaling pathway. Breast Cancer Res Treat 2014; 148: 279-289
  CrossrefPubMedSearch in Google Scholar
• 25 Azuma H, Aizawa Y, Higashitani N, Tsumori T, Kojima-Yuasa A, Matsui-Yuasa I, Nagasaki T. Biological activity of water-soluble inclusion complexes of 1′-acetoxychavicol acetate with cyclodextrins. Bioorg Med Chem 2011; 19: 3855-3863
  CrossrefPubMedSearch in Google Scholar
• 26 Murakami A, Toyota K, Ohura S, Koshimizu K, Ohigashi H. Structure-activity relationships of (1′ S)-1′-acetoxychavicol acetate, a major constituent of a Southeast Asian condiment plant Languas galanga, on the inhibition of tumor-promoter-induced Epstein-Barr virus activation. J Agric Food Chem 2000; 48: 1518-1523
  CrossrefPubMedSearch in Google Scholar
• 27 Lin LY, Shen KH, Yeh XY, Huang BY, Wang HE, Chen KC, Peng RY. Integrated process for production of galangal acetate, the “wasabi-like” spicy compound, and analysis of essential oils of Rhizoma Alpinia officinarum (Hance) Farw. J Food Sci 2016; 81: H1565-H1575
  CrossrefPubMedSearch in Google Scholar
• 28 Kern FG, McLeskey SW, Zhang L, Kurebayashi J, Liu Y, Ding IYF, Kharbanda S, Chen D, Miller D, Cullen K. Transfected MCF-7 cells as a model for breast cancer progression. Breast Canc Res Treat 1994; 31: 153-165
  CrossrefPubMedSearch in Google Scholar
• 29 De Laurentiis M, Arpino G, Massarelli E, Ruggiero A, Carlomagno C, Ciardiello F, Tortora G, DʼAgostino D, Caputo F, Cancello G. A meta-analysis on the interaction between HER-2 expression and response to endocrine treatment in advanced breast cancer. Clin Cancer Res 2005; 11: 4741-4748
  CrossrefPubMedSearch in Google Scholar
• 30 Viedma-Rodríguez R, Baiza-Gutman L, Salamanca-Gómez F, Diaz-Zaragoza M, Martínez-Hernández G, Esparza-Garrido RR, Velázquez-Flores MA, Arenas-Aranda D. Mechanisms associated with resistance to tamoxifen in estrogen receptor-positive breast cancer (review). Oncol Rep 2014; 32: 3-15
  CrossrefPubMedSearch in Google Scholar
• 31 Chen B, Liu J, Ho TT, Ding X, Mo YY. ERK-mediated NF-κB activation through ASIC1 in response to acidosis. Oncogenesis 2016; 5: e279
  CrossrefPubMedSearch in Google Scholar
• 32 Bai D, Ueno L, Vogt PK. Akt-mediated regulation of NFκB and the essentialness of NFκB for the oncogenicity of PI3K and Akt. Int J Cancer 2009; 125: 2863-2870
  CrossrefPubMedSearch in Google Scholar
• 33 Jiang J, Sarwar N, Peston D, Kulinskaya E, Shousha S, Coombes RC, Ali S. Phosphorylation of estrogen receptor-α at Ser167 is indicative of longer disease-free and overall survival in breast cancer patients. Clin Cancer Res 2007; 13: 5769-5776
  CrossrefPubMedSearch in Google Scholar
• 34 Burandt E, Jens G, Holst F, Jänicke F, Müller V, Quaas A, Choschzick M, Wilczak W, Terracciano L, Simon R, Sauter G, Lebeau A. Prognostic relevance of AIB1 (NCoA3) amplification and overexpression in breast cancer. Breast Cancer Res Treat 2013; 137: 745-753
  CrossrefPubMedSearch in Google Scholar
• 35 Giavazzi R, Sennino B, Coltrini D, Garofalo A, Dossi R, Ronca R, Tosatti MPA, Presta M. Distinct role of fibroblast growth factor-2 and vascular endothelial growth factor on tumor growth and angiogenesis. Am J Pathol 2003; 162: 1913-1926
  CrossrefPubMedSearch in Google Scholar
• 36 Sasich LD, Sukkari SR. The US FDAs withdrawal of the breast cancer indication for Avastin (bevacizumab). Saudi Pharm J 2012; 20: 381-385
  CrossrefPubMedSearch in Google Scholar
• 37 Zhao M, Yu Z, Li Z, Tang J, Lai X, Liu L. Expression of angiogenic growth factors VEGF, bFGF and ANG1 in colon cancer after bevacizumab treatment in vitro: a potential self-regulating mechanism. Oncol Rep 2017; 37: 601-607
  CrossrefPubMedSearch in Google Scholar
• 38 Liew SK, Azmi MN, In LLA, Awang K, Nagoor NH. Anti-proliferative, apoptotic induction, and anti-migration effects of hemi-synthetic 1′S-1′-acetoxychavicol acetate analogs on MDA-MB-231 breast cancer cells. Drug Des Devel Ther 2017; 11: 2763
  CrossrefPubMedSearch in Google Scholar
• 39 Ishikawa T, Seto M, Banno H, Kawakita Y, Oorui M, Taniguchi T, Ohta Y, Tamura T, Nakayama A, Miki H. Design and synthesis of novel human epidermal growth factor receptor 2 (HER2)/epidermal growth factor receptor (EGFR) dual inhibitors bearing a pyrrolo [3,2-d] pyrimidine scaffold. J Med Chem 2011; 54: 8030-8050
  CrossrefPubMedSearch in Google Scholar
• 40 Addie M, Ballard P, Buttar D, Crafter C, Currie G, Davies BR, Debreczeni J, Dry H, Dudley P, Greenwood R. Discovery of 4-Amino-N-[(1S)-1-(4-chlorophenyl)-3-hydroxypropyl]-1-(7H-pyrrolo [2,3-d] pyrimidin-4-yl) piperidine-4-carboxamide (AZD5363), an orally bioavailable, potent inhibitor of Akt kinases. J Med Chem 2013; 56: 2059-2073
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  CrossrefPubMedSearch in Google Scholar
• 42 Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA, Greene GL. The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 1998; 95: 927-937
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• 43 Jiang L, Zhang X, Zhou Y, Chen Y, Luo Z, Li J, Yuan C, Huang M. Halogen bonding for the design of inhibitors by targeting the S1 pocket of serine proteases. RSC Adv 2018; 8: 28189-28197
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• 44 Lu H, Chang DJ, Baratte B, Meijer L, Schulze-Gahmen U. Crystal structure of a human cyclin-dependent kinase 6 complex with a flavonol inhibitor, fisetin. J Med Chem 2005; 48: 737-743
  CrossrefPubMedSearch in Google Scholar
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