Expression of nucleotide-binding oligomerization domain 1 and 2 in oral lichen planus.

BACKGROUND/PURPOSE: Oral lichen planus (OLP) is a chronic inflammatory disease of oral mucosa. The present study investigated the expression of nucleotide-binding oligomerization domain (NOD), a pivotal sensor protein of the innate immune system, in OLP. MATERIALS AND METHODS: Oral mucosal biopsies were collected from 20 patients with OLP and 6 individuals with normal oral mucosa (NOM). The expression of NOD1 and NOD2 was determined using RT-PCR and immunohistochemistry in OLP and NOM samples. RESULTS: The mRNA expression of NOD1 and NOD2 was significantly higher in the OLP group than in the NOM group. The protein expression of NOD1 was marginally upregulated in all mucosal layers in the OLP group compared with that of the NOM group; however, the differences were not significant. The expression of NOD2 was elevated in infiltrating lymphocytes of the submucosal layer in the OLP group compared with the NOM group, but was undetected in other inflammatory disease, inflammatory fibrous hyperplasia (IFH). This study revealed the upregulation of NOD2 mRNA and protein in the OLP group, but not in the NOM group. CONCLUSION: These findings suggest that NOD2 may play an important role in the pathogenesis of OLP and represents a new diagnostic and treatment target.

The In vitro Effects of Nano-encapsulated Conjugated Linoleic Acid on Stability of Conjugated Linoleic Acid and Fermentation Profiles in the Rumen.

This study was aimed to evaluate the stability of conjugated linoleic acids (CLAs) by nano-encapsulation against in vitro ruminal biohydrogenation by microbial enzymatic conversion. CLAs (free fatty acid form of CLA [CLA-FFA], nano-encapsulated CLA-FFA, triglyceride form of CLA [CLA-TG], and nano-encapsulated CLA-TG) were used in the in vitro fermentation experiments. When Butyrivibrio fibrisolvens (B. fibrisolvens) was incubated with CLA-FFAs, the concentrations of cis-9, trans-11 CLA and vaccenic acid (VA) slightly was decreased and increased by nano-encapsulation, respectively. When B. fibrisolvens was incubated with CLA-TG, the concentrations of cis-9, trans-11 CLA and VA decreased, but these were increased when B. fibrisolvens was incubated with nano-encapsulated CLA-TG. The nano-encapsulation was more effective against the in vitro biohydrogenation activity of B.fibrisolvens incubated with CLA-FFA than with CLA-TG. In the in vitro ruminal incubation test, the total gas production and concentration of total volatile fatty acids incubated with nano-encapsulated CLA-FFA and CLA-TG were increased significantly after 24 h incubation (p<0.05). Nano-encapsulated CLA-FFA might, thus, improve the ruminal fermentation characteristics without adverse effects on the incubation process. In addition, nano-encapsulated CLA-FFA increased the population of Fibrobacter succinogenes and decreased the population of B. fibrisolvens population. These results indicate that nano-encapsulation could be applied to enhance CLA levels in ruminants by increasing the stability of CLA without causing adverse effects on ruminal fermentation.

Nucleotide-binding oligomerization domain 2 (NOD2) activation induces apoptosis of human oral squamous cell carcinoma cells.

OBJECTIVES: Microbial Pattern-recognition receptors (PRRs), such as nucleotide-binding oligomerization domains (NODs), are essential for mammalian innate immune response. This study was designed to determine the effect of NOD1 and NOD2 agonist on innate immune responses and antitumor activity in oral squamous cell carcinoma (OSCC) cells. MATERIALS AND METHODS: NODs expression was examined by RT-PCR, and IL-8 production by NODs agonist was examined by ELISA. Western blot analysis was performed to determine the MAPK activation in response to their agonist. Cell proliferation was determined by MTT assay. Flow cytometry and Western blot analysis were performed to determine the MDP-induced cell death. RESULTS: The levels of NODs were apparently expressed in OSCC cells. NODs agonist, Tri-DAP and MDP, led to the production of IL-8 and MAPK activation. NOD2 agonist, MDP, inhibited the proliferation of YD-10B cells in a dose-dependent manner. Also, the ratio of Annexin V-positive cells and cleaved PARP was increased by MDP treatment in YD-10B cells, suggesting that MDP-induced cell death in YD-10B cells may be owing to apoptosis. CONCLUSIONS: Our results indicate that NODs are functionally expressed in OSCC cells and can trigger innate immune responses. In addition, NOD2 agonist inhibited cell proliferation and induced apoptosis. These findings provide the potential value of MDP as novel candidates for antitumor agents of OSCC.

