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Zerumbone targets the CXCR4-RhoA and PI3K-mTOR signaling axis to reduce motility and proliferation of oral cancer cells
Phytomedicine 39 (2018) 33–41
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Original Article
Zerumbone targets the CXCR4-RhoA and PI3K-mTOR signaling axis to reduce motility and proliferation of oral cancer cells
T
Nur Syafinaz Zainala, Chai Phei Gana, Beng Fye Laub, Pei San Yeea, Kai Hung Tionga,c, ⁎ Zainal Ariff Abdul Rahmanc,d, Vyomesh Patela, Sok Ching Cheonga,d, a
Cancer Research Malaysia, No. 1, Jalan SS12/1A, 47500 Subang Jaya, Selangor, Malaysia Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia c Oral Cancer Research and Co-ordinating Centre (OCRCC), Faculty of Dentistry, University of Malaya, 50603 Kuala Lumpur, Malaysia d Dept of Oral & Maxillofacial Clinical Sciences, Faculty of Dentistry, University of Malaya, 50603 Kuala Lumpur, Malaysia b
A R T I C L E I N F O
A B S T R A C T
Keywords: Zerumbone Oral squamous cell carcinoma Akt RhoA Proliferation Migration
Background: The CXCR4-RhoA and PI3K-mTOR signaling pathways play crucial roles in the dissemination and tumorigenesis of oral squamous cell carcinoma (OSCC). Activation of these pathways have made them promising molecular targets in the treatment of OSCC. Zerumbone, a bioactive monocyclic sesquiterpene isolated from the rhizomes of tropical ginger, Zingiber zerumbet (L.) Roscoe ex Sm. has displayed promising anticancer properties with the ability to modulate multiple molecular targets involved in carcinogenesis. While the anticancer activities of zerumbone have been well explored across different types of cancer, the molecular mechanism of action of zerumbone in OSCC remains largely unknown. Purpose: Here, we investigated whether OSCC cells were sensitive towards zerumbone treatment and further determined the molecular pathways involved in the mechanism of action. Methods: Cytotoxicity, anti-proliferative, anti-migratory and anti-invasive effects of zerumbone were tested on a panel of OSCC cell lines. The mechanism of action of zerumbone was investigated by analysing the effects on the CXCR4-RhoA and PI3K-mTOR pathways by western blotting. Results: Our panel of OSCC cells was broadly sensitive towards zerumbone with IC50 values of less than 5 µM whereas normal keratinocyte cells were less responsive with IC50 values of more than 25 µM. Representative OSCC cells revealed that zerumbone inhibited OSCC proliferation and induced cell cycle arrest and apoptosis. In addition, zerumbone treatment inhibited migration and invasion of OSCC cells, with concurrent suppression of endogenous CXCR4 protein expression in a time and dose-dependent manner. RhoA-pull down assay showed reduction in the expression of RhoA-GTP, suggesting the inactivation of RhoA by zerumbone. In association with this, zerumbone also inhibited the PI3K-mTOR pathway through the inactivation of Akt and S6 proteins. Conclusion: We provide evidence that zerumbone could inhibit the activation of CXCR4-RhoA and PI3K-mTOR signaling pathways leading to the reduced cell viability of OSCC cells. Our results suggest that zerumbone is a promising phytoagent for development of new therapeutics for OSCC treatment.
Introduction Oral squamous cell carcinoma (OSCC) affects on average 300,400 individuals and accounts for 145,400 deaths every year (Torre et al., 2015). This poor prognosis has largely remained unchanged over the past several decades and is in part, due to limited therapeutic options (Massano et al., 2006; Specenier and Vermorken, 2012). The identification of important signaling pathways driving the development of
OSCC has resulted in the use of cetuximab, a tyrosine kinase inhibitor targeting the epidermal growth factor receptor (EGFR) for the treatment of advanced and recurrent OSCC. However, this treatment combined with chemotherapy only improved the median overall survival by ∼3 months in OSCC patients (Vermorken et al., 2008) underscoring the urgent need to develop new and improved drugs for OSCC. Of note, 50% of approved cancer therapeutic agents are derived from natural products (Newman and Cragg, 2012) and secondary
Abbreviations: Akt, protein kinase B; CXCR4, C-X-C chemokine receptor type 4; EdU, ethynyl deoxyuridine; EGFR, epidermal growth factor receptor; Gα12, G-alpha protein-12; HNSCC, head and neck squamous cell carcinoma; mTOR, mammalian target of rapamycin; OSCC, oral squamous cell carcinoma; PI3K, phosphoinositide 3-kinase; RhoA, Ras homolog gene family member A; S6, ribosomal protein S6 ⁎ Corresponding author: Cheong Sok Ching, Head and Neck Cancer Research Team, Cancer Research Malaysia, No.1, Jalan SS12/1A, 47500 Subang Jaya, Selangor, Malaysia. E-mail address: [email protected] (S.C. Cheong). https://doi.org/10.1016/j.phymed.2017.12.011 Received 15 June 2017; Received in revised form 2 November 2017; Accepted 7 December 2017 0944-7113/ © 2017 Elsevier GmbH. All rights reserved.
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Fig. 1. Zerumbone reduces cell viability of OSCC cell lines. (A) Chemical structure of zerumbone. (B) IC50 of zerumbone across a panel of OSCC cell lines (grey bars) and NOK (white bars) as determined by MTT assay after 72 h of treatment. OSCC cells were more sensitive to zerumbone than NOK (average IC50 is provided in Supplementary Fig. S1). Breast (MCF-7) and colorectal (HCT-116) cell lines (black bars) were added for comparison. (C) The mean IC50 of zerumbone across a panel of OSCC cell lines was compared against two anticancer drugs (cisplatin and gefitinib). The IC50 of cisplatin and gefitinib across OSCC cell line panel is provided in Supplementary Table 1. Results shown are mean ± SD (n = 3). Significant differences between comparative groups were calculated by two-tailed student's T-test. * and *** denotes p < .05 and p < .001 respectively.
consequently the Gα12-RhoA axis in OSCC cells. In addition, our current study also investigated the effect of zerumbone on the PI3K-AktmTOR signaling pathway which is commonly known to be highly activated in OSCC and is associated with poor clinical outcome (Lui et al., 2013; Pickering et al., 2013; Yu et al., 2007).
metabolites from medicinal plants have demonstrated a unique potential as a source of anticancer and chemopreventive agents. Compounds isolated from edible plants have the advantage of low toxicity profiles and are able to simultaneously target multiple signaling pathways (Aggarwal and Shishodia, 2006; Gupta et al., 2010). This could be particularly useful in the context of developing new anticancer agents as cancer development typically involves dysregulation of multiple genes and signaling pathways. In OSCC, treatment and prevention strategies are also moving towards nutraceutical approaches, with green tea extracts and resveratrol showing the most potential in the development of new therapies (Zlotogorski et al., 2013). One natural plant metabolite that has demonstrated potential for the treatment of cancer is zerumbone; (2E,6E,10E)−2,6,9,9-tetramethylcycloundeca-2,6,10-trien-1-one (Fig. 1A), a bioactive monocyclic sesquiterpene isolated from the rhizomes of tropical ginger, Zingiber zerumbet (L.) Roscoe ex Sm. Emerging data have demonstrated anticancer properties of zerumbone in common types of cancers including breast, pancreatic, colon, lung and leukemia (Abdelwahab et al., 2011; Chakraborty et al., 2013; Kim et al., 2009; Sehrawat et al., 2012). Zerumbone is able to suppress key hallmarks of cancer for example, by enhancing apoptosis (Sehrawat et al., 2012), reducing cell proliferation (Shanmugam et al., 2015), and inhibiting migration and invasion (Kang et al., 2015; Sung et al., 2008). With regards to OSCC, preliminary results demonstrated that zerumbone could down-regulate the protein expression of CXCR4 in SCC4 cells (Sung et al., 2008) and reduced NF-κB activity in FaDu and LICR-LON-HN5 cells (Takada et al., 2005). However, the underlying molecular basis of zerumbone's promising anticancer properties remains largely unexplored and represents an opportunity for identifying novel therapies for OSCC. Prior work from our laboratory have shown that the Gα12 axis is enriched in OSCC and RhoA activity is critical in mediating OSCC metastases to the cervical lymph nodes in an orthotopic-tongue mouse model (Gan et al., 2014; Cheong et al., 2009). CXCR4 is able to activate the Gα12 signaling and cause migration and invasion of cells through RhoA (Tan et al., 2006). Therefore in this study, we were interested in discerning whether zerumbone is able to inhibit CXCR4, and
Materials and methods Chemicals Zerumbone was purchased from Sigma Aldrich, MO, USA with reported purity of ≥ 98% by HPLC analysis. Cisplatin and gefitinib were purchased from Selleckchem, TX, USA with reported purity > 99% by HPLC analysis. All drugs were dissolved to 20 mM (zerumbone and gefitinib in dimethyl sulfoxide; DMSO and cisplatin in water) and further diluted in culture medium for immediate use or stored in −20 °C. Cell culture The panel of OSCC lines (ORL series) used in this study were established from oral cancer patients as previously reported (Fadlullah et al., 2016; Hamid et al., 2007). All ORL cell lines were cultured in Dulbecco's Modified Eagle's Medium/Nutrient mixture F12-Ham's medium (DMEM/F12; Hyclone, Utah, USA) supplemented with 10% (v/v) of heat inactivated fetal bovine serum (FBS; Gibco, Auckland, NZ) and 500 ng/ml of hydrocortisone (Sigma-Aldrich, MO, USA). The primary normal oral keratinocytes (NOK; ORL-232 and ORL-235) used in this study were established from healthy gingival biopsies as previously reported (Hamid et al., 2007) and grown in Keratinocyte Serum-Free Medium (KSFM; Gibco, Auckland, NZ) supplemented with 25 µg/ml bovine pituitary extract (BPE), 0.4 ng/ml epidermal growth factor (EGF) and 0.09 mM calcium chloride (CaCl2). HCT-116 and MCF-7 were grown in DMEM (Gibco, Auckland, NZ) supplemented with 10% (v/v) FBS. All cell lines have been authenticated as described previously (Fadlullah et al., 2016). Cells were cultured at 37 °C in a 5% CO2 humidified atmosphere. 34
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DMSO for 24 or 72 h. All floating and attached cells were harvested and subjected to FITC-Annexin V and PI double staining according to the manufacturer's instructions. Cell analysis was conducted using a BD FACSCanto II™ flow cytometer with 10,000 events collected for each read. The percentage of apoptotic cells were obtained from three independent experiments.
3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay The cytotoxic effect of zerumbone, cisplatin and gefitinib were determined by the MTT assay. Briefly, 5 × 103 cells were seeded in triplicates in 96-well plates and following overnight incubation, cells were exposed to zerumbone at concentrations ranging from 0.1–100 µM or vehicle control of 0.5% (v/v) DMSO. After 72 h incubation, 5 mg/ml of MTT solution (Sigma Aldrich, MO, USA) was added into each well and the plates were further incubated for an additional 4 h. The resulting formazan crystals were dissolved in 100 µl of DMSO and absorbance was measured at 570 nm using a Synergy H1 Multi-Mode reader (BioTek Instruments, VT, USA). Dose response curves were generated by plotting log concentrations (µM) against the percentage of cell viability. The half maximal inhibitory concentration (IC50) was expressed as mean ± SD of three independent experiments.
Wound healing assay Wound healing assays were performed as previously described (Chong et al., 2012). Briefly, ORL-48 and ORL-115 were seeded at 8 × 105 cells in each well of 6-well culture plates and incubated for 24 h at 37 °C to form a confluent monolayer. Cells were then treated with 10 µg/ml mitomycin C (Sigma-Aldrich, MO, USA) for 2 h to inhibit cell proliferation. A scratch was made through the monolayer with a P200 pipette tip by applying constant pressure to create an open wound. Cells were rinsed with PBS and then further cultured in DMEM/ F12 complete medium with the addition of zerumbone (3–30 µM) or 0.5% (v/v) DMSO. The open wound areas were microscopically recorded at 0 and 24 h. The percentage of open wound area was obtained using the TScratch analysis software (CSElab, Switzerland) from three independent experiments.
