Differential Anti-Tumor Effects of IFN-Inducible Chemokines CXCL9, CXCL10, and CXCL11 on a Mouse Squamous Cell Carcinoma Cell Line.

Chemokines are a group of cytokines involved in the mobilization of leukocytes, which play a role in host defense and a variety of pathological conditions, including cancer. Interferon (IFN)-inducible chemokines C-X-C motif ligand 9 (CXCL), CXCL10, and CXCL11 are anti-tumor chemokines; however, the differential anti-tumor effects of IFN-inducible chemokines are not completely understood. In this study, we investigated the anti-tumor effects of IFN-inducible chemokines by transferring chemokine expression vectors into a mouse squamous cell carcinoma cell line, SCCVII, to generate a cell line stably expressing chemokines and transplanted it into nude mice. The results showed that CXCL9- and CXCL11-expressing cells markedly inhibited tumor growth, whereas CXCL10-expressing cells did not inhibit growth. The NH2-terminal amino acid sequence of mouse CXCL10 contains a cleavage sequence by dipeptidyl peptidase 4 (DPP4), an enzyme that cleaves the peptide chain of chemokines. IHC staining indicated DPP4 expression in the stromal tissue, suggesting CXCL10 inactivation. These results suggest that the anti-tumor effects of IFN-inducible chemokines are affected by the expression of chemokine-cleaving enzymes in tumor tissues.

Study protocol of brief intervention using gene polymorphism information for excessive drinking among Japanese college students and adults aged 20-30 years: a randomized controlled trial.

BACKGROUND: The alcohol-metabolizing enzyme aldehyde dehydrogenase 2 (ALDH2) is a carcinogenic acetaldehyde-degrading enzyme, and its low activity is a genetic constitution peculiar to East Asians. People with low alcohol dehydrogenase 1B activity (ADH1B*1/*1 genotype) have a high risk of developing head and neck cancer and alcoholism. The study aims to evaluate the effectiveness of brief interventions for excessive drinking among college students and adults in their 20s, including information on five constitutions that combine the ALDH2 and ADH1B genotypes. METHODS: Participants comprised university students and staff aged 20-30 years who had consumed ≥40 g (males) or ≥20 g (females) of pure alcohol; they were classified into intervention and control groups using a simple randomization method. Participants anonymously filled out questionnaires linked to identification numbers and recorded the drinking days and amounts on the drinking calendar. The intervention group will then be tested for genotype testing using saliva (5 types of combinations of ALDH2 and ADH1B enzyme activities); the result report will arrive approximately 1 month later. We will conduct a 30-min face-to-face or online intervention. The control group will be merely given the conventional materials, and genetic testing will be performed voluntarily after 6 months (end of study). The intervention group will undergo questionnaire surveys 1 month after the intervention and 3 and 6 months after baseline. Questionnaire surveys will be conducted 1, 3, and 6 months after baseline for the control group. The average amount of drinking before and after the intervention, attribute/baseline data between the two groups, and time-series data were compared using various analysis tools. For interventions, we engaged in dialog based on intervention materials that added genotyping content to the existing materials, result reports, baseline data, and drinking calendar records. Participants' ingenuity is respected to support their drinking behavior and goal setting. DISCUSSION: Individual information on the genetic makeup of alcohol-metabolizing enzymes provided during the intervention is more personal and objective than general health information, especially in Japan, where the ALDH2 low activity rate is high. This information may be useful for health care and precautionary measures. TRIAL REGISTRATION: R000050379, UMIN000044148, Registered on June 1, 2021. Scientific Title: Examination of simple intervention using genetic polymorphism information for excessive drinking.

Enhanced Cytotoxic Effects in Human Oral Squamous Cell Carcinoma Cells Treated with Combined Methyltransferase Inhibitors and Histone Deacetylase Inhibitors.