5-Nitro-5'-hydroxy-indirubin-3'-oxime (AGM130), an indirubin-3'-oxime derivative, inhibits tumor growth by inducing apoptosis against non-small cell lung cancer in vitro and in vivo.

This study examined the anti-tumor effects of AGM130, a novel indirubin-3'-oxime derivative in A549 human non-small cell lung cancer cells. AGM130 significantly inhibited the proliferation and arrested the cell cycle of G2/M phase. Induction of apoptosis was detected in AGM130-treated A549 cells. The protein levels of Cytochrome c release, Bax, cleaved caspases and PARP were increased in AGM130 treated cells, whereas Bcl-2 levels were decreased. AGM130 inhibited Insulin-like growth factor 1 receptor (IGF1R), AKT/mTOR signaling and inactivated mitogen-activated protein kinases (MAPK). AGM130 also induced slight autophagy as pro-survival function and autophagy inhibition by chloroquine (CQ) induced necrosis. In vivo tumor xenograft model, AGM130 dose-dependently suppressed transplanted A549 cell tumor growth and induced the expression of proliferative cell nuclear antigen (PCNA). AGM130 also increased TUNEL positive apoptotic cell populations and the induction of glandular differentiation with mucin pool compared with vehicle-treated control in tumor tissue. These results suggest that AGM130 is an effective novel indirubin-3'-oxime derivative of anti-cancer drug and may be an attractive candidate for non-small cell lung cancer therapy.

Synthesized Pheophorbide a-mediated photodynamic therapy induced apoptosis and autophagy in human oral squamous carcinoma cells.

BACKGROUND: Pheophorbide a (Pa) is a chlorine-based photosensitizer derived from an ethnopharmacological herb, and our group recently synthesized Pa by the removal of a magnesium ion and a phytyl group from chlorophyll-a. In this study, the effect of photodynamic therapy (PDT) with synthesized Pa was examined in a human oral squamous cell carcinoma (OSCC) cells. METHODS: Cells were treated with PDT with Pa, and reactive oxygen species (ROS) and mitochondrial membrane potential [ΔΨ (m)] were examined. Apoptosis was measured using annexin V staining and immunoblot. Autophagy was characterized by the increase in LC3B-II and the formation of autophagosome and acidic vesicular organelles (AVOs). RESULTS: Pa-PDT inhibited the proliferation of OSCC cells in a dose-dependent manner. Pa-PDT increased the number of apoptotic cells by inactivating ERK pathway. Pa-PDT also induced autophagy in OSCC cells evidenced by the increased levels of LC3 type II expression and the accumulation of AVOs. The inhibition of autophagy enhanced Pa-PDT-mediated cytotoxicity through an increase in necrosis. CONCLUSIONS: These results suggest that synthesized Pa-PDT exerts anti-tumor effects by inducing apoptosis and autophagy and provide novel evidence that Pa-PDT induces autophagy, and autophagy inhibition enhances Pa-PDT-mediated necrosis in OSCC cells.

Toll-like receptor 7 agonist, imiquimod, inhibits oral squamous carcinoma cells through apoptosis and necrosis.

