Bioinformatics analysis of the microRNAs and target genes of microRNAs in salivary adenoid cystic carcinoma / 解剖学报

Objective To identify potential microRNAs (miRNAs) in salivary adenoid cystic carcinoma and to construct a miRNA-mRNA regulatory network to better understand its potential molecular mechanisms. Methods Two microarray datasets of SACC were downloaded from the database Gene Expression Omnibus (GEO), and the differentially expressed miRNAs and mRNA were analyzed by the R language. FunRich 3. 1. 3 software was used to enrich and analyze the transcription factors of differential miRNAs and to predict the target genes of differentially expressed miRNAs. The target genes of differential miRNAs in SACC were utilized to perform Gene Onotology (GO) and Kyoto Encyclopedia of Gene Genomes (KEGG) pathway enrichment analyses, and protein-protein interaction. The miRNA-mRNA regulatory network was constructed in Cytoscape 3.7.0. Results A total of 144 differentially expressed miRNA (DEMs) and 1216 differentially expressed mRNA (DEGs) were screened. The enrichment analysis of KEGG signaling pathway revealed that target genes were mainly involved in the regulation of Rapi signaling pathway, mitogen active protein kinase (MAPK) signaling pathway, phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt) signaling pathway, and regulation of actin cytoskeleton. STRING protein interaction analysis shows that ACSL1, SCD, MGLL, FABP4 may be the key proteins in the protein interaction network. Conclusion Differentially expressed miRNA and mRNA between SACC tissues and normal tissues were screened out and the signaling pathways and functions of these differential molecules were found in our research.

Study on lncRNA ADAMTS9-AS2 promoting invasion and metastasis of salivary adenoid cystic carcinoma / 口腔疾病防治

Objective@#To investigate the role of lncRNAs in the invasion and metastasis of salivary adenoid cystic carcinoma (SACC) cells.@*Methods@#With SACC-LM as the experimental group and SACC83 as the control group, lncRNA chips were used to screen the differentially expressed lncRNAs. The differentially expressed lncRNAs were further verified by real-time quantitative RT-PCR (qRT-PCR). The invasion and migration abilities of the adenoid cystic carcinoma cell lines before and after transfection with lncRNA siRNAs were detected by invasion and migration experiments. The clinicopathological features and prognosis of patients with different expression of lncRNAs and SACC were analyzed.@*Results@#The microarray showed that ADAMTS9-AS2 was highly expressed in the SACC-LM cells. Real-time quantitative RT-PCR further confirmed that ADAMTS9-AS2 was significantly upregulated in the SACC-LM cells. Invasion and migration experiments showed that the invasion and migration were significantly reduced after the expression level of ADAMTS9-AS2 was downregulated (P ＜ 0.001). Analysis of the clinicopathological data showed that ADAMTS9-AS2 was highly expressed in SACC. High expression of ADAMTS9-AS2 was associated with poor prognosis and a high tumor metastasis rate in SACC patients.@*Conclusion @#High expression of ADAMTS9-AS2 promotes the migration and invasion of SACC cells. ADAMTS9-AS2 is upregulated in the SACC tissues and is related to a high metastasis rate and poor prognosis.

Exosomes from salivary adenoid cystic carcinoma promote the expression of PD-L1 in fibroblasts / 中国肿瘤生物治疗杂志

@# Objective: To investigate the effect of salivary adenoid cystic carcinoma (SACC) derived exosomes on PD-L1 expression in fibroblasts. Methods: Exosomes of SACC-83 cell line were extracted by exosome isolation kit, and its particle size, density and phenotypes were identified by electron microscope.After being labeled with PKH67 fluorescence, exosomes were co-incubated with HPLF to observe whether the exosomes could be ingested by fibroblasts under confocal microscope. After co-incubation with the exosomes, the differentially expressed genes (DEGs) in HPLF cells were detected by whole transcriptome sequencing. GO analysis together with KEGG enrichment analysis was used to clarify the biological functions and related signaling pathways related with the DEGs. qPCR, Western blotting and Flow cytometry were used to detect the effect of tumor exosomes on the mRNAand protein expressions of PD-L1, LAG3 and IDO1 in HPLF fibroblasts. Results: SACC exosomes specifically expressed CD63, CD81 and TSG101 molecules, and could be ingested by fibroblasts. After the treatment of fibroblasts by exosomes, the expression of PD-L1 molecule was significantly up-regulated (Fold change=10.19), and the DEGs were significantly enriched in the immune response signaling pathways, such as TNF, NF-κB and cGAS-String pathway, etc. In vitro experiments showed that exosomes could significantly promote the expression of PD-L1 in HPLF cells at both mRNA and protein levels (24.7±4.75 vs 1.03±0.11,P<0.05). Conclusion: Exosomes of SACC can promote the expression of immunocheckpoint ligand PD-L1 in fibroblasts.