Click-iT EdU cell proliferation assay Cell proliferation was determined by the Click-iT EdU assay (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. Briefly, ORL-48 and ORL-115 were seeded at 7 × 104 cells per well, grown overnight on glass coverslips and treated with 3–30 µM zerumbone or 0.5% (v/v) DMSO for 24 h. Cells were incubated with 10 µM 5-ethynyl-2′-deoxyuridine (EdU) for 2–6 h prior to fixation with 3.7% (v/v) formaldehyde. The cells were permeabilized with 0.1% (v/ v) Triton X-100 in phosphate buffer, followed by EdU detection via a copper-catalyzed reaction and nuclei staining by Hoechst 33342 provided in the kit. The coverslips were then mounted on glass slides by using VECTASHIELD® Mounting Medium (Vector Laboratories, Burlingame, CA, USA) and examined on an upright Olympus IX71 microscope (Olympus, Japan) with double bandpass filters to detect fluorescent-stained nuclei (Hoechst 33342: excitation 360−370 nm and emission 420 nm) and Alexa-labelled EdU (Alexa 647: excitation 650 nm and emission 667 nm). Images were captured from 10 randomly chosen fields of each experiment and analyzed with EBImage (Pau et al., 2010). The number of EdU-positive cells and Hoechststained cells were counted and the percentage of EdU-positive cells was calculated (from three independent experiments) using the following formula: number of EdU positive cells/number of Hoechst stained cells × 100. EdU-positive cells broadly represent cells that are undergoing DNA synthesis, whereas Hoechst stained cells represent viable cells in the same field.
Cell invasion assay Cell invasion was assayed with Biocoat Matrigel 24-well invasion chamber (BD Biosciences, MA, USA). Cells were serum starved for 18 h before detachment with Trypsin-EDTA (Gibco, Auckland, NZ). Further, ORL-48 and ORL-115 were suspended at 1 × 105 cells in serum-free medium containing zerumbone (30 µM) or 0.5% (v/v) DMSO, and added into the upper chambers of the transwell inserts while 1 U/ml thrombin (Sigma-Aldrich, MO, USA) was used as chemoattractant in the lower chambers. After 24 h of incubation at 37 °C, cells in the upper surface of the matrigel membrane (non-invaded cells) were removed with a cotton swab. Membranes were fixed with 4% (v/v) formaldehyde for 15 min and stained with 0.2% (w/v) crystal violet for 10 min, and finally rinsed with water. The membranes were air dried, detached from the inserts and were mounted onto glass slides using DPX mountant (Sigma-Aldrich, MO, USA). Stained cells (invaded cells) were counted in 4 randomly chosen microscopic fields for each insert at 100 × magnification and the average value of 3 technical replicates was obtained. Western blot
Cell cycle assay Zerumbone (3–30 µM) and 0.5% (v/v) DMSO-treated cells were lysed on ice in lysis buffer (5 M NaCl, 10% (v/v) NP-40, 1 M Tris pH 8.0, 0.5 mM DTT) supplemented with HALT Protease and Phosphatase Inhibitor Cocktail (Pierce Biotechnology, IL, USA). Cell lysates were then centrifuged at 14,000 rpm for 10 min at 4 ᵒC prior to estimation of protein content using the BCA method (Thermo Scientific, MA, USA). For western blot analysis, 30 µg of total cellular proteins were resolved on a 12% (w/v) SDS-PAGE gel and electro-transferred onto ImmobilonP membrane (PVDF; Millipore, MA, USA) at 100 V for 1 h on ice. Membranes were blocked with 5% (v/v) skimmed milk in Tris-buffered saline with 0.1% (v/v) Tween 20 (TBST; Sigma Aldrich, MO, USA) for 1 h and then probed overnight at 4 °C with the indicated primary antibodies at 1:1000 dilution in 1% (v/v) BSA in TBST (CXCR4: Abcam, Cambridge, UK; Gα12: Santa Cruz Biotechnology, CA, USA; pAkt (S473), total Akt, pS6, total S6, cleaved caspase 3, caspase 3; Cell Signaling Technology, MA, USA; α-tubulin: Sigma-Aldrich, MO, USA; anti-human PARP: BD Pharmingen, CA, USA). After, membranes were washed 3 times in TBST for 5 min each. Membranes were then incubated with the corresponding secondary HRP-conjugated antibodies (Southern Biotech, AL, USA) at 1: 10,000 dilution in 5% (v/v) skimmed milk in TBST for 1 h at room temperature. This was followed by 3
ORL-48 and ORL-115 were seeded at 7 × 104 cells per well in 12well plates, and treated with 3–30 µM zerumbone or 0.5% (v/v) DMSO. After overnight incubation, all floating and attached cells were harvested and fixed in 70% (v/v) ethanol for 16 h at −20 °C. Prior to analysis, fixed cells were pelleted and washed in cold PBS followed by staining with 10 µg/ml propidium iodide solution containing 20 µg/ml RNAse for 30 min at room temperature in the dark. Stained cells were next analysed by BD FACSCanto II™ flow cytometer (BD Biosciences, MA, USA) with 10,000 events collected for each read. The distribution of DNA in different phases was determined using the ModFit software (Verity Software House, USA). The percentage of cells in each phase was obtained from three independent experiments. FITC-Annexin V assay Detection of apoptosis in ORL cell lines treated with zerumbone was carried out using the Annexin V-FITC / Propidium Iodide (PI) binding assay following the manufacturer's recommendations (BD Biosciences, MA, USA). Briefly, ORL-48 and ORL-115 cells were seeded at 2 × 105 in 60 mm culture plates and exposed to 3–30 µM zerumbone or 0.5% (v/v) 35
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Fig. 2. Zerumbone inhibits cell proliferation and induces apoptosis in OSCC cell lines. (A-B) Zerumbone (24 h) exhibited anti-proliferative effects on ORL-48 and ORL-115 by Click-iT EdU assay. (A) Representative fluorescence images showing reduction of proliferating cells upon 10–30 µM zerumbone treatment. Blue (Hoecsht 33342) represents the total number of cells in any field and red (Alexa 647) represents proliferating cells that have incorporated the EdU label. (B) The percentage of EdU-positive cells was significantly reduced by 30 µM zerumbone as compared to DMSO control groups. (C-E) Zerumbone induced G2/M cell cycle arrest in ORL-48 and ORL-115 cells by propidium iodide analysis. (C) Flow histograms showing population of cells in each cell cycle phase (sub-G1, G0/1, S and G2/M) after 24 h of treatment. The percentage of cells in the G2/M phase was recorded as in (D) for 24 h and (E) for 72 h treatment. (F-G) Zerumbone-treated cells showed higher percentage of apoptotic cells (FITC-Annexin V-positive cells) as compared to the DMSO controls. (F) Representative flow histograms depicting early (Q4) and late (Q2) apoptotic fractions of ORL-48 and ORL-115 cells upon 72 h of zerumbone treatment. (G) Percentage of apoptotic cells (early + late apoptosis) in ORL-48 and ORL-115. (H) Zerumbone (72 h) increased the levels of apoptotic markers on ORL-48 and ORL-115 as shown by the western blot analysis of cleaved caspase 3 and cleaved PARP proteins. All quantification results shown are mean ± SD (n = 3). Significant differences from DMSO control groups were calculated by two-tailed student's T-test. * and ** denotes p < .05 and p < .01 respectively. Western blot results shown are representative of at least two independent experiments.
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investigations, we chose ORL-48 and ORL-115 to represent OSCC cells that showed moderate and high sensitivity to zerumbone.
washes in TBST prior to detection by WesternBright Quantum HRP substrate (Advansta Inc, CA, USA) and visualization using the FluorChemTM HD2 imaging systems (ProteinSimple, CA, USA). The visualized protein bands were quantified using ImageJ software (National Institute of Health, Bethesda, MD, USA).
Zerumbone inhibits OSCC via cell cycle arrest and apoptosis As zerumbone was observed to reduce viability of OSCC cells, we then investigated its effect on cell proliferation by measuring DNA synthesis using Click-iT EdU assay. As shown in the representative images in Fig. 2A-B, inhibition of cell proliferation was seen in a dose dependent manner. While low levels of zerumbone (3 µM) conferred a minimal response in ORL-115 (Fig. 2B), further reduction in cell proliferation by ∼10% and 30% was observed in ORL-48 (p = .11) and ORL-115 (p = .04) respectively, following zerumbone treatment at 10 µM. Upon treatment at 30 µM, zerumbone caused reduction of cell proliferation by ∼60% for both lines (p = .01 for both). We next investigated whether the observed effect was due to perturbation in the cell cycle using propidium iodide (PI) staining. As seen in Fig. 2C, both lines treated with zerumbone accumulated at the G2/M phase in a dosedependent manner with a corresponding reduction of cell population in the G0/1 phase. Quantification of the DNA content in the different phases of the cell cycle showed a significant increase in the percentage of cells in G2/M phase at 10 µM and 30 µM compared to the respective DMSO controls in ORL-48 and ORL-115 (p < .05; Fig. 2D). We further tested whether this cytostatic effect of zerumbone was more pronounced and persistent when the experimental treatment time was prolonged to 72 h. Consistently, we saw a significant increase of the percentage of cells in the G2/M phase in cells treated with 10 and 30 µM of zerumbone (p < .05; Fig. 2E). Of interest, we noticed a substantial increase of cell population in the sub-G1 phase, when both lines were treated with 10–30 µM of zerumbone, suggesting the occurrence of cell death at longer treatment period (Supplementary Fig. S2). Correspondingly, as zerumbone has also been reported to induce apoptosis in non-small cell lung cancer (Takada et al., 2005) and liver cancer cells (Sakinah et al., 2007), we explored whether zerumbone induces apoptosis that could account for this sub-G1 population. As shown by the representative flow histograms in Fig. 2F, 72 h of zerumbone exposure (10 and 30 µM) resulted in a marked increase of cells in early (FITC-Annexin V-positive and PI-negative; Q4) and late apoptosis (FITC-Annexin V-positive and PI-positive; Q2) when compared to DMSO controls. Induction of apoptosis by zerumbone was more pronounced in ORL-115 cells with increasing concentrations, where 10 µM was sufficient to increase the percentage of apoptotic cells significantly (p = .04), while 30 µM gave a more robust effect when compared to ORL-48 cells (Fig. 2G). Nonetheless, both lines exhibited a significant increase in the number of apoptotic cells when exposed to 30 µM zerumbone for 72 h (27.1% and 33.9% respectively) when compared with DMSO control (8.1% and 10% respectively; p = .005 and p = .004). To confirm the induction of apoptotic activity by zerumbone, western blot analysis was performed to assess the levels of cleaved caspase 3 and cleaved PARP in zerumbone-treated cells. Caspase 3 and PARP are involved in DNA repair activity and their cleaved forms serve as a marker for cells undergoing apoptosis. We observed increases of cleaved caspase 3 and cleaved PARP levels especially when cells were treated with 30 µM zerumbone, with corresponding decreases of the full-length proteins (Fig. 2H). From the experiments conducted, we verified zerumbone to reduce OSCC cell viability by inhibiting cell growth and further cause apoptosis at a longer exposure.