BACKGROUND: Toll-like receptor (TLR) agonists have anticancer effect by inducing apoptosis or activating immune cells. In this study, we investigated whether imiquimod, TLR7 agonist, inhibits the proliferation of oral cancer cells. METHODS: Toll-like receptor 7 expression and IL-6/8 production by imiquimod were examined using RT-PCR and Enzyme-linked immunosorbent assay, respectively. Cell viability was examined by MTT assay. To examine apoptotic cell death, Annexin V/PI staining for flow cytometry and Western blot analysis were performed. Necrotic cell death was determined by leakage of lactate dehydrogenase (LDH), HMGB1, and PI staining in imiquimod-treated oral squamous cell carcinoma (OSCC) cells. RESULTS: Toll-like receptor 7 mRNA was expressed in OSCC cells. Imiquimod induced IL-6 and IL-8 production in OSCC cells, suggesting the functional expression of TLR7. Imiquimod inhibited cells proliferation in a dose-dependent manner. The ratio of annexin V-positive cells and cleaved caspase-3/7 was increased by imiquimod treatment in OSCC cells, suggesting that imiquimod-induced cell death in OSCC cells may be owing to apoptosis. In addition, LDH secretion and PI staining were detected in OSCC cells treated with imiquimod, showing that imiquimod also induced necrotic cell death in the OSCC cells. CONCLUSIONS: Imiquimod inhibited effectively the growth of OSCC cells by inducing apoptosis and necrosis.

Pheophorbide a-mediated photodynamic therapy induces apoptotic cell death in murine oral squamous cell carcinoma in vitro and in vivo.

Photodynamic therapy (PDT) with several photosensitizers is a promising modality for the treatment of cancer. In this study, the therapeutic effect of PDT using the synthetic photosensitizer pheophorbide a (Pa-PDT) was examined in AT-84 murine oral squamous cell carcinoma (OSCC) cells. The MTT assay revealed that Pa-PDT induced cell growth inhibition in a dose- and time-dependent manner. Pa-PDT treatment significantly induced intracellular ROS generation, which is critical for cell death induced by Pa-PDT. Cell cycle analysis showed the increased sub-G1 proportion of cells in Pa-PDT-treated cells. Induction of apoptotic cell death was confirmed by DAPI staining and the reduction of mitochondrial membrane potential (ΔΨm) on Pa-PDT-treated cells. The changes in apoptosis-related molecules were next examined using western blotting. Cytochrome c release and cleavage of caspase-3 and PAPR were observed in AT-84 cells, whereas Bcl-2 protein levels were decreased. To determine the therapeutic effect of Pa-PDT in vivo, a murine OSCC animal model was used. Treatment of mice with Pa-PDT significantly inhibited tumor growth, especially PDT with Pa intravenous administration (i.v. Pa-PDT), and increased proliferative cell nuclear antigen (PCNA) levels and TUNEL-stained apoptotic cells compared to vehicle-treated controls. The data demonstrate that the in vitro effects of Pa-PDT on the inhibition of tumor cell proliferation and induction of apoptosis correlate to the anticancer activity of Pa-PDT in vivo. Our findings suggest the therapeutic potential of Pa-PDT in OSCC.

Homeobox C5 expression is associated with the progression of 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis.

BACKGROUND: Aberrant expression of homeobox genes (HOX), normally required for the differentiation of a particular tissue, has been reported in several types of cancer, but poorly addressed in oral squamous cell carcinoma (OSCC). The present study investigated the expression of HOXC5 in OSCC and identified molecular biomarker whose expression is associated with the multistep oral carcinogenesis. METHODS: The expression of HOXC5, proliferation cell nuclear antigen (PCNA), and Bcl-2 was examined by RT-PCR and Western blot analysis and confirmed by immunohistochemistry and transferase-mediated dUTP nick end-labeling (TUNEL) assay in a 4-nitroquinoline 1-oxide (4NQO)-induced rat tongue carcinogenesis model. RESULTS: Homeobox genes C5 was overexpressed in SCC tissues, but not in normal tissues by RT-PCR and Western blot analysis. Along with the progress of multistep carcinogenesis, the levels of HOXC5 expression of mRNA and protein significantly increased during the dysplasia (moderate to severe dysplasia) when compared with normal and hyperplasia. The levels of PCNA and Bcl-2 were sequentially increased from hyperplasia to dysplasia and SCC. By immunohistochemistry, HOXC5 expression was significantly increased in dysplasia, whereas PCNA expression was gradually increased during tongue carcinogenesis. TUNEL-positive cells were increased until dysplasia, but reduced in SCC. CONCLUSIONS: These results indicate that overexpression of HOXC5 is correlated with oral carcinogenesis and strongly contributed to the development of OSCC. HOXC5 may be a useful biomarker and has an emerging therapeutic target of OSCC.

