KB Oral Cancer Cells Research Articles

Tanshinone IIA induces apoptosis in human oral cancer KB cells through a mitochondria-dependent pathway.

Tanshinone IIA (Tan IIA), an active phytochemical in the dried root of Salvia miltiorrhiza Bunge, has shown an antiproliferative activity on various human cancer cell lines including nasopharyngeal carcinoma cells. However, the effects of Tan IIA on human oral cancer cells are still unknown. This study aimed to investigate the antiproliferative effects of Tan IIA on human oral cancer KB cells and explored the possible underlying mechanism. Treatment of KB cells with Tan IIA suppressed cell proliferation/viability and induced cell death in a dose-dependent manner through sulforhodamine B colorimetric assay. Observation of cell morphology revealed the involvement of apoptosis in the Tan IIA-induced growth inhibition on KB cells. Cell cycle analysis showed a cell cycle arrest in G2/M phase on Tan IIA-treated cells. The dissipation of mitochondrial membrane potential observed by flow cytometry and the expression of activated caspases with the cleaved poly (ADP-ribose) polymerase under immunoblotting analysis indicated that Tan IIA-induced apoptosis in KB cells was mediated through the mitochondria-dependent caspase pathway. These observations suggested that Tan IIA could be a potential anticancer agent for oral cancer.

Apoptosis-inducing effect of three medicinal plants on oral cancer cells KB and ORL-48.

Brucea javanica, Azadirachta indica, and Typhonium flagelliforme are medicinal plants commonly used to treat conditions associated with tumour formation. This study aimed to determine the antiproliferative activity of these plants extracts on KB and ORL-48 oral cancer cell lines and to suggest their mode of cell death. The concentration producing 50% cell inhibition (IC50) was determined and the activity was examined under an inverted microscope. Immunohistochemistry fluorescent staining method (TUNEL) was performed to indicate the mechanism of cell death and the fragmented DNA band pattern produced was obtained for verification. Compared to Azadirachta sp. and Typhonium sp., the antiproliferative activity of Brucea sp. extract was the most potent on both KB and ORL-48 cells with IC50 of 24.37 ± 1.75 and 6.67 ± 1.15 µg/mL, respectively. Signs of cell attrition were observed 24 hr after treatment. Green fluorescent spots indicating cell death by apoptosis were observed in images of both cells following treatment with all the three extracts. DNA fragments harvested from Brucea-treated cells produced bands in a ladder pattern suggesting the apoptotic effect of the extract. It is thus concluded that Brucea sp. extract exhibited cytotoxic activity on ORL-48 cells and their action mechanism is via apoptosis.

Licochalcone A induces apoptosis in KB human oral cancer cells via a caspase-dependent FasL signaling pathway

Licochalcone A (Lico-A) is a natural phenol licorice compound with multiple bioactivities, including anti-inflammatory, anti-microbial, anti-fungal and osteogenesis-inducing properties. In the present study, we investigated the Lico-A-induced apoptotic effects and examined the associated apoptosis pathway in KB human oral cancer cells. Lico-A decreased the number of viable KB oral cancer cells. However, Lico-A did not have an effect on primary normal human oral keratinocytes. In addition, the IC50 value of Lico-A was determined to be ~50 μM following dose-dependent stimulation. KB oral cancer cells stimulated with Lico-A for 24 h showed chromatin condensation by DAPI staining, genomic DNA fragmentation by agarose gel electrophoresis and a gradually increased apoptotic cell population by FACS analysis. These data suggest that Lico-A induces apoptosis in KB oral cancer cells. Additionally, Lico-A-induced apoptosis in KB oral cancer cells was mediated by the expression of factor associated suicide ligand (FasL) and activated caspase-8 and −3 and poly(ADP-ribose) polymerase (PARP). Furthermore, in the KB oral cancer cells co-stimulation with a caspase inhibitor (Z-VAD-fmk) and Lico-A significantly abolished the apoptotic phenomena. Our findings demonstrated that Lico-A-induced apoptosis in KB oral cancer cells involves the extrinsic apoptotic signaling pathway, which involves a caspase-dependent FasL-mediated death receptor pathway. Our data suggest that Lico-A be developed as a chemotherapeutic agent for the management of oral cancer.

