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1’-Acetoxychavicol acetate inhibits growth of human oral carcinoma xenograft in mice and potentiates cisplatin effect via proinflammatory microenvironment alterations
© In et al.; licensee BioMed Central Ltd. 2012
Received: 12 April 2012
Accepted: 4 October 2012
Published: 9 October 2012
Oral cancers although preventable, possess a low five-year survival rate which has remained unchanged over the past three decades. In an attempt to find a more safe, affordable and effective treatment option, we describe here the use of 1’S-1’-acetoxychavicol acetate (ACA), a component of Malaysian ginger traditionally used for various medicinal purposes.
Whether ACA can inhibit the growth of oral squamous cell carcinoma (SCC) cells alone or in combination with cisplatin (CDDP), was explored both in vitro using MTT assays and in vivo using Nu/Nu mice. Occurrence of apoptosis was assessed using PARP and DNA fragmentation assays, while the mode of action were elucidated through global expression profiling followed by Western blotting and IHC assays.
We found that ACA alone inhibited the growth of oral SCC cells, induced apoptosis and suppressed its migration rate, while minimally affecting HMEC normal cells. ACA further enhanced the cytotoxic effects of CDDP in a synergistic manner as suggested by combination index studies. We also found that ACA inhibited the constitutive activation of NF-κB through suppression of IKKα/β activation. Human oral tumor xenografts studies in mice revealed that ACA alone was as effective as CDDP in reducing tumor volume, and further potentiated CDDP effects when used in combination with minimal body weight loss. The effects of ACA also correlated with a down-regulation of NF-κB regulated gene (FasL and Bim), including proinflammatory (NF-κB and COX-2) and proliferative (cyclin D1) biomarkers in tumor tissue.
Overall, our results suggest that ACA inhibits the growth of oral SCC and further potentiates the effect of standard CDDP treatment by modulation of proinflammatory microenvironment. The current preclinical data could form the basis for further clinical trials to improve the current standards for oral cancer care using this active component from the Malaysian ginger.
Oral cancers are malignancies arising from either tongue, lip, gingivae, palate, salivary glands, buccal mucosa or floor of the mouth, and accounts for an estimated 2.08% (263,900) of total cancer cases worldwide in 2011. About 90% of oral cancers are squamous carcinomas, with main treatment options that include surgery followed by radiotherapy and adjuvant chemotherapy. Even though oral cancers are relatively preventable, diagnosed patients often face a low five-year survival rate of 58%, which has remained unchanged over the past three decades despite recent treatment advances. Presently, platinum-based drugs such as cisplatin (CDDP), remains one of the most commonly used chemotherapeutic agents available for the treatment of advanced oral cancers. While CDDP treatment often results in initial responses and disease stabilization, its long-term success is hindered by the development of drug resistance and dose-limiting toxicities through the occurrence of DNA cross-linking in surrounding non-cancerous cells. Thus, there is an ongoing need for modified CDDP combination regimes that can ideally reduce overall dose-toxicity through chemo-sensitization of oral cancer cells.
Implications of inhibiting the nuclear factor kappa-B (NF-κB) pathway to sensitize cancer cells have been previously reported on various cell types including prostate epithelial cells and bladder cells[8–10]. NF-κB is a transcription factor which is constitutively present in the cytoplasm as an inactive heterotrimer consisting of p50, p52, p65 (RelA) and IκBα subunits. Upon activation by various cytokines and chemokines, IκBα undergoes phosphorylation and subsequent ubiquitination-dependant degradation, allowing NF-κB heterodimers to freely translocate and retain within the nucleus to promote transcription[11, 12]. Overexpression of κB-regulated genes has been linked with most cancers, and can mediate events such as cellular transformation, proliferation, invasion, angigogenesis and metastasis. Agents that suppress NF-κB activation are typically sought for as chemo-sensitizers since it regulates an array of genes governing the sensitivity of cells towards drugs such as glutathione S-transferase (GST), which is an enzyme involved in metal metabolism, whereby its overexpression has been linked to the resistance of cis-platinum drugs in SCCs.