Combined treatment of human oral squamous cell carcinoma (OSCCs) with DNA methyltransferase inhibitors (DNMTis), histone methyltransferase inhibitors (HMTis), and histone deacetylase inhibitors (HDACis), and the molecular mechanisms underlying their anticancer effects, have not been fully elucidated. Herein, we investigated the cytotoxic effects of combined DNMTis (5-Aza-deoxycytidine: 5-Aza-dC, RG108), HMTis (3-deazaneplanocin A: DZNep), and HDACis (trichostatin A: TSA) treatment on human OSCC cells and explored their molecular mechanisms. Combined 5-Aza-dC, or RG108, and TSA treatment significantly decreased HSC-2 and Ca9-22 cell viability. Combinatorial DZNep and TSA treatment also decreased Ca9-22 cell viability. Although caspase 3/7 activation was not observed in HSC-2 cells following combined treatment, caspase activity was significantly increased in Ca9-22 cells treated with DZNep and TSA. Moreover, combined treatment with 5-Aza-dC, RG108, and TSA increased the proportion of HSC-2 and Ca9-22 cells in the S and G2/M phases. Meanwhile, increased phosphorylation of the histone variant H2A.X, a marker of double-stranded DNA breaks, was observed in both cells after combination treatment. Hence, the decreased viability induced by combined treatment with epigenomic inhibitors results from apoptosis and cell cycle arrest in S and G2/M phases. Thus, epigenomic therapy comprising combined low concentrations of DNMTi, HMTi, and HDACi is effective against OSCC.

Simultaneous Expression of Th1- and Treg-Associated Chemokine Genes and CD4+, CD8+, and Foxp3+ Cells in the Premalignant Lesions of 4NQO-Induced Mouse Tongue Tumorigenesis.

Chemokines and cytokines in the tumor microenvironment influence immune cell infiltration and activation. To elucidate their role in immune cell recruitment during oral cancer development, we generated a mouse tongue cancer model using the carcinogen 4-nitroquinoline 1-oxide (4NQO) and investigated the carcinogenetic process and chemokine/cytokine gene expression kinetics in the mouse tongue. C57/BL6 mice were administered 4NQO in drinking water, after which tongues were dissected at 16 and 28 weeks and subjected to analysis using the RT2 Profiler PCR Array, qRT-PCR, and pathologic and immunohistochemical analyses. We found that Th1-associated chemokine/cytokine (Cxcl9, Cxcl10, Ccl5, and Ifng) and Treg-associated chemokine/cytokine (Ccl17, Ccl22, and Il10) mRNA levels were simultaneously increased in premalignant lesions of 4NQO-treated mice at 16 weeks. Additionally, although levels of Gata3, a Th2 marker, were not upregulated, those of Cxcr3, Ccr4, and Foxp3 were upregulated in the tongue tissue. Furthermore, immunohistochemical analysis confirmed the infiltration of CD4+, CD8+, and Foxp3+ cells in the tongue tissue of 4NQO-treated mice, as well as significant correlations between Th1- or Treg-associated chemokine/cytokine mRNA expression and T cell infiltration. These results indicate that CD4+, CD8+, and Foxp3+ cells were simultaneously recruited through the expression of Th1- and Treg-associated chemokines in premalignant lesions of 4NQO-induced mouse tongue tissue.

Recent Progress of Basic Studies of Natural Products and Their Dental Application.