Treatment plan and prognosis of salivary adenoid cystic carcinoma with lung metastasis / 华西口腔医学杂志

Salivary adenoid cystic carcinoma (SACC) is a common malignant tumor in the oral and maxillofacial region and accounts for approximately 3%-5% of all head and neck carcinomas. SACC always occurs in the palatal salivary gland and parotid gland. The tumor has the characteristics of strong invasion, perineural invasion, high hematogenous metastasis, and low lymph node metastasis rate. The biological characteristics of SACC determine the specificity of clinical treatment. Thus far, few clinical trials have investigated the efficacy of systemic therapy owing to the rarity of SACC with lung metastasis. Moreover, long-term results are poor, and no consensus on standard treatment has been reached yet. This systematic review aims to provide a retrospective analysis of treatment options and prognosis for SACC with lung metastasis and evidence for future clinical treatment.

Oral microbiological diversity in patients with salivary adenoid cystic carcinoma / 华西口腔医学杂志

OBJECTIVE@#The aim of this study was to identify the differences in microbial diversity and community in patients with salivary adenoid cystic carcinoma (SACC).@*METHODS@#Saliva was collected from 13 patients with SACC confirmed by histopathological diagnosis and 10 healthy control subjects. Total metagenomic DNA was extracted. The DNA amplicons of the V3-V4 hypervariable regions of the 16S rRNA gene were generated and subjected to high-throughput sequencing. Microbial diversity and community structure were analyzed with Mothur software.@*RESULTS@#A total of 16 genera of dominant bacteria in the SACC group were found, including Streptococcus (36.68%), Neisseria (8.55%), Prevotella_7 (7.53%), and Veillonella (6.37%), whereas 15 dominant bacteria in the control group were found, including Streptococcus (18.41%), Neisseria (18.20%), Prevotella_7 (8.89%), Porphyromonas (6.20%), Fusobacterium (5.86%) and Veillonella (5.82%). The statistically different phyla between the two groups were Firmicutes, Proteobacteria and Fusobacterium (P<0.05). The statistically different genera between the two groups were Streptococcus, Neisseria and Porphyromonas (P<0.05), and Capnocytophaga was only detected in patients with SACC.@*CONCLUSIONS@#Significant differences were observed in the oral microorganisms between the two groups.

Inhibitory effect and underlying mechanism of total saponins from Paris polyphylla var. yunnanensis on the proliferation of salivary adenoid cystic carcinoma ACC-83 cells / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the inhibitory effect and underlying mechanism of total saponins from Paris polyphylla var. yunnanensis on the proliferation of salivary adenoid cystic carcinoma ACC-83 cells.</p><p><b>METHODS</b>In vitro cell culture was performed. The proliferation of ACC-83 cells treated with different concentrations (5, 10, 20, 40, 60, 80, 100 μg·mL⁻¹) of total saponins from Paris polyphylla var. yunnanensis was observed using CCK-8 assay. Meanwhile, the apoptosis of ACC-83 cells treated with different concentrations (25, 50, 100 μg·mL⁻¹) of the total saponins was observed using flow cytometry. The expression levels of macrophage migration inhibitory factor (MIF) and CD74 were measured using Western blot and reverse transcription-polymerase chain reaction.</p><p><b>RESULTS</b>The total saponins from Paris polyphylla var. yunnanensis induced apoptosis and expressed dose-effect relationship. ACC-83 cells expressed MIF and CD74, and the total saponins suppressed MIF and CD74 expression in ACC-83 cells.</p><p><b>CONCLUSIONS</b>The total saponins from Paris polyphylla var. yunnanensis can significantly inhibit the proliferation, suppress MIF and CD74 expression, and promote apoptosis in ACC-83 cells. This study provides a theoretical basis for the treatment of salivary adenoid cystic carcinoma using Paris polyphylla var. yunnanensis.
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Role of CCL5/CCR5 in the perineural invasion of salivary adenoid cystic carcinoma cells / 华西口腔医学杂志

