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

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.

Polygonum cuspidatum (Polygonaceae) has traditionally been used in folk medicine to improve oral public health. However, there are no reports related to its potential of controlling oral cancer. In this study, we evaluated the anti-tumorigenic effects and mechanisms of methanol extract and its fractions separated from Polygonum cuspidatum root in KB human oral cancer cells. The methanol extract of Polygonum cuspidatum (MEPC) inhibited the proliferation of KB cells in a dose- and time-dependent manner by inducing caspase-dependent apoptosis. Protein and mRNA expression levels and the transactivation of Specificity protein 1 (Sp1) were markedly decreased in KB cells treated with MEPC. Ethyl acetate fraction (EA) from MEPC was more potent than aqueous fraction (AQ) from MEPC to induce apoptosis. F2, F3 and F4 from EA differentially inhibited the growth of KB cells and it depends on the amount of Emodin in F2, F3 and F4. Moreover, Emodin inhibited KB cell growth and induced caspse-dependent apoptosis. Thus, the results from this study strongly suggest that MEPC, its fraction and Emodin may be potential bioactive materials to cause apoptosis mechanism via the down-regulation of Sp1 in KB human oral cancer cells. This paper was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund, KRF-2008-331-E00260) and Bio R&D program through the Korea Science and Engineering Foundation funded by the Ministry of Education, Science and Technology (M10870050003-08N7005-00311). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 223.

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.

In the present study, we examined the anticancer properties of berberine in KB oral cancer cells with a specific focus on its cellular mechanism. Berberine did not affect the cell viability of the primary human normal oral keratinocytes that were used as a control. However, the viability of KB cells was found to decrease significantly in the presence of berberine in a dose-dependent manner. Furthermore, in KB cells, berberine induced the fragmentation of genomic DNA, changes in cell morphology, and nuclear condensation. In addition, caspase-3 and -7 activation, and an increase in apoptosis were observed. Berberine was also found to upregulate significantly the expression of the death receptor ligand, FasL. In turn, this upregulation triggered the activation of pro-apoptotic factors such as caspase-8, -9 and -3 and poly(ADP-ribose) polymerase (PARP). Furthermore, pro-apoptotic factors such as Bax, Bad and Apaf-1 were also significantly upregulated by berberine. Anti-apoptotic factors such as Bcl-2 and Bcl-xL were downregulated. Z-VAD-FMK, a cell-permeable pan-caspase inhibitor, suppressed the activation of caspase-3 and PARP. These results clearly indicate that berberine-induced cell death of KB oral cancer cells was mediated by both extrinsic death receptor-dependent and intrinsic mitochondrial-dependent apoptotic signaling pathways. In addition, berberine-induced upregulation of FasL was shown to be mediated by the p38 MAPK signaling pathway. We also found that berberine-induced migration suppression was mediated by downregulation of MMP-2 and MMP-9 through phosphorylation of p38 MAPK. In summary, berberine has the potential to be used as a chemotherapeutic agent, with limited side-effects, for the management of oral cancer.

Effects of diphenyl difluoroketone (EF-24) and curcumin on cell growth and apoptosis induction in KB human oral cancer cells were examined. EF-24 and curcumin inhibited the growth of KB cells in a dose-dependent manner, and the potency of EF-24 was 30 times greater than that of curcumin. Treatment with EF-24 or curcumin resulted in nuclear condensation and fragmentation. EF-24 and curcumin promoted the proteolytic cleavage of procaspases-3, -7, and -9. Activities of caspases-3 and -7 were detected in living KB cells treated with EF-24 or curcumin. These results suggest that EF-24 and curcumin inhibit cell proliferation and induce apoptosis in KB human oral cancer cells, and have potential properties for development of anti oral cancer drug.

Oral squamous cell carcinoma (OSCC), a global threatening disease, is reported mostly in the middle and elderly male population. Even though the exact cause of OSCC was not known, consumption of tobacco in any form has been reported in most of OSCC patients. OSCC is a massive invasive type of cancer which easily spreads to the distant organs. Hence treating it at appropriate time is necessary and the rate of OSCC incidence is also constantly increasing. At present, chemoradiation is the only therapy prescribed for OSCC patients which renders various side effects. Hence, the treatment with lesser side effect was of current research interest. Doxazosin (α1 adrenorecptor antagonist) had been proven to render anticancer effect in prostate, renal, hepatic, and ovarian cancers but its role in oral cancer cells was not been elucidated. Therefore, we have assessed the anticancer effect of doxazosin on oral squamous cancer cells via through the induction of apoptosis, and antioxidant property. The cytoprotective effect of doxazosin on normal Vero cells and anticancer effect on oral cancer KB cells were analyzed with MTT assay. Doxazosin antioxidant activity were analyzed by their reactivity with free radicals and metal ions by the method of FRAP, DPPH, chemilumiscence, and ORAC assay. The antioxidant levels were also assessed by TBARS, SOD, and glutathione levels, and later on apoptosis staining techniques like DCFH-DA, Rhodamine 123, and AO/EtBr stain were conducted. Apoptosis was confirmed by estimating the levels of apoptotic proteins in doxazosin-treated KB human oral cancer cells by ELISA method. The results from our study show that doxazosin is a potent antioxidant and it significantly induces apoptosis in human oral cancer by altering various cellular molecules at downstream signaling which has been depict in the results. Our study proves doxazosin as a potent anticancer drug which may be used in the treatment of oral carcinoma, if it is subjected to further research using human clinical trials.