MicroRNA-205 suppresses the oral carcinoma oncogenic activity via down-regulation of Axin-2 in KB human oral cancer cell

MicroRNA (miRNA) is a small noncoding RNA molecule, 19-25 nucleotides in length, which regulates several pathways including cell development, cell proliferation, carcinogenesis, apoptosis, etc. In this study, the over-expression of microRNA-205 (miR-205) increased the number of apoptotic cells by at least 4 times compared to the control. In addition, over-expressed miRNA in KB oral cancer cells triggered apoptosis via the caspase cascade, including the cleavage of caspase-9, caspase-7, caspase-3, and PARP. Flow cytometry showed that apoptotic cell death was increased significantly by 35.33% in KB oral cancer cells with over-expressed miR-205 compared to the control. The microarray data showed that axis inhibitor protein 2 (Axin2) was down-regulated in KB oral cancer cells transfected with miR-205. In addition, Axin2 was down-regulated by approximately 50% by over-expressed miR-205 at both the mRNA and protein levels. Interestingly, Axin2 was up-regulated in KB oral cancer compared to human normal oral keratinocytes. Furthermore, the cell cytotoxicity and apoptotic population of KB oral cancer cells were increased significantly after Axin2 siRNA transfection. These results suggest that Axin2 is might be as potential oncogene in KB oral cancer cells. The luciferase assay showed that over-expressed miR-205 in KB oral cancer cells suppressed AXIN2 expression through an interaction with its own binding site at AXIN2 3'UTR (64-92). These results suggest that miR-205 is a novel anti-oncogenic miRNA in KB oral cancer cells, and may have potential applications in oral cancer therapy.

Constituents of the Indonesian Epiphytic Medicinal Plant Drynaria rigidula

The rhizome of Drynaria rigidula has been traditionally claimed to have anti-infective properties. A phytochemical study conducted on the rhizome successfully isolated two new aromatic glycosides as well as three known terpenoids, two benzoic acid derivatives and two known flavonoid glycosides. The rhizome methanol extract and its fractions were tested against Mycobacterium tuberculosis, Plasmodium falciparum, herpes simplex virus and KB-oral cancer cell; insignificant activity was found.

Development of peptide inhibitor as a therapeutic agent against head and neck squamous cell carcinoma (HNSCC) targeting p38α MAP kinase

BackgroundThe p38α MAP kinase pathway is involved in inflammation, cell differentiation, growth, apoptosis and production of pro-inflammatory cytokines TNF-α and IL-1β. The overproduction of these cytokines plays an important role in cancer. The aim of this work was to design a peptide inhibitor on the basis of structural information of the active site of p38α. MethodsA tetrapeptide, VWCS as p38α inhibitor was designed on the basis of structural information of the ATP binding site by molecular modeling. The inhibition study of peptide with p38α was performed by ELISA, binding study by Surface Plasmon Resonance and anti-proliferative assays by MTT and flow cytometry. ResultsThe percentage inhibition of designed VWCS against pure p38α protein and serum of HNSCC patients was 70.30 and 71.5%, respectively. The biochemical assay demonstrated the KD and IC50 of the selective peptide as 7.22×10−9M and 20.08nM, respectively. The VWCS as inhibitor significantly reduced viability of oral cancer KB cell line with an IC50 value of 10μM and induced apoptosis by activating Caspase 3 and 7. ConclusionsVWCS efficiently interacted at the ATP binding pocket of p38α with high potency and can be used as a potent inhibitor in case of HNSCC. General significanceVWCS can act as an anticancer agent as it potentially inhibits the cell growth and induces apoptosis in oral cancer cell-line in a dose as well as time dependent manner. Hence, p38α MAP kinase inhibitor can be a potential therapeutic agent for human oral cancer.