Poly I:C inhibits cell proliferation and enhances the growth inhibitory effect of paclitaxel in oral sqaumous cell carcinoma.

OBJECTIVE: Toll-like receptors (TLR) signaling has dual effect of promoting tumor progression and anti-cancer property. This study was designed to determine the effect of polyinosinic-polycytidilic acid (poly I:C), a TLR3 agonist, on the proliferation of oral cancer cells. MATERIALS AND METHODS: Human oral squamous cell carcinoma cell lines, YD-10B and YD-8, were used. TLRs expression was examined by RT-PCR and IL-8 production by poly I:C was examined by ELISA. Cell proliferation was determined by MTT assay. Flow cytometry and Western blot analysis were performed to determine the molecular mechanism of poly I:C-induced cell death. RESULTS: TLR3 was functionally expressed in YD-10B and YD-8 cells. Treatment of poly I:C inhibited the cell growth in a dose-dependent manner. Flow cytometry and Western blot analysis revealed that poly I:C induced apoptosis via a mitochondria-dependent pathway. In addition, combination treatment with poly I:C and paclitaxel more significantly inhibited cell proliferation compared with poly I:C or paclitaxel alone. CONCLUSIONS: Poly I:C effectively inhibits oral cancer cell proliferation and can be considered as a candidate to improve the inhibitory effect of anti-cancer drugs.

The quadruple test for Down syndrome screening in pregnant women of advanced maternal age.

PURPOSE: The purpose of the current study was to determine whether or not the quadruple test for screening Down syndrome is an effective method to replace direct amniocentesis in pregnant women ≥ 35 years of age. METHODS: This study analyzed the screening performance of the quadruple test according to maternal age at delivery among subjects who had a quadruple screening test at 1 of 4 hospitals during a 5-year period and for whom data on fetal chromosomal abnormalities were available. RESULTS: The study population of 9,435 pregnant women was divided into 3 groups according to maternal age: 6,922 women were < 35 years of age; 2,284 were 35-39 years of age; and 229 women ≥ 40 years of age. The detection and false-positive rates of the quadruple screening test for Down or Edward syndrome in the 3 groups of women were 80 and 6.6%, 200 and 15.8%, and 100 and 35.3%, respectively. CONCLUSIONS: Under conditions in which first trimester screening test is not available, the quadruple screening test is a better choice than direct amniocentesis for pregnancies complicated by advanced maternal age. When providing genetic counseling, we need to explain the accurate detection and false-positive rates of the screening test according to maternal age.

Transforming growth factor ß1 promotes migration of human periodontal ligament cells through heat shock protein 27 phosphorylation.

Transforming growth factor ß1 (TGF-ß1) regulates cellular functions including cell growth, differentiation, and migration. However, signal transduction pathways of TGF-ß1 are mostly undefined in human periodontal ligament (hPDL) cells. In this study, we investigated TGF ß1-induced migration focusing on heat shock protein 27 (Hsp27) activation. The cellular response to TGF-ß1 by hPDL cells was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Western blot analysis. Cell migration was determined by counting migrated cells using a Chemotaxis Cell Migration Assay kit. TGF-ß1 induced cell migration and increased the phosphorylation of Hsp27 and p38 mitogen-activated protein kinase (MAPK) in hPDL cells. Inhibiting p38 MAPK with SB203580 blocked TGF-ß1-induced Hsp27 activation and cell migration. These findings suggest that TGF-ß1-induced Hsp27 may promote the migration of hPDL cells via p38 MAPK mediated signaling. Hsp27 may be a potential target for the periodontal regeneration process related to cell migration.

5'-Nitro-indirubinoxime, an indirubin derivative, suppresses metastatic ability of human head and neck cancer cells through the inhibition of Integrin ß1/FAK/Akt signaling.