The use of 1’S-1’-acetoxychavicol acetate (ACA), which is a phenylpropanoid naturally found within various Zingiberaceae family members, has been traditionally associated with a number of various medicinal properties including anti-ulceration, anti-allergic, anti-inflammatory and anti-cancer activities[17, 18]. Previous studies have shown ACA to be associated with the production of intracellular reactive oxygen species (ROS), inhibition of xanthane oxidase (XO) activity, inhibition of nitric oxide synthase (NOS) expression, inhibition of polyamine synthesis, induction of apoptosis via mitochondrial/Fas-mediated dual mechanism and as a potential NF-κB inhibitor[23–25]. Even though the structure-activity relationship of ACA has been thoroughly studied, its intracellular molecular effects on downstream protein candidates involved in sensitization remain unidentified.
In this study, we investigated the role of ACA as a chemo-sensitizer on oral SCC cells and its combined effects with CDDP in vivo to produce an improved chemotherapeutic regime with increased efficacies at lower concentrations, which could hypothetically reduce the occurrence of dose-limiting toxicities.
Rhizomes of Alpinia conchigera Griff were collected from Jeli province of Kelantan, East-coast of Peninsular Malaysia. The sample was identified by Prof. Dr. Halijah Ibrahim from the Institute of Biological Science, Division of Ecology and Biodiversity, Faculty of Science, University of Malaya. A voucher specimen (KL5049) was deposited in the Herbarium of Chemistry Department, Faculty of Science, University of Malaya.
NE-PER protein extraction kit and SuperSignal West Pico chemiluminescent substrate were purchased from Pierce (IL, USA). Suicide Track™ DNA ladder isolation kit, MTT reagent, propidium iodide (PI), mitomycin-C, Suicide TrackTM DNA ladder isolation kit and CDDP were obtained from EMD Chemicals Inc. (CA, USA). Primary NF-κB antibodies p65, IκB-α, IKK-α, IKK-β, histone H3 and β-actin were obtained from Santa Cruz Biotechnology (CA, USA). Antibodies against FasL, Bim, xIAP, poly-(ADP-ribose) polymerase (PARP), SignalStain® Boost IHC detection reagents and IHC antibodies against NF-κB p65, IκBα, phospho-IKKα/β, COX-2, and cyclin D1 were obtained from Cell Signalling (MA, USA). RNeasy® Plus Mini Kit was purchased from Qiagen (Germany), while LIVE/DEAD® Viability/Cytotoxicity kit for mammalian cells was purchased from Molecular Probes, Invitrogen (NY, USA).
Cell lines and culture conditions
Human oral squamous carcinoma cells (HSC-4) were obtained from Dr. Eswary Thirthagiri of the Cancer Research Initiative Foundation (CARIF, Malaysia), while human mammary epithelial cells (HMEC) (Lonza Inc., USA) were used as a normal cell controls. All cells were cultured as monolayers in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10.0% (v/v) FBS, 100 U/ml penicillin and 100.0 μg/ml streptomycin, while HMEC cells were cultured in Mammary Epithelial Growth Medium (MEGM). All cultures were maintained in humidified incubator at 37°C in 5.0% CO2 and 95.0% air.
MTT cell viability assay
The cytotoxic effect of ACA on HSC-4 and HMEC cells was determined by measuring MTT dye uptake and metabolism. ACA was dissolved in dimethyl sufoxide (DMSO) to a final concentration of 10.0 mM. Briefly, 2.0 x 104 cells were treated in triplicates on 96-well plates in the presence or absence of ACA and/or in combination with CDDP at final concentrations of 5.0 μM to 80.0 μM up to 36 h. Final DMSO concentration in each experiment was maintained below 0.5% (v/v) to prevent solvent induced cytotoxicty. 20.0 μl of MTT dye reagent (5.0 mg/ml) was added to each well and cells were incubated in the dark at 37°C. After 2 h of incubation, media containing excess dye was aspirated and 200.0 μl of DMSO was added to dissolve purple formazon precipitates. A microtiter plate reader (Tecan Sunrise®, Switzerland) was used to detect absorbance at a test wavelength of 570 nm, with a reference wavelength of 650 nm.