MicroRNA-205 directly regulates the tumor suppressor, interleukin-24, in human KB oral cancer cells.

MicroRNA (miRNA) is a form of small noncoding RNA that regulates the expression of genes either by inhibiting mRNA translation or by inducing its degradation. Small microRNA play important roles in regulating a large number of cellular processes, including development, proliferation and apoptosis. This study examined the biological functions of miR-205 as a tumor suppressor in KB oral cancer cells. The results showed that miR-205 expression was significantly lower in KB oral cancer cells than in human normal oral keratinocytes. Furthermore, the miR-205 over-expressed in KB oral cancer cells increased the cell cytotoxicity and induced apoptosis through the activation of caspase-3/-7. The transfection of miR-205 into KB oral cancer cells strongly induced IL-24, a well known cytokine that acts as a tumor suppressor in a range of tumor tissues. In addition, miR-205 targeted the IL-24 promoter directly to induce gene expression. Overall, miR-205 has significant therapeutic potential to turn on silenced tumor suppressor genes by targeting them with miRNA.

Microarray analysis in KB human oral cancer cells treated with neuron restrictive silencer factor siRNA

Strong expression of neuron restrictive silencer factor (NRSF) has been observed in many aggressive types of cancer cells and mature neurons. However, the function of the neuron restrictive silencer element (NRSE)/NRSF system in KB human oral cancer cells is unknown. Findings from previous studies in our lab have demonstrated the importance of NRSF as a factor in regulationof cell proliferation of KB human oral cancer cells. Treatment of KB cells with NRSF siRNA resulted in signifi cant inhibitionof cell growth through repression of NRSF expression. In this study, in order to understand the NRSE/NRSF regulatory network in KB cells, we performed microarray analysis in KB cells treated with NRSF specifi c targeted siRNA. The expression profi les of several genes were further validated in KB cells treated with NRSF siRNA. Results of microarray analysis showed upregulation of 117 genes and down-regulation of 215 genes in KB cells treated with NRSF siRNA. Most of the up-regulated genes were involved in signal transduction, cell communication, cell cycle, and apoptosis;down-regulated genes were involved in RNA processing, neurogenesis, transcription factor activity, and synaptogenesis. NRSF is known as a transcriptional repressor for silencingof neuronal genes; however, according to our data, treatment of KB cells with NRSF siRNAresulted in down-regulation of more than 200 genes. As a result, genes identifi ed in this screen represent a novel control pathway via NRSF expression in KB oral cancer cells. Further investigation will be needed in order to defi ne the mechanism of gene regulation by expression of NRSF in KB human oral cancer cells.

The down-regulation of galectin-1 expression is a specific biomarker of arsenic toxicity

Galectin-1 (GAL1) is known as a β-galactoside-binding protein that also can bind with arsenic to regulate cell functions. Using RNA interference technique, we investigated the possible mechanism involved in GAL1 modulation of arsenite-inhibited cell survival in 3T3 fibroblast and KB oral cancer cells. GAL1 gene knockdown significantly attenuated sodium arsenite (NaAsO 2) and arsenic trioxide (As 2O 3) inhibition of cell survival. However, GAL1 gene knockdown did not alter the inhibition of cell survival by antimony chloride, cadmium chloride or nickel sulfate. These results suggested the GAL1 selectively affects particular types of heavy metal elements. Flow cytometric analysis indicated GAL1 gene knockdown also suppressed As(III)-stimulated levels of sub-G1 and G2/M growth arrest in both cells. Moreover, atomic absorption spectrophotometric results showed that GAL1 gene knockdown reduced the total arsenic accumulation of both cells after the NaAsO 2 and As 2O 3 treatment. These results suggested that GAL1 gene knockdown mediates the apoptotic effects of arsenic in 3T3 and KB cells via regulation of the cellular arsenic levels. We propose that down-regulation of GAL1 expression may be a useful and specific biomarker in assessing the toxicity of arsenic exposure.

