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 Track^TM 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).

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.

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.

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.).

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.

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 Track^TM 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).

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.

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).

Assessment of synergistic drug combination treatments between ACA and CDDP were evaluated using MTT assays on HSC-4 cells as previously described 27 . 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 28 .

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.

As our previous studies have characterized the structure of ACA (Figure 1A) and confirmed it as a potent cytotoxic phytocompound on various cancer cell lines 18 , the goal of this follow-up study was, first, to determine whether ACA could induce apoptosis-mediated cell death together with other anti-cancer properties such as anti-migration effects. To determine the effective cytotoxic dose of ACA, MTT viability assays and Live/Dead fluorescence assays were performed on HSC-4 oral SCCs at various incubation intervals over 36 h. Treatment with DMSO was used as a vehicle control to ensure the absence of solvent-induced cytotoxicity, while HMEC cells were used as a normal control to assess the effects of ACA on non-cancerous cells (Figure. 1B & 1C). IC₅₀ values of ACA on HSC-4 cells fell within the range of 10 μM to 20 μM depending on various incubation periods, with minimal cytotoxic effects on HMEC cells where viability remained > 75% after a maximum of 36 h exposure. ACA was also found to reduce HSC-4 cell migration rates whereby the area of scratch wounds healed by 24.9 ± 2.3% compared to 48.3 ± 4.5% in untreated controls (p-value = 0.011) (Figure, 2A & 2B). The occurrence of apoptosis-mediated cell death was confirmed using PARP cleavage assays where full length PARP (116 kDa) was cleaved into a smaller 89 kDa fragment through caspase-3 activity (Figure 2C), while DNA fragmentation assays indicated a 150 kb to 200 kb laddering of DNA as early as 12 h upon ACA exposure, which is a strong hallmark of apoptotic events (Figure 2D).

In order to assess cluster of genes affected upon exposure to ACA, a microarray global expression analysis was performed. Filtered gene expression data sets from HSC-4 cells treated with ACA for 1 h and 2 h were sorted based on top 20 genes related to proliferation, apoptosis and tumorigenesis that were up- and down-regulated as summarized in Table 1. A large portion of genes affected were found to be either directly or indirectly related to the NF-κB pathway, corresponding to 88% of the top 50 genes by fold change. Among the top up-regulated genes were those encoding p53, F-box proteins, cell cycle progression proteins and Bcl-2 family members. In terms of genes down-regulated by ACA, it was observed that a majority of these genes contained the κB binding sequence in its promoter region such as v-fos oncogene, Jun proto-oncogene, lymphotoxin-β, IκB-delta, TNF-R, TRAF-1 and TRADD (Table 1). As our microarray results were centered on the NF-κB pathway, we further investigated the direct relationship between ACA and various NF-κB family members using Western blot analysis.

a Gene Target: contains κB site in promoter region.

b Ubq: involved in ubiquitination process of IκBs.

c Indirect Act: gene is activated by another κB-regulated gene.

Since the DNA binding capability of NF-κB transcription factors are governed by phosphorylation levels upon ubiquitination and subsequent release of IκBs from NF-κB heterodimers, Western blot analysis was conducted on both total and phosphorylated forms of p65, IκB-α/β proteins and the IKK complex. It was found that exposure to ACA reduced Ser536 phosphorylation levels of p65 subunits (Figure 3A & 3B), which suggested that ACA prevented C-terminal phosphorylation required by canonical NF-κB heterodimers for transactivation and commencement of κB gene transcription. Analysis of NF-κB heterodimer translocation between the nucleus and cytoplasm using antibodies against p65 (RelA) also revealed that levels of p65 within the cytoplasm increased corresponding with a reduction in nuclear p65 protein levels (Figure 3A & 3B). This consistently indicated that p65 nuclear localization was perturbed with an increasing ACA exposure time over 4 h. These observations corresponded with up-stream events which indicated that levels of phosphorylated IKKα on Thr23 and IKKβ on Ser176 were reduced, consistent with increasing ACA incubation periods (Figure 3A & 4B). Western blot assays on Ser32/36 phosphorylated forms of IκBα, which is a marker required for ubiquitination signalling, also indicated a reduction in band intensity, resulting in NF-κB heterodimers remaining within the cytoplasm (Figure 4A & 4B).