Live and dead assay
Assessment of cell viability upon treatment with ACA was accomplished using the LIVE/DEAD® Viability/Cytotoxicity kit for mammalian cells according to manufacturer’s protocol. Cancer and normal cell lines were grown as monolayers on cover slips for 24 h and treated with ACA (15.0 μM) for 3 h and 6 h. Staining of cells were done using a dual fluorescence staining system consisting of 150.0 μl of both calcein-AM (2.0 μM) which emits green fluorescence when cleaved by intracellular esterases, and ethidium homodimer (EthD) (4.0 μM) which emits red fluorescence upon binding to nucleic acid in non-viable cells. Excitation and emission wavelengths of both fluoresceins were set at 494/517 nm for calcein-AM and 528/617 nm for EthD respectively. Visualization of samples was done using a Nikon Eclipse TS-100 fluorescence microscope (Nikon, Japan) under 100x magnification with dual pass filters for simultaneous viewing of both stains.
The anti-migration effects of ACA were determined using the wound healing assay. HSC-4 cells were seeded in 6-well plates and allowed to form monolayers overnight. Growth medium was then changed to serum-free medium containing mitomycin-C and further incubated in 37°C for 2 h to halt proliferation of cells. Scratch wounds of equal size were introduced into the monolayer by a sterile pipette tip and cell debris generated from the scratch was washed away with 1x phosphate-buffered saline (PBS). Cells were treated with vehicle or IC20 ACA (10.0 μM) in serum-free medium for 24 h and microscopic images describing speed of wound closure was documented at various time intervals using an inverted fluorescence microscope, Nikon Eclipse TS-100 and analyzed using TScratch software, Version 1.0 (MathWorks Inc.).
PARP cleavage assay
The occurrence of apoptosis was assessed based on the proteolytic cleavage of PARP by caspase 3. Briefly, cells (2.0 x 106/mL) were treated with ACA (15.0 μM) and total proteins were extracted using the NE-PER® nuclear and cytoplasmic extraction kit according to manufacturer’s protocol. Fractionation was done using SDS-PAGE and electro-transferred onto nitrocellulose membranes. Total proteins were incubated with rabbit anti-PARP antibodies and detected using an enhanced chemiluminescence reagent using x-ray films. Apoptosis was represented by cleavage of 116-kDa PARP into an 85-kDa product.
DNA fragmentation assay
Cells were treated with ACA (15.0 μM) for 12 h and 24 h before harvesting, and total DNA was extracted from both untreated and treated cells using the Suicide TrackTM DNA Ladder isolation kit according to the manufacturer’s protocol. Extracted DNA was analysed on a 1.0% (w/v) agarose gel electrophoresis and stained with ethidium bromide. Fragmentation of DNA was observed under UV illumination and visualized using a gel documentation system (Alpha Inotech, USA).
Microarray global gene expression analysis
To investigate changes brought upon by ACA in global gene expression, the Affymetrix GeneChip® Human Gene 1.0 Sense Target (ST) Array (Affymetrix Inc., USA) was used according to manufacturer’s protocol. Briefly, total RNA from HSC-4 cells treated with ACA (15.0 μM) for 60 min and 120 min were extracted using the RNeasy® Plus Mini Kit according to manufacturer’s protocol and analyzed under the Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). RNA samples were then reverse transcribed, labelled and hybridized onto Affymetrix chips containing 764,885 probes representing and spanning across 28,869 human genes. Scanning of all arrays was done using the Affymetrix GeneChip® Scanner (Affymetrix Inc., USA). Statistical and gene expression analysis of triplicate arrays were done using the GeneSpring® GX version 10.0 (Agilent Technologies, CA, USA) software employing principle component analysis plots, p-value and fold-change thresholds.
Western blot analysis
To determine levels of protein expression, cytoplasmic and nuclear extracts from HSC-4 cells treated with ACA at IC50 concentrations for 2 h and 4 h were prepared using the NE-PER® nuclear and cytoplasmic extraction kit according to manufacturer’s protocol. Protein concentration was quantified and normalized using the Quick Start Bradford protein assay kit 2 (Bio-Rad, USA) according to manufacturer’s protocol. Fractionation of proteins were done using a 12.0% (v/v) SDS-PAGE and electrophoretically transferred to a 0.2 μm nitrocellulose membrane using the TransBlot SD Semi Dry Transfer Cell (Bio-Rad, USA). Blots were blocked and incubated with 13 primary antibodies: β-actin, histone H3, FasL, xIAP, Bim, p65, phospho-p65 (Ser536), IκBα, phospho-IκBα (Ser32/36), IKKα, phospho-IKKα (Thr23), IKKβ and phospho-IKKβ (Ser176) overnight at 4°C. Detection of bound antibodies were done using HRP-conjugated secondary antibodies, and visualized using the SuperSignal West Pico chemiluminescent substrate on x-ray films. Normalization of protein concentration was carried against β-actin and histone H3 proteins for cytoplasmic and nuclear components respectively. Relative intensities of all bands were quantified using ImageJ v1.43 analysis software (NIH, USA).