RhoA pull-down assay RhoA activity in ORL-48 and 115 was investigated using a pulldown purification method as previously described (Chikumi et al., 2002; Gan et al., 2014). Briefly, 2 × 105 ORL-48 and ORL-115 cells that were seeded in 60 mm culture plates were treated with 3–30 µM zerumbone or 0.5% (v/v) DMSO and serum starved for 24 h. Prior to protein extraction, cells were replenished with fresh serum-free medium and stimulated with 1 U/ml thrombin for three min as optimized from our previous study (Gan et al., 2014). Following thrombin stimulation, cells were washed with PBS and lysed on ice with cold lysis buffer (as detailed above). Collected lysates were centrifuged at 14,000 rpm for 5 min at 4 °C. The resulting supernatant were immediately incubated with glutathione-sepharose 4B beads (GE Healthcare, Sweden) bound with glutathione-S-transferase (GST)-rhotekinRhoA binding domain for 30 min at 4 °C on a rotator, while the remaining lysates were used to detect total RhoA protein. After incubation, the coated beads were collected by centrifugation at 9000 rpm for 1 min at 4 °C and washed 3 times with cold lysis buffer. The associated GTP-bound RhoA was released with protein loading buffer, and heatinactivated prior to SDS-PAGE and western blotting. Blots were incubated using anti-RhoA (sc-418; Santa Cruz Biotechnology, CA, USA) at 1:1000 dilution for 16 h and further processed according to western blotting conditions as mentioned above. Statistical analyses All data are expressed as mean ± SD from three independent experiments. The significance of differences between groups in each experiment was analysed by Student's t-tests using GraphPad Prism software (GraphPad Software Inc., CA, USA). Differences were considered statistically significant at p < .05. Results Zerumbone reduces OSCC cell viability As previous studies have demonstrated the cytotoxic effect of zerumbone against different human cancer cells, we aimed to determine if a representative panel of OSCC cell lines were sensitive to this compound. To achieve this, we treated a panel of 16 OSCC lines with zerumbone at concentrations ranging from 0 to 100 µM. Two normal oral keratinocytes (NOK) primary cultures (ORL-232 and ORL-235) were also included in the treatment as comparison. The MTT results revealed that the half maximal inhibitory concentration (IC50) of zerumbone ranged from 0.8 to 4.9 µM with an average value of ∼2 µM across this panel of OSCC lines (Fig. 1B). By contrast, the NOK cultures showed significantly higher average IC50 of 25 µM. This shows that zerumbone has more potent effects towards OSCC and was less toxic on normal cells with about eight-fold difference in IC50 (p = .009; supplementary Fig. S1). We also demonstrated that breast (MCF-7) and colorectal (HCT-116) cancer cells were sensitive to zerumbone with IC50 of 8.11 and 1.39 µM respectively (Fig. 1B). To evaluate zerumbone's efficacy against standard anti-cancer drugs, we compared zerumbone with cisplatin (chemotherapy drug) and gefitinib (small molecule inhibitor targeting EGFR). The average IC50 of cisplatin and gefitinib in the same panel of OSCC lines were 8.7 µM (range: 2.7 to 14.5 µM) and 14.49 µM (0.1 to 36.9 µM) respectively, implying that zerumbone could be more potent with lower IC50 concentrations (average 2 µM; range: 0.8 – 4.9 µM) compared to cisplatin and gefitinib (Fig. 1C). For subsequent
Zerumbone reduces cell migration and invasion of OSCC cells We also evaluated the influence of zerumbone on the migration of OSCC cells by performing wound healing assay. Zerumbone, when given at a concentration of 30 µM, inhibited cell migration, where the open wound area of zerumbone-treated ORL-48 and ORL-115 cells were larger as compared to DMSO-treated cells following a 24-h observation (p = .02 and p = .03 respectively; Fig. 3A). To validate that 30 µM of 37
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Fig. 3. Zerumbone suppresses cell migration and invasion in vitro, reduces CXCR4 expression and inactivates RhoA signaling. (A) Migration of ORL-48 and ORL-115 cells was significantly inhibited as indicated by the representative images and the percentage of open wound area after treatment with 30 µM of zerumbone for 24 h. (B) Zerumbone treatment of 30 µM suppressed ORL-48 and ORL-115 cells from invading through the matrigel membrane of transwell inserts as indicated by the representative images and the percentage of cell invasion. Thrombin was used as chemoattractant. (C) Expression of CXCR4 protein was inhibited by 30 µM of zerumbone when cells were treated for 24 h with increasing concentrations. (D) 30 µM of zerumbone caused reduction in CXCR4 protein expression in a time-dependent manner. (E) RhoA inactivation was observed in ORL-48 and ORL-115 after zerumbone treatment at 30 µM for 24 h. Following zerumbone treatment, cells were stimulated by 1 U/ml of thrombin for three min to activate the pathway and whole cell extracts were subjected to RhoA pulldown purification assay and western blot analysis. All quantification results shown are mean ± SD (n = 3). Significant differences from DMSO control groups were calculated by twotailed student's T-test. * denotes p < .05. Western blot results shown are representative of at least two independent experiments. Densitometric quantifications for C-E blots are provided in Supplementary Fig. S4.
Zerumbone reduced CXCR4 protein expression
zerumbone was indeed anti-migratory without inducing cytotoxicity in the two cell lines within the 24-h treatment period, we focused on the cell population specifically in the sub-G1 phase of the cell cycle. After 24 h of 3–30 µM zerumbone treatment, no significant increase in the number of cells in the sub-G1 phase was observed (Supplementary Fig. S3). This indicated that the observed anti-migratory activity of zerumbone was not attributed to the induction of cell death. Following cell migration assay, we further evaluated zerumbone's ability in preventing cell invasion in ORL-48 and ORL-115 cells by transwell invasion assay. We demonstrated that 30 µM zerumbone caused a significant decrease in the percentage of cells invading through the matrigel barrier in response to thrombin as a chemoattractant, compared to the DMSO control in both lines (p = .04 for both; Fig. 3B).
To investigate the potential molecular target of zerumbone that could implicate cell motility, we looked into the expression of CXCR4 which has been known to be involved in the modulation of cancer cell motility, survival and proliferation (Teicher and Fricker, 2010). Our western blot analysis revealed that 30 µM of zerumbone consistently resulted in a reduction of CXCR4 protein levels in both ORL-48 and ORL-115 lines, while lower concentrations had a minimal effect (Fig. 3C). Using 30 µM concentration of zerumbone for subsequent studies, we further investigated the reduction of CXCR4 protein levels through a time-dependent examination by western blot. From our observations (Fig. 3D), reduction of CXCR4 protein levels in both ORL-48 and ORL-115 cells can be observed as early as 8 h and is most pronounce at 24 h of zerumbone treatment. 38
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carcinoma (HNSCC) dissemination. Blockade of CXCR4 signaling axis has been demonstrated to inhibit lymph node metastases in an orthotopic tongue model of OSCC through the inhibition of IL-6 and IL-8 production (Uchida et al., 2011). In another study, EMT biomarkers such as vimentin and integrin β1 were shown to be highly expressed in the metastatic clones of HNSCC and the use of CXCR4 antagonist essentially blocked both primary tumor growth and metastases in the subcutaneous xenograft model (Yoon et al., 2007). Additionally, tumors that have high expression of CXCR4 in OSCC patients correlated with poor overall survival (Albert et al., 2012). Our finding is consistent with Sung et al. (2008), whereby CXCR4 protein expression was downregulated by zerumbone leading to the inhibition of CXCL12-induced invasion of breast and pancreatic cells. However, the effect of the downstream protein activity leading to the inhibition of cell invasion was not ascertained. In this current study, we chose to investigate if the loss of endogenous CXCR4 protein expression was likely to impact RhoA activity as previous reports have demonstrated that CXCR4 activates the Gα12/13 signaling axis and further regulate the migration and invasion of mammalian cells through RhoA (Tan et al., 2006). Our results indicated that RhoA activity was inhibited by zerumbone. This suggests a therapeutic opportunity for OSCC treatment as we have previously reported the over-expression of Gα12 and prevention of metastasis by inhibition of RhoA activation in cell line and orthotopic mouse model (Gan et al., 2014). The aberrancy of RhoA in head and neck cancers has also been described by Abraham et al., in which a subset of motility-related proteins including RhoA were found to be overexpressed in cell lines and patient tissue samples relative to normal specimens (Abraham et al., 2001). For this significance, RhoA was used as one of the molecular markers to characterize potentially malignant tumors for HNSCC. We show that the inhibition of CXCR4-RhoA pathway associates with the inactivation of the PI3K-mTOR pathway by reducing the phosphorylation of Akt and S6 protein kinases. This is a favourable effect as the PI3K-mTOR pathway is known to be widely activated in HNSCC, rendering persistent mitogenic signaling. In particular, 20% of head and neck cancer samples have Akt upregulated while in 80–90% of samples, phosphorylated S6 was present (Iglesias-Bartolome et al., 2013). CXCR4 and Akt have multifaceted roles in tumor development, and both have been implicated in proliferation, cell migration and invasion (Hong et al., 2009) making it difficult to individually determined which phenotypes observed upon zerumbone treatment was due to the inhibition of which individual pathway. Furthermore, the interplay between CXCR4 and Akt signaling has been reported previously, where CXCR4 signaling can result in the activation of Akt (Katayama et al., 2005). In summary, zerumbone is potent at concentrations that are lower than the commonly used cancer therapies tested here, and is selective to OSCC cells. Its ability to inhibit cell proliferation, migration and invasion through well-established pathways that have been implicated in OSCC presents an opportunity for it to be a potential candidate for OSCC treatment.
Zerumbone inactivates RhoA, a downstream target of CXCR4 As previous work had identified RhoA as one of the downstream targets for CXCR4 to promote cancer invasiveness and metastases (Guo et al., 2016), we next investigated if reduced CXCR4 protein levels due to zerumbone resulted in an inhibition of RhoA activity. The activity of RhoA was evaluated using protein pull-down assay by measurement of the active GTP bound RhoA protein. Upon treatment with 30 µM of zerumbone for 24 h, levels of active RhoA decreased in both thrombin-stimulated ORL-48 and ORL-115 cells with no changes in total RhoA expression (Fig. 3E), demonstrating that zerumbone was able to inhibit RhoA activity in these cells. Taken together, zerumbone was able to reduce CXCR4 protein levels and RhoA activation resulting in the inhibition of cell motility in the two OSCC cell lines. Zerumbone downregulates the Akt-mTOR signaling pathway Previous findings have shown that the CXCR4-mediated chemotaxis could occur through PI3K activation (Ward, 2006). In OSCC, preclinical investigation on mouse model revealed the CXCR4/CXCL12 signaling mediates lymph node metastasis through the Akt pathway (Uchida et al., 2004). Here, we further investigated whether inhibition of the CXCR4-RhoA signaling would impact the PI3K-mTOR pathway by firstly looking into whether zerumbone could inactivate EGFR, a tyrosine kinase receptor that is involved in the regulation of PI3KmTOR signaling. As both ORL-48 and ORL-115 do not have constitutive activation of EGFR, the effects of zerumbone were not observed (Fig. 4A & B). As a positive control for data set, we used protein extract of ORL136 cells, which is known to have high levels of phosphorylated EGFR (Fadlullah et al., 2016). While we were not able to see the effect of zerumbone on EGFR activity, we demonstrated that zerumbone could inhibit Akt phosphorylation at 30 µM in both ORL-48 and ORL-115. This was also observed in a time-dependent manner where inactivation of Akt (Ser473) in ORL-48 and ORL-115 were observed at 16 and 8 h post-treatment respectively (Fig. 4B). The inhibition of Akt resulted in the inactivation of S6 (Fig. 4A & B). Of note, significant inactivation of S6 was observed as early as 8 h of treatment in both ORL-48 and 115 (Fig. 4B). Taken together, our data demonstrates that zerumbone impacts multiple proteins of the CXCR4-RhoA and Akt-mTOR pathways, consistent with the observation of inhibition of cell proliferation, migration and invasion of OSCC cells. Discussions Zerumbone, a natural dietary agent is now known to have antitumor properties in different types of human cancer cells (Prasannan et al., 2012). Herein, we demonstrate the bioactivity of zerumbone against OSCC cells with favourable IC50 values (∼2 µM). This sensitivity was comparable to those reported for other cancers for example, colorectal (HT-29, CaCo-2) and breast (MCF-7) with values of ∼10 µM (Kirana et al., 2003), while for cervical (HeLa) and ovarian cancer cells (Caov-3), values were 11.3 µM (Abdul et al., 2008) and 24 µM (Abdelwahab et al., 2012), respectively. Furthermore, we showed zerumbone to have lower average IC50 values against cisplatin and gefitinib, implying a favourable response against standard cancer treatments. Our results demonstrate that zerumbone treatment resulted in the reduction of cell proliferation with a concurrent arrest of cells at the G2/M phase, followed by apoptosis activity at longer exposures. These observations were consistent with a previously reported study showing zerumbone to trigger apoptotic activity following G2/M cell cycle arrest in NB4 leukemia cells (Xian et al., 2007). In our current study, we also noted that zerumbone displayed anti-migration and anti-invasion properties in OSCC cells along with concurrent downregulation of CXCR4 protein expression. Cumulative evidence has suggested that CXCR4 acts as marker of tumor aggressiveness and is involved in head and neck squamous cell
Conflict of interest The authors have no conflict of interests to declare.