Green tea (−)-epigallocatechin-3-gallate inhibits HGF-induced progression in oral cavity cancer through suppression of HGF/c-Met

Hepatocyte growth factor (HGF) and c-Met have recently attracted a great deal of attention as prognostic indicators of patient outcome, and they are important in the control of tumor growth and invasion. Epigallocatechin-3-gallate (EGCG) has been shown to modulate multiple signal pathways in a manner that controls the unwanted proliferation and invasion of cells, thereby imparting cancer chemopreventive and therapeutic effects. In this study, we investigated the effects of EGCG in inhibiting HGF-induced tumor growth and invasion of oral cancer in vitro and in vivo. We examined the effects of EGCG on HGF-induced cell proliferation, migration, invasion, induction of apoptosis and modulation of HGF/c-Met signaling pathway in the KB oral cancer cell line. We investigated the antitumor effect and inhibition of c-Met expression by EGCG in a syngeneic mouse model (C3H/HeJ mice, SCC VII/SF cell line). HGF promoted cell proliferation, migration, invasion and induction of MMP (matrix metalloproteinase)-2 and MMP-9 in KB cells. EGCG significantly inhibited HGF-induced phosphorylation of Met and cell growth, invasion and expression of MMP-2 and MMP-9. EGCG blocked HGF-induced phosphorylation of c-Met and that of the downstream kinases AKT and ERK, and inhibition of p-AKT and p-ERK by EGCG was associated with marked increases in the phosphorylation of p38, JNK, cleaved caspase-3 and poly-ADP-ribose polymerase. In C3H/HeJ syngeneic mice, as an in vivo model, tumor growth was suppressed and apoptosis was increased by EGCG. Our results suggest that EGCG may be a potential therapeutic agent to inhibit HGF-induced tumor growth and invasion in oral cancer.

Synthesis and cytotoxicity evaluation of (tetrahydro-β-carboline)-1,3,5-triazine hybrids as anticancer agents

A series of tetrahydro-β-carbolines and 1,3,5-triazine hybrids have been synthesized and evaluated for their cytotoxicity against a panel of eight human cancer cell lines and normal human fibroblasts (NIH3T3). It led us to discovery of racemic compounds 69, 71 and 75, which are selectively cytotoxic towards KB (oral cancer) cell line with IC 50 values of 105.8, 664.7 and 122.2 nM, respectively; while their enantiopure forms are less active and not selective. Enantiopure compound 42 showed 2.5 times more selectivity towards MCF7 cells over normal fibroblast NIH3T3 cells with an IC 50 value of 740 nM, also arrests cell cycle in G 1 phase and induces apoptosis in MCF7 and MDA MB231cell lines.

Inhibition of L-Type Amino Acid Transporter Modulates the Expression of Cell Cycle Regulatory Factors in KB Oral Cancer Cells

The purpose of this study was to examine the effect of 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), an inhibitor of L-type amino acid transporters, on the cell growth suppression in KB human oral cancer cells and to study the roles of cell cycle regulatory factors in the BCH-induced growth inhibition. The effect of BCH on cell growth suppression and the influence of BCH to cell cycle regulatory factors in KB cell growth inhibition were examined using cell cycle analysis, immunoblotting and immunoprecipitation. The BCH treatment induced cell cycle arrest at G1 phase in KB cells. The expression of cyclin D3 was remarkably decreased by BCH treatment. The BCH inhibited the expression of cyclin-dependent protein kinase 6 (CDK6) in a time-dependent manner. In addition, the expression of CDK inhibitor p27 was increased by BCH treatment in KB cells, but not CDK inhibitors p21 and p15. These results suggest that, in KB cells, the inhibition of LAT1 by BCH causes cell cycle arrest at G1 phase by inhibiting cyclin D3-CDK6 complex whereas increasing expression of a CDK inhibitor p27.