Combination effects of ACA and CDDP
Assessment of synergistic drug combination treatments between ACA and CDDP were evaluated using MTT assays on HSC-4 cells as previously described. A total of 2.0 x 104 cells were plated in triplicates and treated with standalone ACA, standalone CDDP, and ACA in combination with CDDP at various concentration ratios for duration of 24 h and 48 h exposure. In groups where ACA were held constant, a sub-optimal IC25 of dose 5.0 μM was used, while for CDDP constant groups, a sub-optimal IC25 dose of 30.0 μg/ml was used. After incubation, 5.0 mg/ml MTT reagent was added into each well, incubated for 2 h in the dark at 37°C until a purple formazan precipitate was clearly visible and absorbance measured at 570 nm wavelength with a 650 nm reference wavelength using the Tecan Sunrise® microtitre plate reader (Tecan, Switzerland). Assessment on the type of combination relationship was done using an isobologram analysis, while the degree of synergy was assessed based on calculated combination index (CI) values, where CI values of >1.0 implies antagonism, 1.0 implies additivity, and <1.0 implies synergistic type relationships between two drugs. All calculations were based upon the CI equation adapted from previous literature.
Effects of ACA in vivo
Athymic nude mice (Nu/Nu) were obtained from Biolasco Taiwan Co. Ltd. and used for all human oral SCC tumor xenografts. Male nude mice 6-weeks-old, weighing 27 g to 30 g were used and fed ad libitum with sterilized food pellets and sterile water. Tumor induction was done by injecting suspensions of 100.0 μl HSC-4 cells (1 x 107cells/ml) in 1x PBS subcutaneously (s.c.) at the lateral neck region of Nu/Nu mice using 25 gauge needles. Both ACA (1.9 μg/ml) and CDDP (8.0 μg/ml for combination or 35.0 μg/ml for standalone) were dissolved in 0.9% (w/v) sodium chloride solution and administered via s.c. locally at tumor induction sites once tumor reached above 100.0 mm3 in volume. Standalone and combination treatments were administered three times a week at two day intervals via in situ s.c. injections, and sterile PBS solutions were used as placebo controls. Tumor volumes were assessed by measuring length x width x height with a Traceable Digital Calliper (Fisher Scientific) every 7-days post-treatment, and net body weight minus weight of tumors were measured. All animal studies were conducted in specific pathogen free (SPF) facilities with HEPA filtered air provided by Genetic Improvement and Farm Technologies Pte. Ltd. (GIFT) and were in accordance with the guidelines for the Veterinary Surgeons Act 1974 and Animal Act 1953. Housing and husbandry management were conducted according to guidelines by Institute of Laboratory Animal Resources (ILAR), while termination of specimens was done using purified CO2 gas according to the American Veterinary Medical Association (AVMA) Guidelines on Euthanasia.
Paraffin-embedded tumor biopsies were harvested, fixed in 10% (v/v) neutral buffered formalin (NBF) and embedded in paraffin for IHC analyses. Removal of paraffin from tissue sections were done using xylol followed by rehydration in a graded alcohol series. Epitope retrieval was achieved by boiling the tissue sections in sodium citrate buffer (0.01 M, pH 6.0) for 10 min. Endogenous peroxidase activity was blocked using 3% (v/v) hydrogen peroxide and washed. All sections were blocked with TBST and 5% (v/v) normal goat serum for 1 h. IHC was performed using antibodies specific for NF-κB p65 (1:400), IκBα (1:50), phospho-IKKα/β (1:300), COX-2 (1:200) and cyclin D1 (1:25). SignalStain® Boost IHC Detection Reagent (HRP, Mouse/Rabbit) were used for signal detection according to the manufacturer's protocol and further developed with DAB solution. Counter-staining was done using hematoxylin and embedded with DPX mounting medium. Images were captured using an inverted fluorescence microscope Nikon Eclipse TS 100 (Nikon Instruments, Japan) and quantified using the Nikon NIS-BR Element software (Nikon Instruments, Japan).