The aim of the present study was to investigate the effect of Solanum xanthocarpum on KB human oral cancer cells by analyzing its anti-proliferative and apoptotic properties as well as its inhibitory effect on cell adhesion. In this study, the leaves extract of S. xanthocarpum was prepared using the maceration method. Cytotoxic effect of different doses of the S. xanthocarpum extract was assessed using MTT assay. Measurements of reactive oxygen species (ROS), lipid peroxidation and antioxidant enzymes were also performed. In addition, we also studied the impacts of S. xanthocarpum on the apoptosis and mitochondrial membrane potential of KB cells. Determination of antioxidant enzymes and lipid peroxidation was performed using biochemical methods. The S. xanthocarpum showed cytotoxic activity against KB cells with IC50 (200 μg/mL). Besides, DCFH-DA staining and acridine orange/ethidium bromide staining results demonstrated that S. xanthocarpum induced the generation of ROS and apoptosis in KB cells, respectively. Based on the Rh-123 staining results, S. xanthocarpum decreased mitochondrial depolarization in KB cells. Furthermore, the S. xanthocarpum treatment contributed to increased lipid peroxidation, accompanied by reduced activities of superoxide dismutase and catalase, as well as decreased glutathione content. Taken together, these findings indicate that S. xanthocarpum extract might comprise bioactive compounds of therapeutic significance, which can inhibit the growth of KB cells.

Resveratrol (trans-3,4′s,5,-trihydroxystilbene), a phytoalexin present in grape skin and red wine, suppresses many types of cancers by regulating cell proliferation and apoptosis through a variety of mechanisms. However, the effects of resveratrol on oral cancer are not completely understood. Thus, effects of resveratrol on cell growth and apoptosis induction were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, DNA fragmentation, immunoblotting, and determination of caspase activation in KB human oral cancer cells. Treatment with resveratrol induced inhibition of cell growth depending on the resveratrol treatment time and concentration in KB cells. Treatment with resveratrol induced DNA ladder formation in KB cells and promoted proteolytic cleavage of procaspase-3 and procaspase-7 with increases in the amount of cleaved caspases-3 and -7. Proteolytic processing of caspase-9 in KB cells was increased by resveratrol treatment. Activation of caspase-3/-7 was detected in living KB cells by fluorescence microscopy. These results suggest that the resveratrol can suppress cell growth and induce cell apoptosis in KB human oral cancer cells, and may have potential as an anti-cancer drug.

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.

Background Oral squamous cell carcinoma (OSCC) signifies a major global health issue, defined by its destructive nature and often delayed diagnosis, leading to suboptimal prognoses. The global case and mortality rates of OSCC continue to increase, especially among younger populations. Purpose This work aims to study the anti-cancer properties of crebanine on oral cancer KB cells by inducing apoptosis via suppressing PI3K/AKT/mTOR and JAK-2/STAT-3 pathways. Materials and Methods Crebanine at various doses (0.5–25 µM) was evaluated for its in vitro free-radical scavenging properties, including 2,2-diphenyl-1-picrylhydrazyl (DPPH), peroxyl, and superoxide radicals. The impact of crebanine on the growth of oral cancer KB and normal Vero cells was evaluated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. The mitochondrial membrane potential (MMP) level in untreated and crebanine-treated KB cells was assessed using a fluorescent staining assay. The oxidative stress markers, apoptosis-related proteins, and PI3K/AKT/mTOR and JAK-2/STAT-3 pathway proteins were evaluated in untreated and crebanine-treated KB cells. Results The findings of the free radical scavenging experiments demonstrated the in vitro anti-oxidant properties of crebanine. The findings of the MTT experiment revealed that crebanine considerably inhibited the viability of KB cells without significantly affecting the normal Vero cells. The crebanine treatment reduced the MMP level in KB cells, as demonstrated by the findings of the fluorescent staining assay. The crebanine-treated KB cells exhibited elevated thiobarbituric acid reactive substances (TBARS) levels, alongside decreased glutathione (GSH) and superoxide dismutase (SOD) levels. Furthermore, crebanine treatment enhanced the pro-apoptotic proteins Bax and caspase-3/9 levels, while concurrently inhibiting the PI3K/AKT/mTOR and JAK-2/STAT-3 signaling protein levels in KB cells. Conclusion The current study demonstrates that crebanine treatment can impede cellular proliferation, trigger oxidative stress, and facilitate apoptosis in KB cells via downregulating PI3K/AKT/mTOR and JAK-2/STAT-3 pathways.