Cell Death and Intracellular Distribution of Hematoporphyrin in a KB Cell Line

The objective of this study is to investigate the effect of intracellular photosensitizer distribution on tumor cell death after photodynamic therapy (PDT). The photosensitizer accumulates in tumor tissue during PDT, and generates intracellular reactive oxygen species (ROS), resulting in tumor cell death. This study was carried out to elucidate the effects of PDT in a KB oral cancer cell line using hematoporphyrin with irradiation at 635 nm and 5 mW/cm(2). After irradiation, the MTT reduction method, agarose gel electrophoresis, flow cytometry, and Diff-Quick staining were performed. The intracellular ROS level was measured by DCF-DA. Intracellular hematoporphyrin was monitored with a confocal microscope, and Western blot and caspase activity assays were performed. In our study, cell survival was reduced by about 50% after 3 h of hematoporphyrin incubation time. In DNA fragmentation, flow cytometry, and Diff-Quick assay, necrosis was identified within 12 h and apoptosis soon thereafter. Confocal microscopy revealed that hematoporphyrin was localized in the cell membrane, cytoplasm, and nucleus as time passed. The quantities of intracellular ROS correlated with the time of hematoporphyrin accumulation. Additionally, Western blot analysis of Bcl-2/Bax, the release of cytochrome C, and activity of caspase-3 and caspase-9 showed that apoptosis followed the mitochondria-dependent pathway. PDT with hematoporphyrin in the KB cell line showed morphological changes of cell necrosis and apoptosis, which were associated with the time of distribution and localization of hematoporphyrin. Also, the apoptosis evoked followed the mitochondria-dependent pathway.

412 POSTER A first-in-man phase I study of TH-302, a hypoxia-activated cytotoxic prodrug

Hepatocyte growth factor (HGF) and c-Met have recently attracted a great deal of attention as prognostic indicators of patient outcome, and they are important in the control of tumor growth and invasion. Epigallocatechin-3-gallate (EGCG) has been shown to modulate multiple signal pathways in a manner that controls the unwanted proliferation and invasion of cells, thereby imparting cancer chemopreventive and therapeutic effects. In this study, we investigated the effects of EGCG in inhibiting HGF-induced tumor growth and invasion of oral cancer in vitro and in vivo. We examined the effects of EGCG on HGF-induced cell proliferation, migration, invasion, induction of apoptosis and modulation of HGF/c-Met signaling pathway in the KB oral cancer cell line. We investigated the antitumor effect and inhibition of c-Met expression by EGCG in a syngeneic mouse model (C3H/HeJ mice, SCC VII/SF cell line). HGF promoted cell proliferation, migration, invasion and induction of MMP (matrix metalloproteinase)-2 and MMP-9 in KB cells. EGCG significantly inhibited HGF-induced phosphorylation of Met and cell growth, invasion and expression of MMP-2 and MMP-9. EGCG blocked HGF-induced phosphorylation of c-Met and that of the downstream kinases AKT and ERK, and inhibition of p-AKT and p-ERK by EGCG was associated with marked increases in the phosphorylation of p38, JNK, cleaved caspase-3 and poly-ADP-ribose polymerase. In C3H/HeJ syngeneic mice, as an in vivo model, tumor growth was suppressed and apoptosis was increased by EGCG. Our results suggest that EGCG may be a potential therapeutic agent to inhibit HGF-induced tumor growth and invasion in oral cancer.

Expression of Cytosolic Phospholipase A2 and Cyclooxygenase 2 and Their Significance in Human Oral Mucosae, Dysplasias and Squamous Cell Carcinomas

