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.