In recent years, population-based studies and retrospective analyses of clinical studies have shown that metformin treatment is associated with reduced cancer incidence and a decrease in cancer‑associated mortality. However, its mechanism of action remains to be fully understood. The present study demonstrates the effects of metformin on KB human oral cancer cells and explores the role of myeloid cell leukaemia‑1 (Mcl‑1) in metformin‑induced mitochondria‑dependent cellular apoptosis. It was demonstrated that metformin exposure caused significant suppression of KB cell proliferation and induced cell death. Furthermore, metformin induced apoptosis through the downregulation of Mcl‑1 in KB human oral cancer cells, and the overexpression of Mcl‑1 in metformin‑treated KB cells significantly increased cell viability. Consistently, Bax and Bim were upregulated in metformin‑treated cells. The results also reveal that microRNA (miR)‑26a expression was markedly increased by metformin. Subsequent to enforced miR‑26a expression in KB cells using miR‑26a mimics, cell viability and the level of Mcl‑1 decreased. These results suggest that the anti‑proliferative effects of metformin in KB cells may result partly from induction of apoptosis by miR-26a-induced downregulation of Mcl-1.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are known to inhibit cancer growth by inhibiting the activity of cyclooxygenase (COX). However, there is increasing evidence that the COX-independent pathway may be also involved in the inhibitory effect of NSAIDs against tumor progression. Tolfenamic acid is a NSAID that exhibits anticancer activity in pancreatic and colorectal cancer models. In the present study, the anti-tumor effect of tolfenamic acid in KB human oral cancer cells is investigated. The results showed that tolfenamic acid does not alter the expression of the COX proteins, but it inhibits cell growth and induces apoptosis as evidenced by the annexin V positivity, sub-G1 population, nuclear fragmentation and the cleavage of poly ADP-ribose polymerase. In addition, tolfenamic acid also leads to a loss of the mitochondrial membrane potential in KB cells. These effects are related to the activation of p38 mitogen-activated protein kinase (MAPK) pathway. These results suggest that tolfenamic acid-induced apoptotic cell death inhibits cancer growth by activating the p38 MAPK pathway for cancer prevention.

Context: Saussurea lappa Dence (Compositae) is used as a traditional herbal medicine to treat abdominal pain and tenesmus in East Asia. Current studies have shown that S. lappa has anticancer activity in divergent of cancer cells. However, the effects of S. lappa on oral cancer and its mechanisms of action have yet to be elucidated.Objective: To explore its potential chemotherapeutic effects and mechanism of cell growth inhibition on human oral cancer cells.Materials and methods: The dried roots of S. lappa were used in this study. Cell viability of KB cells was evaluated by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assay after treatment with 30 µg/ml of methanol extract from the dried roots of S. lappa. To understand whether its effect on cell death is related with apoptosis pathway, we performed DNA fragmentation assay, western blot, caspase activity assay and fluorescence-activated cell sorting (FACS) analysis.Results: Treatment of S. lappa extract onto KB cells reduced cell viability significantly with an IC50 value of 30 µg/ml. The formation of a DNA ladder was observed starting at the 24 h treatment. In western blotting analysis, the S. lappa extract induced the proteolytic processing of caspase-3, -9 and poly (ADP-ribose) polymerase, a significant increase of Bax and marked reduction of Bcl-2. We also confirmed the activation of caspase-3/-7 in living KB cells by fluorescence microscopy.Conclusion: These results suggested that S. lappa extract inhibited cell proliferation through the apoptosis pathway in KB human oral cancer cells.

Diindolylmethane (DIM), an isothiocyanates found in cruciferous vegetables, has been shown to have cancer chemopreventive effects. A series of synthetic C-substituted DIMs (C-DIMs) analogs was developed, including DIM-C-pPhtBu and DIM-C-pPhC6H5, which exhibited better inhibitory activity in cancer cells than DIM. This study examined the effects of C-DIMs on the growth of KB human oral cancer cells. DIM-C-pPhtBu and DIM-C-pPhC6H5 decreased the number of viable cells and induced caspase-dependent apoptosis. The apoptotic cell death was accompanied by a change in Bax/Bcl-2 ratio and damage to mitochondrial membrane potential through the induction of Death Receptor 5 and the cleavage of bid and caspase 8. Mechanistic studies demonstrated that the apoptotic cell death induced by DIM-C-pPhtBu and DIM-C-pPhC6H5 was mediated by endoplasmic reticulum (ER) stress. This provides the first evidence that synthetic C-DIMs originating from cruciferous vegetables induce apoptosis in KB cells via the ER stress pathway. This study was supported by Bio R&D program through the Korea Science and Engineering Foundation funded by the Ministry of Education, Science and Technology (M10870050003-08N7005-00311) and National Research Foundation of Korea Grant funded by the Korean Government (KRF-2008-331-E00260). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 224.

miR-203 downregulates Yes-1 and suppresses oncogenic activity in human oral cancer cells