The present article reviews the research progress of three major polyphenols (tannins, flavonoids and lignin carbohydrate complexes), chromone (backbone structure of flavonoids) and herbal extracts. Chemical modified chromone derivatives showed highly specific toxicity against human oral squamous cell carcinoma cell lines, with much lower toxicity against human oral keratinocytes, as compared with various anticancer drugs. QSAR analysis suggests the possible correlation between their tumor-specificity and three-dimensional molecular shape. Condensed tannins in the tea extracts inactivated the glucosyltransferase enzymes, involved in the biofilm formation. Lignin-carbohydrate complexes (prepared by alkaline extraction and acid-precipitation) and crude alkaline extract of the leaves of Sasa species (SE, available as an over-the-counter drug) showed much higher anti-HIV activity, than tannins, flavonoids and Japanese traditional medicine (Kampo). Long-term treatment with SE and several Kampo medicines showed an anti-inflammatory and anti-oxidant effects in small size of clinical trials. Although the anti-periodontitis activity of synthetic angiotensin II blockers has been suggested in many papers, natural angiotensin II blockers has not yet been tested for their possible anti-periodontitis activity. There should be still many unknown substances that are useful for treating the oral diseases in the natural kingdom.

Tumor-associated macrophages in oral premalignant lesions coexpress CD163 and STAT1 in a Th1-dominated microenvironment.

BACKGROUND: Tumor-associated macrophages (TAMs) are implicated in the growth, invasion and metastasis of various solid tumors. However, the phenotype of TAMs in premalignant lesions of solid tumors has not been clarified. In the present study, we identify the phenotype of TAMs in leukoplakia, an oral premalignant lesion, by immunohistochemical analysis and investigate the involvement of infiltrated T cells that participate in the polarization of TAMs. METHODS: The subjects included 30 patients with oral leukoplakia and 10 individuals with normal mucosa. Hematoxylin and eosin slides were examined for the histological grades, and immunohistochemical analysis was carried out using antibodies against CD68 (pan-MΦ), CD80 (M1 MΦ), CD163 (M2 MΦ), CD4 (helper T cells: Th), CD8 (cytotoxic T cells), CXCR3, CCR5 (Th1), CCR4 (Th2), signal transducer and activator of transcription (STAT1), phosphorylated STAT1 (pSTAT1) and chemokine CXCL9. The differences in the numbers of positively stained cells among the different histological grades were tested for statistical significance using the Kruskal-Wallis test. Correlations between different types of immune cells were determined using Spearman's rank analysis. RESULTS: An increase in the rate of CD163(+) TAM infiltration was observed in mild and moderate epithelial dysplasia, which positively correlated with the rate of intraepithelial CD4(+) Th cell infiltration. Although CCR4(+) cells rarely infiltrated, CXCR3(+) and CCR5(+) cells were observed in these lesions. Cells positive for STAT1 and chemokine CXCL9, interferon- (IFN)-induced gene products, and pSTAT1 were also observed in the same lesions. Double immunofluorescence staining demonstrated that the cells that were positive for CD163 were also positive for STAT1. CONCLUSIONS: CD163(+) TAMs in oral premalignant lesions coexpress CD163 and STAT1, suggesting that the TAMs in oral premalignant lesions possess an M1 phenotype in a Th1-dominated micromilieu.

Infiltration of m2 tumor-associated macrophages in oral squamous cell carcinoma correlates with tumor malignancy.

Tumor-associated macrophages (TAMs) are a major cellular component in the tumor microenvironment of many solid tumors. The functional competence of TAMs varies depending on the type of tumors and their respective microenvironments. The classically activated M1 macrophages exhibit antitumor functions, whereas the alternatively activated M2 macrophages exhibit protumor functions that contribute to tumor development and progression. Although TAMs have been detected in oral squamous cell carcinoma (OSCC), little is known about their phenotype. In the present study, we performed an immunohistochemical analysis to identify TAMs in surgically resected specimens from 50 patients with OSCC and evaluated the relationship between infiltrated TAMs and the pathological grade of OSCC. Positive staining for CD163, which has been used as a marker for M2 macrophages, was observed in OSCC specimens, and the percentages of CD163+ cells were significantly increased based on the pathological grade. CD163+ cells were detected in the tumor stroma in grade I tumors, whereas an increase in the CD163+ cells in the tumor nest was observed in higher grades of tumors. Although infiltrated CD4+ and CD8+ T cells were detected in all pathological grades of OSCC, no correlation between the infiltrated T cells and the CD163+ TAMs was observed. These results indicate that the infiltrated TAMs in OSCC have an M2 phenotype and that the M2 macrophages may participate in the development of OSCC.