Acknowledgments This work was supported by High Impact Research, Ministry of Higher Education (HIR-MOHE) from University of Malaya (UM.C/625/ 1/HIR/MOHE/DENT-03) and other sponsors of Cancer Research Malaysia. Cancer Research Malaysia is a non-profit research organization. We are committed to an understanding of cancer prevention, diagnosis and treatment through a fundamental research program. 39
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Fig. 4. Zerumbone inactivates Akt-mTOR signaling. (A) Dose-dependent analysis of zerumbone showed inactivation of Akt and S6 in OSCC cells at 30 µM concentration. Cells were treated with the indicated concentrations for 24 h. (B) Time-course analysis of 30 µM zerumbone on ORL-48 and 115 cells showed inactivation of Akt and S6 at 8 h post treatment. Western blot results shown are representative of at least two independent experiments. Densitometric quantifications for A-B blots are provided in Supplementary Fig. S5.
Supplementary materials
Albert, S., Hourseau, M., Halimi, C., et al., 2012. Prognostic value of the chemokine receptor CXCR4 and epithelial-to-mesenchymal transition in patients with squamous cell carcinoma of the mobile tongue. Oral Oncol. 48, 1263–1271. Chakraborty, A., Coffman, R., Jorvig, J., 2013. Zerumbone, a phytochemical from Asian ginger is a novel inhibitor of Jak2/Stat3 inhibits promigratory gene expression, growth and migration of pancreatic cancer cells. Pancreatology 13, e18–e19. Cheong, S., Chandramouli, G., Saleh, A., et al., 2009. Gene expression in human oral squamous cell carcinoma is influenced by risk factor exposure. Oral Oncol. 45, 712–719. Chikumi, H., Vázquez-Prado, J., Servitja, J.-M., et al., 2002. Potent activation of RhoA by Gαq and Gq-coupled receptors. J. Biol. Chem. 277, 27130–27134. Chong, C.E., Lim, K.P., Gan, C.P., et al., 2012. Over-expression of MAGED4B increases cell migration and growth in oral squamous cell carcinoma and is associated with poor disease outcome. Cancer Lett. 321, 18–26. Fadlullah, M., Chiang, I., Dionne, K.R., et al., 2016. Genetically-defined novel oral squamous cell carcinoma cell lines for the development of molecular therapies. Oncotarget 7, 27802. Gan, C.P., Patel, V., Mikelis, C.M., et al., 2014. Heterotrimeric G-protein alpha-12 (G'12) subunit promotes oral cancer metastasis. Oncotarget 5, 9626–9640. Guo, J., Yu, X., Gu, J., et al., 2016. Regulation of CXCR4/AKT-signaling-induced cell invasion and tumor metastasis by RhoA, Rac-1, and Cdc42 in human esophageal
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2017.12.011. References Abdelwahab, S.I., Abdul, A.B., Mohan, S., et al., 2011. Zerumbone induces apoptosis in Tacute lymphoblastic leukemia cells. Leukemia Res. 35, 268–271. Abdelwahab, S.I., Abdul, A.B., Zain, Z.N.M., et al., 2012. Zerumbone inhibits interleukin6 and induces apoptosis and cell cycle arrest in ovarian and cervical cancer cells. Int. Immunopharmacol. 12, 594–602. Abdul, A.B., Al-Zubairi, A.S., Tailan, N.D., et al., 2008. Anticancer activity of natural compound (Zerumbone) extracted from Zingiber zerumbet in human HeLa cervical cancer cells. Int. J. Pharmacol. 4, 160–168. Abraham, M.T., Kuriakose, M.A., Sacks, P.G., et al., 2001. Motility‐Related Proteins as Markers for Head and Neck Squamous Cell Cancer. Laryngoscope 111, 1285–1289. Aggarwal, B.B., Shishodia, S., 2006. Molecular targets of dietary agents for prevention and therapy of cancer. Biochem. Pharmacol. 71, 1397–1421.
40
Phytomedicine 39 (2018) 33–41
N.S. Zainal et al.
mediated apoptosis in human breast cancer cells and inhibits orthotopic xenograft growth in vivo. Breast Cancer Res. Treatment 136, 429–441. Shanmugam, M.K., Rajendran, P., Li, F., et al., 2015. Abrogation of STAT3 signaling cascade by zerumbone inhibits proliferation and induces apoptosis in renal cell carcinoma xenograft mouse model. Mol. Carcinog. 54, 971–985. Specenier, P., Vermorken, J.B., 2012. Biologic therapy in head and neck cancer: a road with hurdles. ISRN Oncol. 2012. Sung, B., Jhurani, S., Ahn, K.S., et al., 2008. Zerumbone down-regulates chemokine receptor CXCR4 expression leading to inhibition of CXCL12-induced invasion of breast and pancreatic tumor cells. Cancer Res. 68, 8938–8944. Takada, Y., Murakami, A., Aggarwal, B.B., 2005. Zerumbone abolishes NF-κB and IκBα kinase activation leading to suppression of antiapoptotic and metastatic gene expression, upregulation of apoptosis, and downregulation of invasion. Oncogene 24, 6957–6969. Tan, W., Martin, D., Gutkind, J.S., 2006. The Gα13-Rho signaling axis is required for SDF1-induced migration through CXCR4. J. Biol. Chem. 281, 39542–39549. Teicher, B.A., Fricker, S.P., 2010. CXCL12 (SDF-1)/CXCR4 pathway in cancer. Clinical Cancer Res. 16, 2927–2931. Torre, L.A., Bray, F., Siegel, R.L., et al., 2015. Global cancer statistics, 2012. CA Cancer J Clin 65, 87–108. Uchida, D., Begum, N.-M., Tomizuka, Y., et al., 2004. Acquisition of lymph node, but not distant metastatic potentials, by the overexpression of CXCR4 in human oral squamous cell carcinoma. Lab. Invest. 84, 1538–1546. Uchida, D., Onoue, T., Kuribayashi, N., et al., 2011. Blockade of CXCR4 in oral squamous cell carcinoma inhibits lymph node metastases. Eur. J. Cancer 47, 452–459. Vermorken, J.B., Mesia, R., Rivera, F., et al., 2008. Platinum-based chemotherapy plus cetuximab in head and neck cancer. New Eng. J. Med. 359, 1116–1127. Ward, S.G., 2006. T lymphocytes on the move: chemokines, PI 3-kinase and beyond. Trends Immunol. 27, 80–87. Xian, M., Ito, K., Nakazato, T., et al., 2007. Zerumbone, a bioactive sesquiterpene, induces G2/M cell cycle arrest and apoptosis in leukemia cells via a Fas‐and mitochondria‐mediated pathway. Cancer Sci. 98, 118–126. Yoon, Y., Liang, Z., Zhang, X., et al., 2007. CXC chemokine receptor-4 antagonist blocks both growth of primary tumor and metastasis of head and neck cancer in xenograft mouse models. Cancer Res. 67, 7518–7524. Yu, Z., Weinberger, P.M., Sasaki, C., et al., 2007. Phosphorylation of Akt (Ser473) Predicts Poor Clinical Outcome in Oropharyngeal Squamous Cell Cancer. Cancer Epidemiol. Biomarkers Preven. 16, 553–558. Zlotogorski, A., Dayan, A., Dayan, D., et al., 2013. Nutraceuticals as new treatment approaches for oral cancer: II. Green tea extracts and resveratrol. Oral Oncol. 49, 502–506.
cancer. Tumor Biol. 37, 6371–6378. Gupta, S.C., Kim, J.H., Prasad, S., et al., 2010. Regulation of survival, proliferation, invasion, angiogenesis, and metastasis of tumor cells through modulation of inflammatory pathways by nutraceuticals. Cancer Metastasis Rev. 29, 405–434. Hamid, S., Lim, K.P., Zain, R.B., et al., 2007. Establishment and characterization of Asian oral cancer cell lines as in vitro models to study a disease prevalent in Asia. Int. J.Mol. Med. 19, 453–460. Hong, J.S., Pai, H.K., Hong, K.O., et al., 2009. CXCR‐4 knockdown by small interfering RNA inhibits cell proliferation and invasion of oral squamous cell carcinoma cells. J. Oral Pathol. Med. 38, 214–219. Iglesias-Bartolome, R., Martin, D., Gutkind, J.S., 2013. Exploiting the head and neck cancer oncogenome: widespread PI3K-mTOR pathway alterations and novel molecular targets. Cancer Discovery 3, 722–725. Kang, C.G., Lee, H.-J., Kim, S.-H., et al., 2015. Zerumbone Suppresses OsteopontinInduced Cell Invasion Through Inhibiting the FAK/AKT/ROCK Pathway in Human Non-Small Cell Lung Cancer A549 Cells. J. Natural Products. Katayama, A., Ogino, T., Bandoh, N., et al., 2005. Expression of CXCR4 and its downregulation by IFN-γ in head and neck squamous cell carcinoma. Clinical Cancer Res. 11, 2937–2946. Kim, M., Miyamoto, S., Yasui, Y., et al., 2009. Zerumbone, a tropical ginger sesquiterpene, inhibits colon and lung carcinogenesis in mice. Int. J. Cancer 124, 264–271. Kirana, C., McIntosh, G.H., Record, I.R., et al., 2003. Antitumor activity of extract of Zingiber aromaticum and its bioactive sesquiterpenoid zerumbone. Nutrition Cancer 45, 218–225. Lui, V.W., Hedberg, M.L., Li, H., et al., 2013. Frequent mutation of the PI3K pathway in head and neck cancer defines predictive biomarkers. Cancer Discovery 3, 761–769. Massano, J., Regateiro, F.S., Januário, G., et al., 2006. Oral squamous cell carcinoma: review of prognostic and predictive factors. Oral Surgery Oral Med. Oral Pathol. Oral Radiol. Endodontol. 102, 67–76. Newman, D.J., Cragg, G.M., 2012. Natural products as sources of new drugs over the 30 years from 1981 to 2010. J. Natural Prod. 75, 311–335. Pau, G., Fuchs, F., Sklyar, O., et al., 2010. EBImage—an R package for image processing with applications to cellular phenotypes. Bioinformatics 26, 979–981. Pickering, C.R., Zhang, J., Yoo, S.Y., et al., 2013. Integrative genomic characterization of oral squamous cell carcinoma identifies frequent somatic drivers. Cancer Discovery 3, 770–781. Prasannan, R., Kalesh, K.A., Shanmugam, M.K., et al., 2012. Key cell signaling pathways modulated by zerumbone: role in the prevention and treatment of cancer. Biochem. Pharmacol. 84, 1268–1276. Sakinah, S.S., Handayani, S.T., Hawariah, L.A., 2007. Zerumbone induced apoptosis in liver cancer cells via modulation of Bax/Bcl-2 ratio. Cancer Cell Int. 7, 1. Sehrawat, A., Arlotti, J.A., Murakami, A., et al., 2012. Zerumbone causes Bax-and Bak-
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Phytomedicine journal homepage: www.elsevier.com/locate/phymed
Original Article
Zerumbone targets the CXCR4-RhoA and PI3K-mTOR signaling axis to reduce motility and proliferation of oral cancer cells
T
Nur Syafinaz Zainala, Chai Phei Gana, Beng Fye Laub, Pei San Yeea, Kai Hung Tionga,c, ⁎ Zainal Ariff Abdul Rahmanc,d, Vyomesh Patela, Sok Ching Cheonga,d, a
Cancer Research Malaysia, No. 1, Jalan SS12/1A, 47500 Subang Jaya, Selangor, Malaysia Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia c Oral Cancer Research and Co-ordinating Centre (OCRCC), Faculty of Dentistry, University of Malaya, 50603 Kuala Lumpur, Malaysia d Dept of Oral & Maxillofacial Clinical Sciences, Faculty of Dentistry, University of Malaya, 50603 Kuala Lumpur, Malaysia b
A R T I C L E I N F O
A B S T R A C T
Keywords: Zerumbone Oral squamous cell carcinoma Akt RhoA Proliferation Migration
Background: The CXCR4-RhoA and PI3K-mTOR signaling pathways play crucial roles in the dissemination and tumorigenesis of oral squamous cell carcinoma (OSCC). Activation of these pathways have made them promising molecular targets in the treatment of OSCC. Zerumbone, a bioactive monocyclic sesquiterpene isolated from the rhizomes of tropical ginger, Zingiber zerumbet (L.) Roscoe ex Sm. has displayed promising anticancer properties with the ability to modulate multiple molecular targets involved in carcinogenesis. While the anticancer activities of zerumbone have been well explored across different types of cancer, the molecular mechanism of action of zerumbone in OSCC remains largely unknown. Purpose: Here, we investigated whether OSCC cells were sensitive towards zerumbone treatment and further determined the molecular pathways involved in the mechanism of action. Methods: Cytotoxicity, anti-proliferative, anti-migratory and anti-invasive effects of zerumbone were tested on a panel of OSCC cell lines. The mechanism of action of zerumbone was investigated by analysing the effects on the CXCR4-RhoA and PI3K-mTOR pathways by western blotting. Results: Our panel of OSCC cells was broadly sensitive towards zerumbone with IC50 values of less than 5 µM whereas normal keratinocyte cells were less responsive with IC50 values of more than 25 µM. Representative OSCC cells revealed that zerumbone inhibited OSCC proliferation and induced cell cycle arrest and apoptosis. In addition, zerumbone treatment inhibited migration and invasion of OSCC cells, with concurrent suppression of endogenous CXCR4 protein expression in a time and dose-dependent manner. RhoA-pull down assay showed reduction in the expression of RhoA-GTP, suggesting the inactivation of RhoA by zerumbone. In association with this, zerumbone also inhibited the PI3K-mTOR pathway through the inactivation of Akt and S6 proteins. Conclusion: We provide evidence that zerumbone could inhibit the activation of CXCR4-RhoA and PI3K-mTOR signaling pathways leading to the reduced cell viability of OSCC cells. Our results suggest that zerumbone is a promising phytoagent for development of new therapeutics for OSCC treatment.