Data from all experiments were presented as mean ± SEM. Student’s two-tailed t-test was used to determine the statistical significance of results with p ≤ 0.05 or p ≤ 0.10 in some in vivo experiments. Migration assay experiments were performed in triplicates and all data were also reported as mean ± SEM of four sub-sections per replicate. All global gene expression and in vitro drug combination experiments were carried out in triplicates. All in vivo data were calculated based on five replicates per treatment or placebo group.
ACA induces apoptosis-mediated cell death and suppresses the proliferation and migration rate of oral SCC in vitro
ACA dysregulated NF-ÎºB related genes as indicated through microarray global expression analysis
Summary of the top 20 cancer and apoptosis-related gene expression changes in HSC-4 cells following ACA treatment for 1 h and 2 h. Genes were selected based on triplicates with p -values ≤ 0.05; and mean fold changes ≥ 1.50
Fold change (0 h vs. 1h)
Fold change (0 h vs. 2 h)
Tumour protien p53
F-box protien 21
BCL2-associated X (Bax)
Tumour necrosis factor alpha (TNF-α)
Poly (ADP-ribose) polymerase (PARP)
Caspase 6 (CASP-6)
Cyclin-dependent kinase 2 (CDK-2)
v-fos FBJ murine osteosarcoman viral oncogene
Fibroblast growth factor 2 (FBF-2)
Lymphotoxin beta (LTB)
Nuclear factor of kappa inhibitor delta (lkB-δ)
Mitogen-activated protien 3-kinase (MAP3K)
Tumour necrosis factor receptor (TNF-R)
Tumour protien 73 (p73)
TNFR-associated death domain (TRADD)
TNF receptor-associated factor 1 (TRAF-1)
ACA inhibits IKKÎ±/β-based phosphorylation and subsequent NF-ÎºB activation in HSC-4 cells
ACA potentiates the cytotoxic effects of CDDP in HSC-4 SCC cells
ACA increases the efficacy of CDDP on HSC-4 oral SCC xenografts in vivo
ACA potentiates the efficacy of CDDP in vivo by down-regulating the NF-ÎºB pathway and NF-ÎºB regulated genes
In the current study, we demonstrated the natural ginger compound ACA’s ability to inhibit the growth of oral SCC cells alone and in combination with CDDP both in vitro and in vivo. Various natural compounds can and has been shown to sensitize cancer cells through various ways. For example, curcumin (diferuloymethane) has been shown to potentiate the apoptotic effects of chemotherapeutic agents such as gemcitabine and paclitaxel in human bladder cancer cells through the deactivation of the NF-κB pathway[9, 10]. Recent reports have also shown that multi-targeted therapy has a higher success rate against cancer compared to mono-targeted therapies[29, 30]. This newly emerging form of combination chemotherapy involving chemo-sensitizers and anti-cancer drugs have been gaining vast popularity among oncologist worldwide, whereby new combination regimes are continuously being developed to reduce drug resistance and with increased efficacies. Of note, our in vivo data have showed that ACA on its own or in combination with CDDP was able to reduce tumor volumes and toxicity levels, resulting in reduced body weight loss compared to CDDP on its own.