Head and neck cancer is a malignant cancer and has the high infiltrative potential leading to metastasis. The objective of the study was to investigate the effects of 5'-nitro-indirubinoxime (5'-NIO), an indirubin derivative, on metastasis of head and neck cancer cells and to explore the underlying molecular mechanisms involved in this process. After treatment of head and neck cancer cells with 5'-NIO, cell metastatic behaviors such as colony formation, invasion, and migration were inhibited in a concentration-dependent manner. 5'-NIO inhibited the beta1 Integrin/FAK/Akt pathway which can then facilitate invasion and/or migration of cancer cells through the extracellular matrix (ECM). Moreover, treatment of head and neck cancer cell with Integrin ß1 siRNA or FAK inhibitor effectively inhibited the invasion and migration, suggesting their regulatory role at invasiveness and migratory of head and neck cancer cells. In vivo CAM assay, treatment with 5'-NIO reduced the angiogenesis in FaDu cells xenograft fertilized chicken eggs, primarily by inhibiting expression of VEGF. We conclude that 5'-NIO inhibits the metastatic ability of head and neck cancer cells by blocking the Integrin ß1/FAK/Akt pathway.

p-HPEA-EDA, a phenolic compound of virgin olive oil, activates AMP-activated protein kinase to inhibit carcinogenesis.

Phenolic constituents of virgin olive oil are reported to have antitumor activity. However, the underlying molecular mechanisms and specific target proteins of virgin olive oil remain to be elucidated. Here, we report that dialdehydic form of decarboxymethyl ligstroside aglycone (p-HPEA-EDA), a phenolic compound of virgin olive oil, inhibits tumor promoter-induced cell transformation in JB6 Cl41 cells and suppress cyclooxygenase-2 (COX-2) and tumorigenicity by adenosine monophosphate-activated protein kinase (AMPK) activation in HT-29 cells. p-HPEA-EDA inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced phosphorylation of extracellular signal-regulated kinases 1/2 and p90RSK in JB6 Cl41 cells, resulting in the inhibition of cell proliferation, activator protein-1 transactivation and cell transformation promoted by TPA. Moreover, p-HPEA-EDA strongly inhibited the cell viability and COX-2 expression by activation of AMPK activity in HT-29 cells, resulted from depletion of intracellular adenosine triphosphate. p-HPEA-EDA-induced activation of caspase-3 and poly-adenosine diphosphate-ribose polymerase, phosphorylation of p53 (Ser15) and DNA fragmentation in HT-29 cells, leading to apoptosis. Importantly, p-HPEA-EDA suppressed the colony formation of HT-29 cells in soft agar. In contrast, Compound C, an AMPK inhibitor, and Z-DEVD-FMK, a caspase-3 inhibitor, blocked the p-HPEA-EDA-inhibited colony formation in HT-29 cells. In vivo chorioallantoic membrane assay also showed that p-HPEA-EDA-inhibited tumorigenicity of HT-29 cells. These findings revealed that targeted activation of AMPK and inhibition of COX-2 expression by p-HPEA-EDA contribute to the chemopreventive and chemotherapeutic potential of virgin olive oil against colon cancer cells.

Lipopolysaccharide promotes adhesion and migration of murine dental papilla-derived MDPC-23 cells via TLR4.

Odontoblasts and/or dental pulp cells are responsible for tooth repair and dentin formation. Furthermore, adhesion and migration are critical processes for tissue regeneration. This study was performed to clarify whether lipopolysaccharide (LPS) modulates adhesion and migration of the murine odontoblast-like cell line MDPC-23, and whether Toll-like receptor 4 (TLR4) signaling is engaged in this process. TLR4 expression in MDPC-23 cells was examined by RT-PCR. Adhesion assay was performed using type I collagen-coated plates. Migration ability was determined by a commercial assay kit. Phosphorylation of IκB-α, FAK, AKT, and ERK was examined by Western blot analysis. TLR4 was functionally expressed in MDPC-23 cells. LPS treatment enhanced adhesion and migration of MDPC-23 cells in a dose-dependent manner. Blockade of TLR4 using its antibody restored LPS-induced adhesion and migration of MDPC-23 cells. These findings indicate that LPS, an immune activator from Gram-negative bacteria, can promote the adhesion and migration ability of MDPC-23 cells via TLR4.