Introduction Oral squamous cell carcinoma (OSCC) affects on average 300,400 individuals and accounts for 145,400 deaths every year (Torre et al., 2015). This poor prognosis has largely remained unchanged over the past several decades and is in part, due to limited therapeutic options (Massano et al., 2006; Specenier and Vermorken, 2012). The identification of important signaling pathways driving the development of
OSCC has resulted in the use of cetuximab, a tyrosine kinase inhibitor targeting the epidermal growth factor receptor (EGFR) for the treatment of advanced and recurrent OSCC. However, this treatment combined with chemotherapy only improved the median overall survival by ∼3 months in OSCC patients (Vermorken et al., 2008) underscoring the urgent need to develop new and improved drugs for OSCC. Of note, 50% of approved cancer therapeutic agents are derived from natural products (Newman and Cragg, 2012) and secondary
Abbreviations: Akt, protein kinase B; CXCR4, C-X-C chemokine receptor type 4; EdU, ethynyl deoxyuridine; EGFR, epidermal growth factor receptor; Gα12, G-alpha protein-12; HNSCC, head and neck squamous cell carcinoma; mTOR, mammalian target of rapamycin; OSCC, oral squamous cell carcinoma; PI3K, phosphoinositide 3-kinase; RhoA, Ras homolog gene family member A; S6, ribosomal protein S6 ⁎ Corresponding author: Cheong Sok Ching, Head and Neck Cancer Research Team, Cancer Research Malaysia, No.1, Jalan SS12/1A, 47500 Subang Jaya, Selangor, Malaysia. E-mail address: [email protected] (S.C. Cheong). https://doi.org/10.1016/j.phymed.2017.12.011 Received 15 June 2017; Received in revised form 2 November 2017; Accepted 7 December 2017 0944-7113/ © 2017 Elsevier GmbH. All rights reserved.
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Fig. 1. Zerumbone reduces cell viability of OSCC cell lines. (A) Chemical structure of zerumbone. (B) IC50 of zerumbone across a panel of OSCC cell lines (grey bars) and NOK (white bars) as determined by MTT assay after 72 h of treatment. OSCC cells were more sensitive to zerumbone than NOK (average IC50 is provided in Supplementary Fig. S1). Breast (MCF-7) and colorectal (HCT-116) cell lines (black bars) were added for comparison. (C) The mean IC50 of zerumbone across a panel of OSCC cell lines was compared against two anticancer drugs (cisplatin and gefitinib). The IC50 of cisplatin and gefitinib across OSCC cell line panel is provided in Supplementary Table 1. Results shown are mean ± SD (n = 3). Significant differences between comparative groups were calculated by two-tailed student's T-test. * and *** denotes p < .05 and p < .001 respectively.
consequently the Gα12-RhoA axis in OSCC cells. In addition, our current study also investigated the effect of zerumbone on the PI3K-AktmTOR signaling pathway which is commonly known to be highly activated in OSCC and is associated with poor clinical outcome (Lui et al., 2013; Pickering et al., 2013; Yu et al., 2007).
metabolites from medicinal plants have demonstrated a unique potential as a source of anticancer and chemopreventive agents. Compounds isolated from edible plants have the advantage of low toxicity profiles and are able to simultaneously target multiple signaling pathways (Aggarwal and Shishodia, 2006; Gupta et al., 2010). This could be particularly useful in the context of developing new anticancer agents as cancer development typically involves dysregulation of multiple genes and signaling pathways. In OSCC, treatment and prevention strategies are also moving towards nutraceutical approaches, with green tea extracts and resveratrol showing the most potential in the development of new therapies (Zlotogorski et al., 2013). One natural plant metabolite that has demonstrated potential for the treatment of cancer is zerumbone; (2E,6E,10E)−2,6,9,9-tetramethylcycloundeca-2,6,10-trien-1-one (Fig. 1A), a bioactive monocyclic sesquiterpene isolated from the rhizomes of tropical ginger, Zingiber zerumbet (L.) Roscoe ex Sm. Emerging data have demonstrated anticancer properties of zerumbone in common types of cancers including breast, pancreatic, colon, lung and leukemia (Abdelwahab et al., 2011; Chakraborty et al., 2013; Kim et al., 2009; Sehrawat et al., 2012). Zerumbone is able to suppress key hallmarks of cancer for example, by enhancing apoptosis (Sehrawat et al., 2012), reducing cell proliferation (Shanmugam et al., 2015), and inhibiting migration and invasion (Kang et al., 2015; Sung et al., 2008). With regards to OSCC, preliminary results demonstrated that zerumbone could down-regulate the protein expression of CXCR4 in SCC4 cells (Sung et al., 2008) and reduced NF-κB activity in FaDu and LICR-LON-HN5 cells (Takada et al., 2005). However, the underlying molecular basis of zerumbone's promising anticancer properties remains largely unexplored and represents an opportunity for identifying novel therapies for OSCC. Prior work from our laboratory have shown that the Gα12 axis is enriched in OSCC and RhoA activity is critical in mediating OSCC metastases to the cervical lymph nodes in an orthotopic-tongue mouse model (Gan et al., 2014; Cheong et al., 2009). CXCR4 is able to activate the Gα12 signaling and cause migration and invasion of cells through RhoA (Tan et al., 2006). Therefore in this study, we were interested in discerning whether zerumbone is able to inhibit CXCR4, and
Materials and methods Chemicals Zerumbone was purchased from Sigma Aldrich, MO, USA with reported purity of ≥ 98% by HPLC analysis. Cisplatin and gefitinib were purchased from Selleckchem, TX, USA with reported purity > 99% by HPLC analysis. All drugs were dissolved to 20 mM (zerumbone and gefitinib in dimethyl sulfoxide; DMSO and cisplatin in water) and further diluted in culture medium for immediate use or stored in −20 °C. Cell culture The panel of OSCC lines (ORL series) used in this study were established from oral cancer patients as previously reported (Fadlullah et al., 2016; Hamid et al., 2007). All ORL cell lines were cultured in Dulbecco's Modified Eagle's Medium/Nutrient mixture F12-Ham's medium (DMEM/F12; Hyclone, Utah, USA) supplemented with 10% (v/v) of heat inactivated fetal bovine serum (FBS; Gibco, Auckland, NZ) and 500 ng/ml of hydrocortisone (Sigma-Aldrich, MO, USA). The primary normal oral keratinocytes (NOK; ORL-232 and ORL-235) used in this study were established from healthy gingival biopsies as previously reported (Hamid et al., 2007) and grown in Keratinocyte Serum-Free Medium (KSFM; Gibco, Auckland, NZ) supplemented with 25 µg/ml bovine pituitary extract (BPE), 0.4 ng/ml epidermal growth factor (EGF) and 0.09 mM calcium chloride (CaCl2). HCT-116 and MCF-7 were grown in DMEM (Gibco, Auckland, NZ) supplemented with 10% (v/v) FBS. All cell lines have been authenticated as described previously (Fadlullah et al., 2016). Cells were cultured at 37 °C in a 5% CO2 humidified atmosphere. 34
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DMSO for 24 or 72 h. All floating and attached cells were harvested and subjected to FITC-Annexin V and PI double staining according to the manufacturer's instructions. Cell analysis was conducted using a BD FACSCanto II™ flow cytometer with 10,000 events collected for each read. The percentage of apoptotic cells were obtained from three independent experiments.
3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay The cytotoxic effect of zerumbone, cisplatin and gefitinib were determined by the MTT assay. Briefly, 5 × 103 cells were seeded in triplicates in 96-well plates and following overnight incubation, cells were exposed to zerumbone at concentrations ranging from 0.1–100 µM or vehicle control of 0.5% (v/v) DMSO. After 72 h incubation, 5 mg/ml of MTT solution (Sigma Aldrich, MO, USA) was added into each well and the plates were further incubated for an additional 4 h. The resulting formazan crystals were dissolved in 100 µl of DMSO and absorbance was measured at 570 nm using a Synergy H1 Multi-Mode reader (BioTek Instruments, VT, USA). Dose response curves were generated by plotting log concentrations (µM) against the percentage of cell viability. The half maximal inhibitory concentration (IC50) was expressed as mean ± SD of three independent experiments.
Wound healing assay Wound healing assays were performed as previously described (Chong et al., 2012). Briefly, ORL-48 and ORL-115 were seeded at 8 × 105 cells in each well of 6-well culture plates and incubated for 24 h at 37 °C to form a confluent monolayer. Cells were then treated with 10 µg/ml mitomycin C (Sigma-Aldrich, MO, USA) for 2 h to inhibit cell proliferation. A scratch was made through the monolayer with a P200 pipette tip by applying constant pressure to create an open wound. Cells were rinsed with PBS and then further cultured in DMEM/ F12 complete medium with the addition of zerumbone (3–30 µM) or 0.5% (v/v) DMSO. The open wound areas were microscopically recorded at 0 and 24 h. The percentage of open wound area was obtained using the TScratch analysis software (CSElab, Switzerland) from three independent experiments.