Prostaglandin E₂ (PGE₂) plays an important role in promoting carcinogenesis. Cytosolic phospholipase A₂ (cPLA₂) and cyclooxygenase 2 (COX-2) are both key enzymes for PGE₂ biosynthesis. Recent evidence suggests that the coordinated function of cPLA₂ and COX-2 in the arachidonic acid pathway may contribute to the process of carcinogenesis in various tissue types. However, the concomitant effect of these enzymes on oral carcinogenesis remains unclear. In this study,we evaluated the expression of cPLA₂ and COX-2 in normal oral mucosa, dysplastic oral mucosa and squamous carcinoma (SCC) using immunohistochemistry. In an in vitro assay, Tca8113 and KB oral cancer cells were treatedwith NS-398 (a selective inhibitor of COX-2) for varying time intervals: 6, 12, 24, 48 and 72 h. The levels of cPLA₂ and COX-2 expression were evaluated by Western blot, and PGE₂ production was analyzed by radioimmunoassay. We found that cPLA₂ and COX-2 were expressed at higher levels in oral dysplasia and SCC than in normal oral mucosa. cPLA₂ expression was also found to correlate closely with COX-2 expression. Moreover, the enzymatic activities of cPLA₂ and COX-2 were gradually downregulated with longer durations of treatment with NS-398, as demonstrated by a reduction in the amount of PGE₂ production over time. Our data suggest that the coordinated activity of cPLA₂ and COX-2 contribute to the process of oral carcinogenesis, thus identifying cPLA₂ as a potential target for the prevention and treatment of oral cancers.

A novel peroxisome proliferator-activated receptor α/γ agonist, BPR1H0101, inhibits topoisomerase II catalytic activity in human cancer cells

Peroxisome proliferator-activated receptor (PPAR) gamma agonists are used clinically for treating diabetes mellitus and cancer. 2-Methyl-2[(1-{3-phenyl-7-propylbenzol[d]isoxazol-6-yl}oxy)propyl]-1H-4-indolyl) oxy]propanoic acid (BPR1H0101) is a novel synthetic indole-based compound, discovered through research to identify new PPARgamma agonists, and it acts as a dual agonist for PPARgamma and PPARalpha. Isobologram analysis demonstrated that BPR1H0101 is capable of antagonistic interaction with the topoisomerase (topo) II poison, VP16. A study of its mechanism showed that BPR1H0101 could inhibit the catalytic activity of topo II in vitro, but did not produce detectable topo II-mediated DNA strand breaks in human oral cancer KB cells. Furthermore, BPR1H0101 could inhibit VP16-induced topo II-mediated DNA cleavage and ataxia-telangiectasia-mutated phosphorylation in KB cells. The results suggest that BPR1H0101 can interfere with the topo II reaction by inhibiting catalytic activity before the formation of the intermediate cleavable complex; consequently, it can impede VP16-induced topo II-mediated DNA cleavage and cell death. This is the first identified PPARalpha/gamma agonist that can serve as a topo II catalytic inhibitor, to interfere with VP16-induced cell death. The result might have relevance to the clinical use of the PPARalpha/gamma agonist in combination chemotherapy.

Carotenoids suppress proliferating cell nuclear antigen and cyclin D 1 expression in oral carcinogenic models

The purpose of this study was to investigate the chemopreventive effect of carotenoids on proliferating cell nuclear antigen (PCNA) and cyclin D 1 expression in betel ( Areca catechu) quid extract (BQE)-induced hamster oral cancer and human KB cell models, respectively. In the in vivo animal study, 41 hamsters were divided into six groups and treated with 0.3 ml of 0.5% 9,10-dimethyl-1,2-benz[a]-anthracene, BQE, α-tocopherol, β-carotene, lycopene, lutein and mixed carotenoids for 12 weeks. After treatment, the pouches were excised and graded using an immunohistochemical assay of PCNA. In the in vitro cell experiment, KB cells were cultured, and the inhibitory effect of carotenoids (β-carotene, lycopene and lutein) on cell proliferation was evaluated. Cyclin D 1 and PCNA were evaluated in terms of cell differentiation. In the results, most of the animal lesions showed no overexpression of PCNA. However, in dysplastic lesions, PCNA expressions by the β-carotene, lutein, lycopene, mixed and vitamin E groups were less than that of the control group. In papilloma lesions, PCNA expressions by the β-carotene, mixed and vitamin E groups were less severe than that of the control group. PCNA expression by the vitamin E-treated group was less severe than that of the control group. No carcinoma was found in the lycopene or mixed groups. In the cell study, all carotenoids exerted a significant inhibitory effect on KB cell proliferation. Although lycopene suppressed KB cell proliferation at the G 0/G 1 phase with a significant decrease in PCNA expression, β-carotene and lutein possessed less of an inhibitory effect and even exhibited elevated cell proliferation at the G 2/M phase. These results indicate that different carotenoids present various suppressive abilities against PCNA and cyclin D 1 expressions in cell proliferation. In conclusion, carotenoids suppressed the carcinogenesis of induced hamster oral cancer and a cancer cell line by acting as a suppressor which inhibited the expressions of PCNA and cyclin D 1.