Amurensin G, a potent natural SIRT1 inhibitor, rescues doxorubicin responsiveness via down-regulation of multidrug resistance 1.

The transition from a chemotherapy-responsive cancer to a chemotherapy-resistant one is accompanied by increased expression of multidrug resistance 1 (MDR1, p-glycoprotein), which plays an important role in the efflux from the target cell of many anticancer agents. We recently showed that a Forkhead box-containing protein of the O subfamily 1 (FoxO1) is a key regulator of MDR1 gene transcription. Because nuclear localization of FoxO1 is regulated by silent information regulator two ortholog 1 (SIRT1) deacetylase, we wondered whether SIRT1 dominates MDR1 gene expression in breast cancer cells. Overexpression of SIRT1 enhanced both FoxO reporter activity and nuclear levels of FoxO1. Protein expression of MDR1 and gene transcriptional activity were also up-regulated by SIRT1 overexpression. In addition, SIRT1 inhibition reduced both nuclear FoxO1 levels and MDR1 expression in doxorubicin-resistant breast cancer cells (MCF-7/ADR) cells. A potent SIRT1 inhibitor, amurensin G (from Vitis amurensis), was identified by screening plant extracts and bioassay-guided fractionation. The compound suppressed FoxO1 activity and MDR1 expression in MCF-7/ADR cells. Moreover, pretreatment of MCF-7/ADR cells with 1 µg/ml amurensin G for 24 h increased cellular uptake of doxorubicin and restored the responsiveness of MCF-7/ADR cells to doxorubicin. In xenograft studies, injection of 10 mg/kg i.p. amurensin G substantially restored the ability of doxorubicin to inhibit MCF-7/ADR-induced tumor growth. These results suggest that SIRT1 is a potential therapeutic target of MDR1-mediated chemoresistance and that it may be possible to develop amurensin G as a useful agent for chemoresistance reversal.

Transforming growth factor beta1-induced heat shock protein 27 activation promotes migration of mouse dental papilla-derived MDPC-23 cells.

INTRODUCTION: Transforming growth factor beta1 (TGFbeta1) regulates cellular functions including cell growth, differentiation, angiogenesis, migration, and metastasis. The TGFbeta1 signal transduction pathways are mostly undefined in mouse dental papilla-derived MDPC-23 cells. In this study, we investigated TGFbeta1-induced migration focusing on heat shock protein 27 (Hsp27) activation. METHODS: Cellular responses mediated by TGFbeta1 in MDPC-23 cells were measured by Western blot and MTT assays. Cell migration was determined by counting migrated cells using the chemotaxis cell migration assay. RESULTS: TGFbeta1 induced cell migration and increased the phosphorylation of Hsp27 and p38 MAPK in MDPC-23 cells. However, TGFbeta1 did not affect Akt/NF-kappaB signaling to regulate the migration of MDPC-23 cells. Inhibiting p38 MAPK with SB203580 blocked TGFbeta1-induced Hsp27 activation and cell migration. CONCLUSION: Hsp27 phosphorylation followed by p38 MAPK activation was required for TGFbeta1-induced migration, and Hsp27 itself contributed to MDPC-23 cell migration.

5,5'-substituted indirubin-3'-oxime derivatives as potent cyclin-dependent kinase inhibitors with anticancer activity.

To enhance the ability of indirubin derivatives to inhibit CDK2/cyclin E, a target of anticancer agents, we designed and synthesized a new series of indirubin-3'-oxime derivatives with combined substitutions at the 5 and 5' positions. A molecular docking study predicted the binding of derivatives with OH or halogen substitutions at the 5' position to the ATP binding site of CDK2, revealing the critical interactions that may explain the improved CDK2 inhibitory activity of these derivatives. Among the synthesized derivatives, the 5-nitro-5'-hydroxy analogue 3a and the 5-nitro-5'-fluoro analogue 5a displayed potent inhibitory activity against CDK2, with IC(50) values of 1.9 and 1.7 nM, respectively. These derivatives also showed antiproliferative activity against several human cancer cell lines, with IC(50) values of 0.2-3.3 microM. A representative analogue, 3a, showed greater than 500-fold selectivity for CDK relative to selected kinase panel and potent in vivo anticancer activity.