Since natural compounds that inhibited the NF-κB pathway are often sought for as sensitizing potentiators, we next investigated the combined synergistic in vitro anti-proliferative effects of ACA with CDDP in HSC-4 SCC cells. Based on MTT assay isobologram-illustrated results, it was found that ACA was able to reduce the viability levels of HSC-4 cells to a higher extent when used in combination with CDDP (Figure 5A). CI analysis indicates that synergistic effects (CI < 1.0) were observed for all combinations where ACA was maintained at a constant concentration and CDDP at varying concentrations for both 24 h and 48 h. However, as significantly synergistic CI value thresholds were set at CI < 0.75, only sequential regimes where cells were pre-treated with ACA for 12 h followed by CDDP for 24 h yielded synergistic drug interactions of CI = 0.53 ± 0.11. Regimes where ACA was held constant with varying CDDP also yielded similar CI values at 24 h, but was no longer synergistically significant at 48 h. Inversely, a shift towards an antagonistic effect (CI=1.23 ± 0.06) was observed when CDDP was maintained at a constant concentration, while ACA was allowed to vary after 48 h of incubation (Figure 5B). This concluded that ACA’s action through NF-κB down-regulation was successful in transiently sensitizing HSC-4 cells, hence, potentiating the cytotoxic effects of CDDP.

We next assessed the in vivo combined effects of ACA/CDDP on oral cancer xenografts in nude mice models. Following three weeks of a combined ACA/CDDP regime, where a lower CDDP dose (8.0 μg/ml) was used in comparison to a higher standalone CDDP dose (35.0 μg/ml), tumor volumes were found to be greatly reduced by 93.2 ± 5.2% compared to placebo groups. In reference to standalone groups, tumor volume reductions were 79.8 ± 9.5% and 86.5 ± 8.2% for ACA and CDDP respectively (Figure 6A to 6C), which corresponded to an efficacy improvement of 13.4% and 6.7% respectively in comparison to combined treatments. The use of ACA (1.9 μg/ml) to reduce effective CDDP dosage was also successful in reducing the extent of mean body weight loss by 2.5 ± 1.8% when compared to CDDP standalone treatments (Figure 6D). In addition, all mice appeared healthy during treatment, while histopathological analysis at autopsy revealed no ACA-induced tissue changes in any of the organs. Therefore, these results supported ACA’s potential to overcome dose-limiting toxicities brought upon by CDDP, while maintaining a similar, if not, higher efficacy level.

To evaluate the consistency of ACA’s chemo-sensitizing and apoptosis-inducing effects in vivo, IHC assays were conducted on members of the NF-κB pathway as well as NF-κB regulated proteins. A reduction in p65 and phospho-IKKα/β protein levels were observed upon ACA treatment compared to placebo sections, but remained relatively similar after the incorporation of CDDP in ACA combination treatments (Figure 7 & 8A). Subsequent effects on NF-κB regulated proinflammatory genes following alterations in NF-κB activation were also observed between placebo and ACA treated sections, indicating slight reductions in COX-2 and cyclin D1 protein levels. Protein levels of IκBα were also shown to increase in the presence of ACA, which were consistent with a reduction in IKK phosphorylation and subsequent prevention of 26S proteasomal degradation of IκBα as observed under in vitro conditions (Figure 7 & 8A). Observation on other NF-κB regulated genes also indicate a reduction in Fas-ligand and Bim protein levels, however, xIAP levels remained unchanged following ACA treatment, thus suggesting the involvement of other post-transcriptional regulatory elements (Figure 8B).

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).