Identification of a structural motif in the tumor-suppressive protein GRIM-19 required for its antitumor activity.

We have previously isolated GRIM-19, a novel growth suppressor, using a genetic method. GRIM-19 ablates cell growth by inhibiting the transcription factor signal transducer and activator of transcription 3 (STAT3). Up-regulation of STAT3 and growth promotion were observed in a number of human tumors. Although the tumor-suppressive actions of GRIM-19 are known, the structural elements required for its antitumor actions are not understood. Mutational and protein sequence analyses identified a motif in the N terminus of GRIM-19 that exhibited similarity to certain RNA viral proteins. We show that disruption of specific amino acids within this motif cripples the antitumor actions of GRIM-19. These mutants fail to interact with STAT3 efficiently and consequently do not inhibit growth-promoting gene expression. More importantly, we show that a clinically observed mutation in the N terminus of GRIM-19 also weakened its interaction with STAT3 and antitumor action. Together, these studies identify a major role for the N terminus of GRIM-19 in mediating its tumor-suppressive actions.

STAT1 represses hypoxia-inducible factor-1-mediated transcription.

Tumor hypoxia is associated with tumor promotion, malignant progression, and resistance to cancer therapy. The hypoxia-induced phenotypic changes in tumors result, at least partially, from the induction of hypoxia-responsive genes, such as chemokine receptor CXCR4. Hypoxia-inducible factor 1 (HIF-1) is a critical transcription factor involved in the transcriptional regulation of these genes. Although interferon-gamma (IFNgamma) exerts anti-tumor responses against various tumors, the effect of IFNgamma on HIF-1-dependent transcription remains to be determined. In this study, we examined the inhibitory effect of IFNgamma on hypoxia-induced CXCR4 gene expression in human glioblastoma cell lines and explored the mechanism of inhibition. Hypoxia (1% O(2)) and the iron chelator deferoxamine (DFX), a hypoxia mimetic, increased the levels of CXCR4 mRNA in A172 and T98G cells, and treatment with IFNgamma inhibited the expression of CXCR4 mRNA. Although hypoxia and DFX induced the expression of HIF-1alpha protein and its hypoxia response element (HRE) DNA-binding activity, IFNgamma failed to inhibit its expression or DNA-binding activity. The results of a luciferase reporter assay using a heterologous promoter construct containing the HRE sequence revealed that IFNgamma suppressed the hypoxia- and DFX-induced reporter activities. Lentivirus-mediated RNAi of signal transducer and activator of transcription 1 (STAT1) expression abolished the inhibitory effect of IFNgamma on hypoxia-induced reporter gene activity. Furthermore, this activity was not detected in a stable cell line expressing dominant-negative STAT1. These results indicate that IFNgamma-activated STAT1 functions as a negative regulator of HIF-1-dependent transcription in tumor cells.

Mechanisms of resistance to interferon-gamma-mediated cell growth arrest in human oral squamous carcinoma cells.

Interferon-gamma (IFNgamma) has an antiproliferative effect on a variety of tumor cells. However, many tumor cells resist treatment with IFNs. Here, we show that IFNgamma fails to inhibit the growth of some types of oral squamous cell carcinoma (OSCC) cells that possess a fully functional IFNgamma/STAT1 (signal transducer and activator of transcription-1) signaling pathway. IFNgamma inhibited the growth of the HSC-2, HSC-3, and HSC-4 OSCC cell lines. However, Ca9-22 cells were resistant to IFNgamma despite having intact STAT1-dependent signaling, such as normal tyrosine phosphorylation, DNA binding activity, and transcriptional activity of STAT1. The growth inhibition of HSC-2 cells resulted from S-phase arrest of the cell cycle. IFNgamma inhibited cyclin A2 (CcnA2)-associated kinase activity, which correlated with the IFNgamma-mediated down-regulation of CcnA2 and Cdk2 expression at both the transcriptional and post-transcriptional level in HSC-2 cells but not in Ca9-22 cells. RNAi-mediated knockdown of CcnA2 and Cdk2 resulted in growth inhibition in both cell lines. These results indicate that the resistance of OSCC to IFNgamma is not due simply to the deficiency in STAT1-dependent signaling but results from a defect in the signaling component that mediates this IFNgamma-induced down-regulation of CcnA2 and Cdk2 expression at the transcriptional and post-transcriptional levels.