Click-iT EdU cell proliferation assay Cell proliferation was determined by the Click-iT EdU assay (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. Briefly, ORL-48 and ORL-115 were seeded at 7 × 104 cells per well, grown overnight on glass coverslips and treated with 3–30 µM zerumbone or 0.5% (v/v) DMSO for 24 h. Cells were incubated with 10 µM 5-ethynyl-2′-deoxyuridine (EdU) for 2–6 h prior to fixation with 3.7% (v/v) formaldehyde. The cells were permeabilized with 0.1% (v/ v) Triton X-100 in phosphate buffer, followed by EdU detection via a copper-catalyzed reaction and nuclei staining by Hoechst 33342 provided in the kit. The coverslips were then mounted on glass slides by using VECTASHIELD® Mounting Medium (Vector Laboratories, Burlingame, CA, USA) and examined on an upright Olympus IX71 microscope (Olympus, Japan) with double bandpass filters to detect fluorescent-stained nuclei (Hoechst 33342: excitation 360−370 nm and emission 420 nm) and Alexa-labelled EdU (Alexa 647: excitation 650 nm and emission 667 nm). Images were captured from 10 randomly chosen fields of each experiment and analyzed with EBImage (Pau et al., 2010). The number of EdU-positive cells and Hoechststained cells were counted and the percentage of EdU-positive cells was calculated (from three independent experiments) using the following formula: number of EdU positive cells/number of Hoechst stained cells × 100. EdU-positive cells broadly represent cells that are undergoing DNA synthesis, whereas Hoechst stained cells represent viable cells in the same field.
Cell invasion assay Cell invasion was assayed with Biocoat Matrigel 24-well invasion chamber (BD Biosciences, MA, USA). Cells were serum starved for 18 h before detachment with Trypsin-EDTA (Gibco, Auckland, NZ). Further, ORL-48 and ORL-115 were suspended at 1 × 105 cells in serum-free medium containing zerumbone (30 µM) or 0.5% (v/v) DMSO, and added into the upper chambers of the transwell inserts while 1 U/ml thrombin (Sigma-Aldrich, MO, USA) was used as chemoattractant in the lower chambers. After 24 h of incubation at 37 °C, cells in the upper surface of the matrigel membrane (non-invaded cells) were removed with a cotton swab. Membranes were fixed with 4% (v/v) formaldehyde for 15 min and stained with 0.2% (w/v) crystal violet for 10 min, and finally rinsed with water. The membranes were air dried, detached from the inserts and were mounted onto glass slides using DPX mountant (Sigma-Aldrich, MO, USA). Stained cells (invaded cells) were counted in 4 randomly chosen microscopic fields for each insert at 100 × magnification and the average value of 3 technical replicates was obtained. Western blot
Cell cycle assay Zerumbone (3–30 µM) and 0.5% (v/v) DMSO-treated cells were lysed on ice in lysis buffer (5 M NaCl, 10% (v/v) NP-40, 1 M Tris pH 8.0, 0.5 mM DTT) supplemented with HALT Protease and Phosphatase Inhibitor Cocktail (Pierce Biotechnology, IL, USA). Cell lysates were then centrifuged at 14,000 rpm for 10 min at 4 ᵒC prior to estimation of protein content using the BCA method (Thermo Scientific, MA, USA). For western blot analysis, 30 µg of total cellular proteins were resolved on a 12% (w/v) SDS-PAGE gel and electro-transferred onto ImmobilonP membrane (PVDF; Millipore, MA, USA) at 100 V for 1 h on ice. Membranes were blocked with 5% (v/v) skimmed milk in Tris-buffered saline with 0.1% (v/v) Tween 20 (TBST; Sigma Aldrich, MO, USA) for 1 h and then probed overnight at 4 °C with the indicated primary antibodies at 1:1000 dilution in 1% (v/v) BSA in TBST (CXCR4: Abcam, Cambridge, UK; Gα12: Santa Cruz Biotechnology, CA, USA; pAkt (S473), total Akt, pS6, total S6, cleaved caspase 3, caspase 3; Cell Signaling Technology, MA, USA; α-tubulin: Sigma-Aldrich, MO, USA; anti-human PARP: BD Pharmingen, CA, USA). After, membranes were washed 3 times in TBST for 5 min each. Membranes were then incubated with the corresponding secondary HRP-conjugated antibodies (Southern Biotech, AL, USA) at 1: 10,000 dilution in 5% (v/v) skimmed milk in TBST for 1 h at room temperature. This was followed by 3
ORL-48 and ORL-115 were seeded at 7 × 104 cells per well in 12well plates, and treated with 3–30 µM zerumbone or 0.5% (v/v) DMSO. After overnight incubation, all floating and attached cells were harvested and fixed in 70% (v/v) ethanol for 16 h at −20 °C. Prior to analysis, fixed cells were pelleted and washed in cold PBS followed by staining with 10 µg/ml propidium iodide solution containing 20 µg/ml RNAse for 30 min at room temperature in the dark. Stained cells were next analysed by BD FACSCanto II™ flow cytometer (BD Biosciences, MA, USA) with 10,000 events collected for each read. The distribution of DNA in different phases was determined using the ModFit software (Verity Software House, USA). The percentage of cells in each phase was obtained from three independent experiments. FITC-Annexin V assay Detection of apoptosis in ORL cell lines treated with zerumbone was carried out using the Annexin V-FITC / Propidium Iodide (PI) binding assay following the manufacturer's recommendations (BD Biosciences, MA, USA). Briefly, ORL-48 and ORL-115 cells were seeded at 2 × 105 in 60 mm culture plates and exposed to 3–30 µM zerumbone or 0.5% (v/v) 35
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Fig. 2. Zerumbone inhibits cell proliferation and induces apoptosis in OSCC cell lines. (A-B) Zerumbone (24 h) exhibited anti-proliferative effects on ORL-48 and ORL-115 by Click-iT EdU assay. (A) Representative fluorescence images showing reduction of proliferating cells upon 10–30 µM zerumbone treatment. Blue (Hoecsht 33342) represents the total number of cells in any field and red (Alexa 647) represents proliferating cells that have incorporated the EdU label. (B) The percentage of EdU-positive cells was significantly reduced by 30 µM zerumbone as compared to DMSO control groups. (C-E) Zerumbone induced G2/M cell cycle arrest in ORL-48 and ORL-115 cells by propidium iodide analysis. (C) Flow histograms showing population of cells in each cell cycle phase (sub-G1, G0/1, S and G2/M) after 24 h of treatment. The percentage of cells in the G2/M phase was recorded as in (D) for 24 h and (E) for 72 h treatment. (F-G) Zerumbone-treated cells showed higher percentage of apoptotic cells (FITC-Annexin V-positive cells) as compared to the DMSO controls. (F) Representative flow histograms depicting early (Q4) and late (Q2) apoptotic fractions of ORL-48 and ORL-115 cells upon 72 h of zerumbone treatment. (G) Percentage of apoptotic cells (early + late apoptosis) in ORL-48 and ORL-115. (H) Zerumbone (72 h) increased the levels of apoptotic markers on ORL-48 and ORL-115 as shown by the western blot analysis of cleaved caspase 3 and cleaved PARP proteins. All quantification results shown are mean ± SD (n = 3). Significant differences from DMSO control groups were calculated by two-tailed student's T-test. * and ** denotes p < .05 and p < .01 respectively. Western blot results shown are representative of at least two independent experiments.
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investigations, we chose ORL-48 and ORL-115 to represent OSCC cells that showed moderate and high sensitivity to zerumbone.
washes in TBST prior to detection by WesternBright Quantum HRP substrate (Advansta Inc, CA, USA) and visualization using the FluorChemTM HD2 imaging systems (ProteinSimple, CA, USA). The visualized protein bands were quantified using ImageJ software (National Institute of Health, Bethesda, MD, USA).
Zerumbone inhibits OSCC via cell cycle arrest and apoptosis As zerumbone was observed to reduce viability of OSCC cells, we then investigated its effect on cell proliferation by measuring DNA synthesis using Click-iT EdU assay. As shown in the representative images in Fig. 2A-B, inhibition of cell proliferation was seen in a dose dependent manner. While low levels of zerumbone (3 µM) conferred a minimal response in ORL-115 (Fig. 2B), further reduction in cell proliferation by ∼10% and 30% was observed in ORL-48 (p = .11) and ORL-115 (p = .04) respectively, following zerumbone treatment at 10 µM. Upon treatment at 30 µM, zerumbone caused reduction of cell proliferation by ∼60% for both lines (p = .01 for both). We next investigated whether the observed effect was due to perturbation in the cell cycle using propidium iodide (PI) staining. As seen in Fig. 2C, both lines treated with zerumbone accumulated at the G2/M phase in a dosedependent manner with a corresponding reduction of cell population in the G0/1 phase. Quantification of the DNA content in the different phases of the cell cycle showed a significant increase in the percentage of cells in G2/M phase at 10 µM and 30 µM compared to the respective DMSO controls in ORL-48 and ORL-115 (p < .05; Fig. 2D). We further tested whether this cytostatic effect of zerumbone was more pronounced and persistent when the experimental treatment time was prolonged to 72 h. Consistently, we saw a significant increase of the percentage of cells in the G2/M phase in cells treated with 10 and 30 µM of zerumbone (p < .05; Fig. 2E). Of interest, we noticed a substantial increase of cell population in the sub-G1 phase, when both lines were treated with 10–30 µM of zerumbone, suggesting the occurrence of cell death at longer treatment period (Supplementary Fig. S2). Correspondingly, as zerumbone has also been reported to induce apoptosis in non-small cell lung cancer (Takada et al., 2005) and liver cancer cells (Sakinah et al., 2007), we explored whether zerumbone induces apoptosis that could account for this sub-G1 population. As shown by the representative flow histograms in Fig. 2F, 72 h of zerumbone exposure (10 and 30 µM) resulted in a marked increase of cells in early (FITC-Annexin V-positive and PI-negative; Q4) and late apoptosis (FITC-Annexin V-positive and PI-positive; Q2) when compared to DMSO controls. Induction of apoptosis by zerumbone was more pronounced in ORL-115 cells with increasing concentrations, where 10 µM was sufficient to increase the percentage of apoptotic cells significantly (p = .04), while 30 µM gave a more robust effect when compared to ORL-48 cells (Fig. 2G). Nonetheless, both lines exhibited a significant increase in the number of apoptotic cells when exposed to 30 µM zerumbone for 72 h (27.1% and 33.9% respectively) when compared with DMSO control (8.1% and 10% respectively; p = .005 and p = .004). To confirm the induction of apoptotic activity by zerumbone, western blot analysis was performed to assess the levels of cleaved caspase 3 and cleaved PARP in zerumbone-treated cells. Caspase 3 and PARP are involved in DNA repair activity and their cleaved forms serve as a marker for cells undergoing apoptosis. We observed increases of cleaved caspase 3 and cleaved PARP levels especially when cells were treated with 30 µM zerumbone, with corresponding decreases of the full-length proteins (Fig. 2H). From the experiments conducted, we verified zerumbone to reduce OSCC cell viability by inhibiting cell growth and further cause apoptosis at a longer exposure.