The antitumor effect of PLK1 and HSF1 double knockdown on human oral carcinoma cells.

High levels of mitotic progression-associated PLK1 and stress-associated HSF1 have been observed in various human cancers. In the present study, we investigated the effects of PLK1 and HSF1 knockdown on the proliferation of oral cancer cells using small interfering RNA. In human oral squamous cell carcinoma (SCC) tissues, the levels of PLK1 and HSF1 were higher compared to normal tissues. The expression levels of PLK1 and HSF1 were also elevated in the human oral SCC cell lines FaDu and HEp-2. Disruption of PLK1 induced cell cycle arrest at G2/M phase as well as apoptosis in oral cancer cells. Interestingly, knockdown of both PLK1 and HSF1 expression decreased cell proliferation while increasing apoptotic cell death in synergistic fashion. These results establish the potential value of PLK1 and HSF1 as targets for oral cancer therapy.

5'-Nitro-indirubinoxime induces G1 cell cycle arrest and apoptosis in salivary gland adenocarcinoma cells through the inhibition of Notch-1 signaling.

BACKGROUND: 5'-Nitro-indirubinoxime (5'-NIO) is a new derivative of indirubin that exhibits anti-cancer activity in a variety of human cancer cells. However, its mechanism has not been fully clarified. METHODS: Human salivary gland adenocarcinoma (SGT) cells were used in this study. Western blot and RT-PCR analyses were performed to determine cellular Notch levels. The cell cycle stage and level of apoptosis were analyzed using flow cytometry analysis. RESULTS: 5'-NIO significantly inhibited the mRNA levels of Notch-1 and Notch-3 and their ligands (Delta1, 2, 3, and Jagged-2) in SGT cells. Immunocytochemistry analysis showed that 5'-NIO specifically decreased the level of Notch-1 in the nucleus. In addition, 5'-NIO induced G1 cell cycle arrest by reducing levels of CDK4 and CDK6 in SGT cells. Using flow cytometry and immunoblotting analysis, we found that 5'-NIO induces apoptosis following the secretion of cytochrome c and the activation of caspase-3 and caspase-7. Intracellular Notch-1 overexpression led to a decrease in G1 phase arrest and an inhibition of 5'-NIO-induced apoptosis. CONCLUSION: These observations suggest that 5'-NIO induces cell cycle arrest and apoptosis by down-regulating Notch-1 signaling. GENERAL SIGNIFICANCE: This study identifies a new mechanism of 5'-NIO-mediated anti-tumor properties. Thus, 5'-NIO could be used as a candidate for salivary gland adenocarcinoma therapeutics.

Efficient preparation of highly pure chlorin e6 and its photodynamic anti-cancer activity in a rat tumor model.

Photodynamic therapy (PDT) is currently being used as an alternative therapeutic modality for a variety of malignant tumors. This study was performed to show an efficient preparation of second generation of photosensitizer chlorin e6 (Ce6) with high yield and purity, and to test antitumor activity of Ce6-induced PDT (Ce6-PDT) both in vitro and in vivo using a rat tumor model. Three-week-old male Sprague-Dawley (SD) rats were inoculated s.c. on the right flank with 5x10(6) RK3E-ras cells. The animals were administered i.v. with Ce6 (10 mg/kg) and 24 h later, PDT was performed using a laser diode at a light dose of 100 J/cm2. Ce6-PDT generated reactive oxygen species and led to significant growth inhibition in RK3E-ras cell. In addition, Ce6-PDT induced apoptosis through the activation of caspase-3 and its downstream target, PARP cleavage. The protein level of anti-apoptotic bcl-2 was also reduced by Ce6-PDT in RK3E-ras cells. In in vivo experiments, application of Ce6-PDT led to a significant reduction of tumor size. PCNA immunostaining and TUNEL assay revealed that Ce6-PDT inhibited tumor cell proliferation and increased apoptosis. These findings suggest that the newly purified Ce6-PDT can effectively arrest tumor growth by inhibiting cell proliferation and inducing apoptosis.