Atherosclerotic plaque characterization by 0.5-mm-slice multislice computed tomographic imaging.

BACKGROUND: It has been proposed that 0.5-mm-slice multislice computed tomography (MSCT) is a noninvasive tool for the detection of atherosclerotic plaque, but the validity of such an assessment has not been demonstrated by an invasive investigation. The present study was performed to compare the 0.5-mm-slice MSCT density of plaques with intravascular ultrasound (IVUS) findings. METHODS AND RESULTS: Atherosclerotic plaques were characterized in 37 consecutive patients undergoing percutaneous interventions. Based on the IVUS echogenecity, the plaques were classified as soft (n=18), fibrous (n=40) or calcified (n=40). In these 98 plaques, 0.5-mm-slice MSCT plaque density was calculated in 443 regions-of-interest, including 331 lesional foci and 112 luminal cross-sections, and represented as Hounsfield units (HU). MSCT density of the 3 types of plaque was 11+/-12 HU, 78+/-21 HU, and 516 +/-198 HU respectively. Computed tomography density of the (contrast-filled) lumen was 258+/-43 HU. There were statistically highly significant differences in the densitometric characteristics among the 4 groups (soft, fibrous, calcified plaque and lumen) by nonparametric Kruskal-Wallis test (p<0.0001). CONCLUSIONS: The IVUS-based coronary plaque configuration can be accurately identified by 0.5-mm slice MSCT. Noninvasive assessment of plaque characterization will ensure emphasis on the vessel wall beyond the vascular lumen.

Cytotoxic activity of selected trifluoromethyl ketones against oral tumor cells.

Several trifluoromethyl ketones (TF1-4) and related non-fluorinated ketones (TF5 and 6) were tested for their relative cytotoxicity on four human tumor cell lines (oral squamous cell carcinoma HSC-2, HSC-3, HSC-4 and promyelocytic leukemia HL-60) and three normal human cells [gingival fibroblasts (HGF), pulp cells (HPC) and periodontal ligament fibroblasts (HPLF)]. Trifluoromethylated a-diketone (TF1, CF3COCOPh) and alpha-hydroxy ketones (TF2, CF3CH(OH)COPh; TF3, CF3CH(OH)COCH2Ph) showed higher tumor-specific cytotoxic activity than the corresponding non-fluorinated analogs (TF5, CH3COCOPh; TF6, CH3CH(OH)COPh), while the anti-tumor potency of trifluoromethyl ketone (TF4, CF3COCH2Ph) was lower. Among four tumor cell lines, HL-60 cells were the most sensitive to TF1-4, followed by HSC-2 and HSC-3 cells. HSC-4 cells were the most resistant in most cases. Agarose gel electrophoresis showed that TF1-3 did not induce intemucleosomal DNA fragmentation nor activated caspase-3. The cytotoxic activities of TF1-3 were not significantly affected by FeCl3. Electron microscopy of TF2- or 3-treated HL-60 cells showed the development of autophagosomes in HL-60 cells, without the production of an apoptotic body, or affecting the mitochondria and cell surface microvilli. The autophagy inhibitor, 3-methyladenine (3-MA), partially inhibited the TF2- or 3-induced cytotoxicity. These data suggest the induction of non-apoptotic cell death by TF2 or 3.