RhoA pull-down assay RhoA activity in ORL-48 and 115 was investigated using a pulldown purification method as previously described (Chikumi et al., 2002; Gan et al., 2014). Briefly, 2 × 105 ORL-48 and ORL-115 cells that were seeded in 60 mm culture plates were treated with 3–30 µM zerumbone or 0.5% (v/v) DMSO and serum starved for 24 h. Prior to protein extraction, cells were replenished with fresh serum-free medium and stimulated with 1 U/ml thrombin for three min as optimized from our previous study (Gan et al., 2014). Following thrombin stimulation, cells were washed with PBS and lysed on ice with cold lysis buffer (as detailed above). Collected lysates were centrifuged at 14,000 rpm for 5 min at 4 °C. The resulting supernatant were immediately incubated with glutathione-sepharose 4B beads (GE Healthcare, Sweden) bound with glutathione-S-transferase (GST)-rhotekinRhoA binding domain for 30 min at 4 °C on a rotator, while the remaining lysates were used to detect total RhoA protein. After incubation, the coated beads were collected by centrifugation at 9000 rpm for 1 min at 4 °C and washed 3 times with cold lysis buffer. The associated GTP-bound RhoA was released with protein loading buffer, and heatinactivated prior to SDS-PAGE and western blotting. Blots were incubated using anti-RhoA (sc-418; Santa Cruz Biotechnology, CA, USA) at 1:1000 dilution for 16 h and further processed according to western blotting conditions as mentioned above. Statistical analyses All data are expressed as mean ± SD from three independent experiments. The significance of differences between groups in each experiment was analysed by Student's t-tests using GraphPad Prism software (GraphPad Software Inc., CA, USA). Differences were considered statistically significant at p < .05. Results Zerumbone reduces OSCC cell viability As previous studies have demonstrated the cytotoxic effect of zerumbone against different human cancer cells, we aimed to determine if a representative panel of OSCC cell lines were sensitive to this compound. To achieve this, we treated a panel of 16 OSCC lines with zerumbone at concentrations ranging from 0 to 100 µM. Two normal oral keratinocytes (NOK) primary cultures (ORL-232 and ORL-235) were also included in the treatment as comparison. The MTT results revealed that the half maximal inhibitory concentration (IC50) of zerumbone ranged from 0.8 to 4.9 µM with an average value of ∼2 µM across this panel of OSCC lines (Fig. 1B). By contrast, the NOK cultures showed significantly higher average IC50 of 25 µM. This shows that zerumbone has more potent effects towards OSCC and was less toxic on normal cells with about eight-fold difference in IC50 (p = .009; supplementary Fig. S1). We also demonstrated that breast (MCF-7) and colorectal (HCT-116) cancer cells were sensitive to zerumbone with IC50 of 8.11 and 1.39 µM respectively (Fig. 1B). To evaluate zerumbone's efficacy against standard anti-cancer drugs, we compared zerumbone with cisplatin (chemotherapy drug) and gefitinib (small molecule inhibitor targeting EGFR). The average IC50 of cisplatin and gefitinib in the same panel of OSCC lines were 8.7 µM (range: 2.7 to 14.5 µM) and 14.49 µM (0.1 to 36.9 µM) respectively, implying that zerumbone could be more potent with lower IC50 concentrations (average 2 µM; range: 0.8 – 4.9 µM) compared to cisplatin and gefitinib (Fig. 1C). For subsequent
Zerumbone reduces cell migration and invasion of OSCC cells We also evaluated the influence of zerumbone on the migration of OSCC cells by performing wound healing assay. Zerumbone, when given at a concentration of 30 µM, inhibited cell migration, where the open wound area of zerumbone-treated ORL-48 and ORL-115 cells were larger as compared to DMSO-treated cells following a 24-h observation (p = .02 and p = .03 respectively; Fig. 3A). To validate that 30 µM of 37
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Fig. 3. Zerumbone suppresses cell migration and invasion in vitro, reduces CXCR4 expression and inactivates RhoA signaling. (A) Migration of ORL-48 and ORL-115 cells was significantly inhibited as indicated by the representative images and the percentage of open wound area after treatment with 30 µM of zerumbone for 24 h. (B) Zerumbone treatment of 30 µM suppressed ORL-48 and ORL-115 cells from invading through the matrigel membrane of transwell inserts as indicated by the representative images and the percentage of cell invasion. Thrombin was used as chemoattractant. (C) Expression of CXCR4 protein was inhibited by 30 µM of zerumbone when cells were treated for 24 h with increasing concentrations. (D) 30 µM of zerumbone caused reduction in CXCR4 protein expression in a time-dependent manner. (E) RhoA inactivation was observed in ORL-48 and ORL-115 after zerumbone treatment at 30 µM for 24 h. Following zerumbone treatment, cells were stimulated by 1 U/ml of thrombin for three min to activate the pathway and whole cell extracts were subjected to RhoA pulldown purification assay and western blot analysis. All quantification results shown are mean ± SD (n = 3). Significant differences from DMSO control groups were calculated by twotailed student's T-test. * denotes p < .05. Western blot results shown are representative of at least two independent experiments. Densitometric quantifications for C-E blots are provided in Supplementary Fig. S4.
Zerumbone reduced CXCR4 protein expression
zerumbone was indeed anti-migratory without inducing cytotoxicity in the two cell lines within the 24-h treatment period, we focused on the cell population specifically in the sub-G1 phase of the cell cycle. After 24 h of 3–30 µM zerumbone treatment, no significant increase in the number of cells in the sub-G1 phase was observed (Supplementary Fig. S3). This indicated that the observed anti-migratory activity of zerumbone was not attributed to the induction of cell death. Following cell migration assay, we further evaluated zerumbone's ability in preventing cell invasion in ORL-48 and ORL-115 cells by transwell invasion assay. We demonstrated that 30 µM zerumbone caused a significant decrease in the percentage of cells invading through the matrigel barrier in response to thrombin as a chemoattractant, compared to the DMSO control in both lines (p = .04 for both; Fig. 3B).
To investigate the potential molecular target of zerumbone that could implicate cell motility, we looked into the expression of CXCR4 which has been known to be involved in the modulation of cancer cell motility, survival and proliferation (Teicher and Fricker, 2010). Our western blot analysis revealed that 30 µM of zerumbone consistently resulted in a reduction of CXCR4 protein levels in both ORL-48 and ORL-115 lines, while lower concentrations had a minimal effect (Fig. 3C). Using 30 µM concentration of zerumbone for subsequent studies, we further investigated the reduction of CXCR4 protein levels through a time-dependent examination by western blot. From our observations (Fig. 3D), reduction of CXCR4 protein levels in both ORL-48 and ORL-115 cells can be observed as early as 8 h and is most pronounce at 24 h of zerumbone treatment. 38
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carcinoma (HNSCC) dissemination. Blockade of CXCR4 signaling axis has been demonstrated to inhibit lymph node metastases in an orthotopic tongue model of OSCC through the inhibition of IL-6 and IL-8 production (Uchida et al., 2011). In another study, EMT biomarkers such as vimentin and integrin β1 were shown to be highly expressed in the metastatic clones of HNSCC and the use of CXCR4 antagonist essentially blocked both primary tumor growth and metastases in the subcutaneous xenograft model (Yoon et al., 2007). Additionally, tumors that have high expression of CXCR4 in OSCC patients correlated with poor overall survival (Albert et al., 2012). Our finding is consistent with Sung et al. (2008), whereby CXCR4 protein expression was downregulated by zerumbone leading to the inhibition of CXCL12-induced invasion of breast and pancreatic cells. However, the effect of the downstream protein activity leading to the inhibition of cell invasion was not ascertained. In this current study, we chose to investigate if the loss of endogenous CXCR4 protein expression was likely to impact RhoA activity as previous reports have demonstrated that CXCR4 activates the Gα12/13 signaling axis and further regulate the migration and invasion of mammalian cells through RhoA (Tan et al., 2006). Our results indicated that RhoA activity was inhibited by zerumbone. This suggests a therapeutic opportunity for OSCC treatment as we have previously reported the over-expression of Gα12 and prevention of metastasis by inhibition of RhoA activation in cell line and orthotopic mouse model (Gan et al., 2014). The aberrancy of RhoA in head and neck cancers has also been described by Abraham et al., in which a subset of motility-related proteins including RhoA were found to be overexpressed in cell lines and patient tissue samples relative to normal specimens (Abraham et al., 2001). For this significance, RhoA was used as one of the molecular markers to characterize potentially malignant tumors for HNSCC. We show that the inhibition of CXCR4-RhoA pathway associates with the inactivation of the PI3K-mTOR pathway by reducing the phosphorylation of Akt and S6 protein kinases. This is a favourable effect as the PI3K-mTOR pathway is known to be widely activated in HNSCC, rendering persistent mitogenic signaling. In particular, 20% of head and neck cancer samples have Akt upregulated while in 80–90% of samples, phosphorylated S6 was present (Iglesias-Bartolome et al., 2013). CXCR4 and Akt have multifaceted roles in tumor development, and both have been implicated in proliferation, cell migration and invasion (Hong et al., 2009) making it difficult to individually determined which phenotypes observed upon zerumbone treatment was due to the inhibition of which individual pathway. Furthermore, the interplay between CXCR4 and Akt signaling has been reported previously, where CXCR4 signaling can result in the activation of Akt (Katayama et al., 2005). In summary, zerumbone is potent at concentrations that are lower than the commonly used cancer therapies tested here, and is selective to OSCC cells. Its ability to inhibit cell proliferation, migration and invasion through well-established pathways that have been implicated in OSCC presents an opportunity for it to be a potential candidate for OSCC treatment.
Zerumbone inactivates RhoA, a downstream target of CXCR4 As previous work had identified RhoA as one of the downstream targets for CXCR4 to promote cancer invasiveness and metastases (Guo et al., 2016), we next investigated if reduced CXCR4 protein levels due to zerumbone resulted in an inhibition of RhoA activity. The activity of RhoA was evaluated using protein pull-down assay by measurement of the active GTP bound RhoA protein. Upon treatment with 30 µM of zerumbone for 24 h, levels of active RhoA decreased in both thrombin-stimulated ORL-48 and ORL-115 cells with no changes in total RhoA expression (Fig. 3E), demonstrating that zerumbone was able to inhibit RhoA activity in these cells. Taken together, zerumbone was able to reduce CXCR4 protein levels and RhoA activation resulting in the inhibition of cell motility in the two OSCC cell lines. Zerumbone downregulates the Akt-mTOR signaling pathway Previous findings have shown that the CXCR4-mediated chemotaxis could occur through PI3K activation (Ward, 2006). In OSCC, preclinical investigation on mouse model revealed the CXCR4/CXCL12 signaling mediates lymph node metastasis through the Akt pathway (Uchida et al., 2004). Here, we further investigated whether inhibition of the CXCR4-RhoA signaling would impact the PI3K-mTOR pathway by firstly looking into whether zerumbone could inactivate EGFR, a tyrosine kinase receptor that is involved in the regulation of PI3KmTOR signaling. As both ORL-48 and ORL-115 do not have constitutive activation of EGFR, the effects of zerumbone were not observed (Fig. 4A & B). As a positive control for data set, we used protein extract of ORL136 cells, which is known to have high levels of phosphorylated EGFR (Fadlullah et al., 2016). While we were not able to see the effect of zerumbone on EGFR activity, we demonstrated that zerumbone could inhibit Akt phosphorylation at 30 µM in both ORL-48 and ORL-115. This was also observed in a time-dependent manner where inactivation of Akt (Ser473) in ORL-48 and ORL-115 were observed at 16 and 8 h post-treatment respectively (Fig. 4B). The inhibition of Akt resulted in the inactivation of S6 (Fig. 4A & B). Of note, significant inactivation of S6 was observed as early as 8 h of treatment in both ORL-48 and 115 (Fig. 4B). Taken together, our data demonstrates that zerumbone impacts multiple proteins of the CXCR4-RhoA and Akt-mTOR pathways, consistent with the observation of inhibition of cell proliferation, migration and invasion of OSCC cells. Discussions Zerumbone, a natural dietary agent is now known to have antitumor properties in different types of human cancer cells (Prasannan et al., 2012). Herein, we demonstrate the bioactivity of zerumbone against OSCC cells with favourable IC50 values (∼2 µM). This sensitivity was comparable to those reported for other cancers for example, colorectal (HT-29, CaCo-2) and breast (MCF-7) with values of ∼10 µM (Kirana et al., 2003), while for cervical (HeLa) and ovarian cancer cells (Caov-3), values were 11.3 µM (Abdul et al., 2008) and 24 µM (Abdelwahab et al., 2012), respectively. Furthermore, we showed zerumbone to have lower average IC50 values against cisplatin and gefitinib, implying a favourable response against standard cancer treatments. Our results demonstrate that zerumbone treatment resulted in the reduction of cell proliferation with a concurrent arrest of cells at the G2/M phase, followed by apoptosis activity at longer exposures. These observations were consistent with a previously reported study showing zerumbone to trigger apoptotic activity following G2/M cell cycle arrest in NB4 leukemia cells (Xian et al., 2007). In our current study, we also noted that zerumbone displayed anti-migration and anti-invasion properties in OSCC cells along with concurrent downregulation of CXCR4 protein expression. Cumulative evidence has suggested that CXCR4 acts as marker of tumor aggressiveness and is involved in head and neck squamous cell
Conflict of interest The authors have no conflict of interests to declare.