Aloe-emodin induces in vitro G2/M arrest and alkaline phosphatase activation in human oral cancer KB cells

Aloe-emodin is a natural anthraquinone compound from the root and rhizome of Rheum palmatum. In this study, KB cells were treated with 2.5, 5, 10, 20, and 40 microM aloe-emodin for 1 to 5 days. The results showed that aloe-emodin inhibited cancer cells in a dose-dependent manner. Treatment with aloe-emodin at 10 to 40 microM resulted in cell cycle arrest at G2/M phase. The alkaline phosphatase (ALP) activity in KB cells increased upon treatment with aloe-emodin when compared to controls. This is one of the first studies to focus on the expression of ALP in human oral carcinomas cells treated with aloe-emodin. These results indicate that aloe-emodin has anti-cancer effect on oral cancer, which may lead to its use in chemotherapy and chemopreventment of oral cancer.

Inhibition of growth and increase of alkaline phosphatase activity in cultured human oral cancer cells by all- trans retinoic acid

In this study, the effects of all- trans retinoic acid (ATRA) on human oral cancer cells with regard to cell growth, the cell cycle, and alkaline phosphatase (ALP) activity were evaluated. Human oral cancer KB cells were treated with various concentrations of ATRA, and cell growth was then determined using the MTT viability assay. The cell-cycle distribution and ALP activity were analysed using a flow cytometer and chemical analyser, respectively. The KB cells were inhibited by ATRA at concentrations of 1–16 μM (1 μM, P < 0.05; 2 μM, P < 0.01; 4, 8 and 16 μM, P < 0.001) in a dose-dependent manner. ATRA arrested KB cells in the G0/G1 phase. The ALP activity in KB cells was increased by ATRA. This is one of the first studies to focus on the expression of ALP in human head-and-neck carcinoma cells treated with retinoids. These findings suggest that the anti-tumour effects of ATRA on human oral cancer are associated with G0/G1 phase arrest and an increase in ALP activity.

The anti-inflammatory potential of berberine in vitro and in vivo

Berberine, an isoquinoline alkaloid, has a wide range of pharmacological effects, including anti-inflammation, yet the exact mechanism is unknown. Because cyclooxygenase-2 (COX-2) plays a key role in prostaglandins (PGs) synthesis, which is elevated in inflammation, we examined whether the anti-inflammatory mechanism of berberine is mediated through COX-2 regulation. In oral cancer cell line OC2 and KB cells, a 12 h berberine treatment (1, 10, and 100 μM) reduced prostaglandin E 2 (PGE 2) production dose-dependently with or without 12- O-tetradecanoylphorbol-13-acetate (TPA, 10 nM) induction. This berberine induced effect occurred rapidly (3 h) as a result of reduced COX-2 protein, but not enzyme activity. The electrophoretic mobility shift assay revealed that activator protein 1 (AP-1) binding was decreased in oral cancer cells treated with berberine for 2 h. Further analysis showed that berberine inhibited AP-1 binding directly. These anti-inflammatory effects paralleled to the in vivo results where berberine pretreatment of Wistar rat inhibited the production of exudates and PGE 2 in carrageenan induced air pouch.