The activation extent of various signal transduction pathways involved in chemo-sensitivity such as the NF-κB pathway, explains how resistant or susceptible a cancer type is towards drugs. Since activation of the NF-κB pathway also protects cells from undergoing apoptosis, it is theoretically viable that the successful blocking of this pathway would have a reverse effect on tumor cells through the induction of apoptosis and increased susceptibility towards other drugs. One of the early evidence describing this hypothesis was presented when studies on p65-deficient mice hepatocytes with an inactive NF-κB pathway was shown to induce massive levels of apoptosis. Since then, there have been reports on various chemotherapeutic agents that were able to cause dysregulation of NF-κB and NF-κB target genes, leading to sensitization and apoptosis[12, 33–35]. In addition to its anti-apoptotic role, NF-κB also induces cell proliferation and cell-cycle progression by regulating the expression of target genes including growth factors such as IL-2, COX-2 and cell-cycle regulators such as cyclin D1[34, 36]. Here, our IHC results has provided evidence indicating that ACA was not only able to down-regulate NF-κB activation, but also reduce the expression of NF-κB-regulated genes such as proinflammatory (NF-κB and COX-2) and proliferative (cyclin D1), which are up-regulated in most human oral neoplasia[37, 38]. This was found to be a favourable observation based on past reports, where higher levels of cyclin D1 expression exhibited higher resistance to CDDP, and a reduction in its expression resulted in increased sensitivity.
Key regulatory steps in IKK activation involve phosphorylation of several sites on the catalytic IKKα/β subunit, as well as polyubiquitination-based activation of its NEMO subunit. Based on Figures4 and5, it was observed that ACA prevented the site-specific phosphorylation of IKKα/β at Thr23 and Ser176. This led to the assumption that ACA may either obstruct site-specific phosphorylation through a direct interaction with IKK, or modulate further upstream signalling kinases such as MEKK3, TAK1 and NIK[40, 41]. Inactivation of the IKK complex in turn, prevented the phosphorylation of RelA/p50 bound IκB-α and its subsequent ubiquitination and degradation. The inability to remove of IκB-α from the heterodimer prevented RelA/p50 phosphorylation, and its localization within the nucleus, therefore inhibiting the canonical mode of NF-κB activation and the expression of downstream κB-regulated genes.
In normal cells, even though NF-κB is rarely constitutively expressed, with the exception of proliferating B cells, T cells, thymocytes, monocytes and astrocytes, basal levels of NF-κB expression still remains detectable. Therefore, the incomplete inhibitory effects of ACA on the NF-κB pathway as shown in this study is ideal, since a complete shutdown will result in the loss of peripheral immunogenic properties linked to immunodeficiency symptoms, which will subsequently make ACA a non-viable drug candidate. Observations indicating that the chemo-sensitizing effects of ACA were momentary, with synergism diminishing after 24 h of exposure, suggested that phenylpropanoids such as ACA can be either metabolized or chemically modified within the cell to an unstable structure. This unstable state which does not accumulate within cells can also be viewed as a desirable trait of ACA, which in turn prevents a toxicity build-up within an in vivo system.
Despite current findings pointing towards the use of ACA as a viable drug candidate, several problem-arising issues such as solubility factors and the non-specific nature surrounding organic compounds such as ACA should be further investigated. However, they can be addressed through the manipulation of delivery methods to include soluble protein partners with tumor receptor specificity. Nevertheless, our results support further research on ACA to improve its shortcomings as well as its inclusion into clinical trials in patients with oral cancer.
Overall, our studies have proven that ACA can inhibit the growth of human oral cancer and further potentiate the effect of standard (CDDP) treatment by modulation of proinflammatory microenvironment. The current preclinical data could form the basis for further clinical trials to improve the current standards of care for oral malignancies, and perhaps other malignancies, using this active component of Malaysian ginger with an overall improved efficacy coupled with a lower effective CDDP dose.
We declare that all in vivo experiments were approved by the University of Malaya ethical committee, and were reported according to the ARRIVE guidelines as set by the National Centre for the Replacement, Refinement and Reduction of Animal in Research (NC3Rs). Animal care including housing, husbandry and termination were in accordance with the Veterinary Surgeons Act 1974 and Animal Act 1953, and guidelines by the Institute of Laboratory Animal Resources (ILAR) and American Veterinary Medical Association (AVMA).
Acknowledgement and grant support
This study was supported by the University of Malaya Postgraduate Research Grant (PPP) (PS127-2008A and PS302-2010A), the Ministry of Higher Education (MOHE) through the Fundamental Research Grant Scheme (FRGS) (FP024-2008C, FP031-2010A and KPT1060-2012), the Ministry of Science, Technology and Innovation (MOSTI) eScience Grant (12-02-03-2022) and the Centre for Natural Product Research and Drug Discovery (CENAR) Grant (4911274).
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