Acknowledgments This work was supported by High Impact Research, Ministry of Higher Education (HIR-MOHE) from University of Malaya (UM.C/625/ 1/HIR/MOHE/DENT-03) and other sponsors of Cancer Research Malaysia. Cancer Research Malaysia is a non-profit research organization. We are committed to an understanding of cancer prevention, diagnosis and treatment through a fundamental research program. 39
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Fig. 4. Zerumbone inactivates Akt-mTOR signaling. (A) Dose-dependent analysis of zerumbone showed inactivation of Akt and S6 in OSCC cells at 30 µM concentration. Cells were treated with the indicated concentrations for 24 h. (B) Time-course analysis of 30 µM zerumbone on ORL-48 and 115 cells showed inactivation of Akt and S6 at 8 h post treatment. Western blot results shown are representative of at least two independent experiments. Densitometric quantifications for A-B blots are provided in Supplementary Fig. S5.
Supplementary materials
Albert, S., Hourseau, M., Halimi, C., et al., 2012. Prognostic value of the chemokine receptor CXCR4 and epithelial-to-mesenchymal transition in patients with squamous cell carcinoma of the mobile tongue. Oral Oncol. 48, 1263–1271. Chakraborty, A., Coffman, R., Jorvig, J., 2013. Zerumbone, a phytochemical from Asian ginger is a novel inhibitor of Jak2/Stat3 inhibits promigratory gene expression, growth and migration of pancreatic cancer cells. Pancreatology 13, e18–e19. Cheong, S., Chandramouli, G., Saleh, A., et al., 2009. Gene expression in human oral squamous cell carcinoma is influenced by risk factor exposure. Oral Oncol. 45, 712–719. Chikumi, H., Vázquez-Prado, J., Servitja, J.-M., et al., 2002. Potent activation of RhoA by Gαq and Gq-coupled receptors. J. Biol. Chem. 277, 27130–27134. Chong, C.E., Lim, K.P., Gan, C.P., et al., 2012. Over-expression of MAGED4B increases cell migration and growth in oral squamous cell carcinoma and is associated with poor disease outcome. Cancer Lett. 321, 18–26. Fadlullah, M., Chiang, I., Dionne, K.R., et al., 2016. Genetically-defined novel oral squamous cell carcinoma cell lines for the development of molecular therapies. Oncotarget 7, 27802. Gan, C.P., Patel, V., Mikelis, C.M., et al., 2014. Heterotrimeric G-protein alpha-12 (G'12) subunit promotes oral cancer metastasis. Oncotarget 5, 9626–9640. Guo, J., Yu, X., Gu, J., et al., 2016. Regulation of CXCR4/AKT-signaling-induced cell invasion and tumor metastasis by RhoA, Rac-1, and Cdc42 in human esophageal
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2017.12.011. References Abdelwahab, S.I., Abdul, A.B., Mohan, S., et al., 2011. Zerumbone induces apoptosis in Tacute lymphoblastic leukemia cells. Leukemia Res. 35, 268–271. Abdelwahab, S.I., Abdul, A.B., Zain, Z.N.M., et al., 2012. Zerumbone inhibits interleukin6 and induces apoptosis and cell cycle arrest in ovarian and cervical cancer cells. Int. Immunopharmacol. 12, 594–602. Abdul, A.B., Al-Zubairi, A.S., Tailan, N.D., et al., 2008. Anticancer activity of natural compound (Zerumbone) extracted from Zingiber zerumbet in human HeLa cervical cancer cells. Int. J. Pharmacol. 4, 160–168. Abraham, M.T., Kuriakose, M.A., Sacks, P.G., et al., 2001. Motility‐Related Proteins as Markers for Head and Neck Squamous Cell Cancer. Laryngoscope 111, 1285–1289. Aggarwal, B.B., Shishodia, S., 2006. Molecular targets of dietary agents for prevention and therapy of cancer. Biochem. Pharmacol. 71, 1397–1421.
40
Phytomedicine 39 (2018) 33–41
N.S. Zainal et al.
mediated apoptosis in human breast cancer cells and inhibits orthotopic xenograft growth in vivo. Breast Cancer Res. Treatment 136, 429–441. Shanmugam, M.K., Rajendran, P., Li, F., et al., 2015. Abrogation of STAT3 signaling cascade by zerumbone inhibits proliferation and induces apoptosis in renal cell carcinoma xenograft mouse model. Mol. Carcinog. 54, 971–985. Specenier, P., Vermorken, J.B., 2012. Biologic therapy in head and neck cancer: a road with hurdles. ISRN Oncol. 2012. Sung, B., Jhurani, S., Ahn, K.S., et al., 2008. Zerumbone down-regulates chemokine receptor CXCR4 expression leading to inhibition of CXCL12-induced invasion of breast and pancreatic tumor cells. Cancer Res. 68, 8938–8944. Takada, Y., Murakami, A., Aggarwal, B.B., 2005. Zerumbone abolishes NF-κB and IκBα kinase activation leading to suppression of antiapoptotic and metastatic gene expression, upregulation of apoptosis, and downregulation of invasion. Oncogene 24, 6957–6969. Tan, W., Martin, D., Gutkind, J.S., 2006. The Gα13-Rho signaling axis is required for SDF1-induced migration through CXCR4. J. Biol. Chem. 281, 39542–39549. Teicher, B.A., Fricker, S.P., 2010. CXCL12 (SDF-1)/CXCR4 pathway in cancer. Clinical Cancer Res. 16, 2927–2931. Torre, L.A., Bray, F., Siegel, R.L., et al., 2015. Global cancer statistics, 2012. CA Cancer J Clin 65, 87–108. Uchida, D., Begum, N.-M., Tomizuka, Y., et al., 2004. Acquisition of lymph node, but not distant metastatic potentials, by the overexpression of CXCR4 in human oral squamous cell carcinoma. Lab. Invest. 84, 1538–1546. Uchida, D., Onoue, T., Kuribayashi, N., et al., 2011. Blockade of CXCR4 in oral squamous cell carcinoma inhibits lymph node metastases. Eur. J. Cancer 47, 452–459. Vermorken, J.B., Mesia, R., Rivera, F., et al., 2008. Platinum-based chemotherapy plus cetuximab in head and neck cancer. New Eng. J. Med. 359, 1116–1127. Ward, S.G., 2006. T lymphocytes on the move: chemokines, PI 3-kinase and beyond. Trends Immunol. 27, 80–87. Xian, M., Ito, K., Nakazato, T., et al., 2007. Zerumbone, a bioactive sesquiterpene, induces G2/M cell cycle arrest and apoptosis in leukemia cells via a Fas‐and mitochondria‐mediated pathway. Cancer Sci. 98, 118–126. Yoon, Y., Liang, Z., Zhang, X., et al., 2007. CXC chemokine receptor-4 antagonist blocks both growth of primary tumor and metastasis of head and neck cancer in xenograft mouse models. Cancer Res. 67, 7518–7524. Yu, Z., Weinberger, P.M., Sasaki, C., et al., 2007. Phosphorylation of Akt (Ser473) Predicts Poor Clinical Outcome in Oropharyngeal Squamous Cell Cancer. Cancer Epidemiol. Biomarkers Preven. 16, 553–558. Zlotogorski, A., Dayan, A., Dayan, D., et al., 2013. Nutraceuticals as new treatment approaches for oral cancer: II. Green tea extracts and resveratrol. Oral Oncol. 49, 502–506.
cancer. Tumor Biol. 37, 6371–6378. Gupta, S.C., Kim, J.H., Prasad, S., et al., 2010. Regulation of survival, proliferation, invasion, angiogenesis, and metastasis of tumor cells through modulation of inflammatory pathways by nutraceuticals. Cancer Metastasis Rev. 29, 405–434. Hamid, S., Lim, K.P., Zain, R.B., et al., 2007. Establishment and characterization of Asian oral cancer cell lines as in vitro models to study a disease prevalent in Asia. Int. J.Mol. Med. 19, 453–460. Hong, J.S., Pai, H.K., Hong, K.O., et al., 2009. CXCR‐4 knockdown by small interfering RNA inhibits cell proliferation and invasion of oral squamous cell carcinoma cells. J. Oral Pathol. Med. 38, 214–219. Iglesias-Bartolome, R., Martin, D., Gutkind, J.S., 2013. Exploiting the head and neck cancer oncogenome: widespread PI3K-mTOR pathway alterations and novel molecular targets. Cancer Discovery 3, 722–725. Kang, C.G., Lee, H.-J., Kim, S.-H., et al., 2015. Zerumbone Suppresses OsteopontinInduced Cell Invasion Through Inhibiting the FAK/AKT/ROCK Pathway in Human Non-Small Cell Lung Cancer A549 Cells. J. Natural Products. Katayama, A., Ogino, T., Bandoh, N., et al., 2005. Expression of CXCR4 and its downregulation by IFN-γ in head and neck squamous cell carcinoma. Clinical Cancer Res. 11, 2937–2946. Kim, M., Miyamoto, S., Yasui, Y., et al., 2009. Zerumbone, a tropical ginger sesquiterpene, inhibits colon and lung carcinogenesis in mice. Int. J. Cancer 124, 264–271. Kirana, C., McIntosh, G.H., Record, I.R., et al., 2003. Antitumor activity of extract of Zingiber aromaticum and its bioactive sesquiterpenoid zerumbone. Nutrition Cancer 45, 218–225. Lui, V.W., Hedberg, M.L., Li, H., et al., 2013. Frequent mutation of the PI3K pathway in head and neck cancer defines predictive biomarkers. Cancer Discovery 3, 761–769. Massano, J., Regateiro, F.S., Januário, G., et al., 2006. Oral squamous cell carcinoma: review of prognostic and predictive factors. Oral Surgery Oral Med. Oral Pathol. Oral Radiol. Endodontol. 102, 67–76. Newman, D.J., Cragg, G.M., 2012. Natural products as sources of new drugs over the 30 years from 1981 to 2010. J. Natural Prod. 75, 311–335. Pau, G., Fuchs, F., Sklyar, O., et al., 2010. EBImage—an R package for image processing with applications to cellular phenotypes. Bioinformatics 26, 979–981. Pickering, C.R., Zhang, J., Yoo, S.Y., et al., 2013. Integrative genomic characterization of oral squamous cell carcinoma identifies frequent somatic drivers. Cancer Discovery 3, 770–781. Prasannan, R., Kalesh, K.A., Shanmugam, M.K., et al., 2012. Key cell signaling pathways modulated by zerumbone: role in the prevention and treatment of cancer. Biochem. Pharmacol. 84, 1268–1276. Sakinah, S.S., Handayani, S.T., Hawariah, L.A., 2007. Zerumbone induced apoptosis in liver cancer cells via modulation of Bax/Bcl-2 ratio. Cancer Cell Int. 7, 1. Sehrawat, A., Arlotti, J.A., Murakami, A., et al., 2012. Zerumbone causes Bax-and Bak-
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