<p><b>OBJECTIVE</b>This study aimed the role of the CCL5/CCR5 axis in the perineural invasion of salivary adenoid cystic carcinoma (SACC) cells.</p><p><b>METHODS</b>Immunohistochemical analysis and flow cytometric analysis were conducted to detect the expression of the chemokine receptor CCR5 in SACC cells. Enzyme linked immunosorbent assay (ELISA) was performed to determine the expression of CCL5 in the supernate of human nerve cells. The flow cytometric analysis was applied to observe the changes in F-actin in SACC-LM cells, which were pretreated with CCL5. To assess the effects of the CCL5/CCR5 axis on the migration and invasion of SACC-LM cells, we performed a scratch test and invasion assay under CCL5 stimulation.</p><p><b>RESULTS</b>CCR5 was highly expressed in SACC cells. The concentration of CCL5 in the supernatant of human nerve cells was (359.2±15.8), (696.4±22.6) pg·mL⁻¹. The CCL5/CCR5 axis promoted the migration and invasion of SACC-LM cells.</p><p><b>CONCLUSIONS</b>The CCL5/CCR5 axis may be involved in the perineural invasion of SACC cells.</p>

BRD4 inhibition suppresses cell growth, migration and invasion of salivary adenoid cystic carcinoma

BACKGROUND: Bromodomain-containing protein 4 (BRD4) inhibition is a new therapeutic strategy for many malignancies. In this study, we aimed to explore the effect of BRD4 inhibition by JQ1 on in vitro cell growth, migration and invasion of salivary adenoid cystic carcinoma (SACC). METHODS: The human normal epithelial cells and SACC cells (ACC-LM and ACC-83) were treated with JQ1 at concentrations of 0, 0.1, 0.5 or 1 µM. Cell Counting Kit-8 (CCK-8) assay was performed to evaluate cell proliferation. Cell apoptosis and cell cycle distribution was evaluated by Flow cytometry. Immunofluorescence staining was used to examine the expression of BRD4 in SACC cells. The quantitative real-time polymerase chain reaction (qRT-PCR) assay and western blot assay were performed to examine messenger RNA (mRNA) and protein levels in SACC cells. Wound- healing assay and transwell assay were used to evaluate the activities of migration and invasion of SACC cells. RESULTS: JQ1 exhibits no adverse effects on proliferation, cell cycle and cell apoptosis of the normal human epithelial cells, while suppressed proliferation and cell cycle, and induced apoptosis of SACC cells, down-regulated the mRNA and protein levels of BRD4 in SACC cells, meanwhile reduced protein expressions of c-myc and BCL-2, two known target genes of BRD4. Moreover, JQ1 inhibited SACC cell migration and invasion by regulating key epithelial-mesenchymal transition (EMT) characteristics including E-cadherin, Vimentin and Twist. CONCLUSIONS: BRD4 is an important transcription factor in SACC and BRD4 inhibition by JQ1 may be a new strategy for SACC treatment.

The effects of 5-aza-2-deoxycytidine on RECK gene expression and invasion of salivary adenoid cystic car-cinoma cells / 实用口腔医学杂志

Objective:To investigate the effects of 5-aza-2′deoxycytidine(5-aza-dC),a DNA methyltransferase (DNMT)inhibitor, on the methylation status of the RECK gene and the invasion of salivary adenoid cystic carcinoma cell lines.Methods:Methylation-specific PCR,Western blot analysis and quantitative real-time PCR were used to investigate the methylation status of RECK gene and the expression of RECK mRNA and protein in SACC cell lines.The invasive ability of SACC cells was examined by transwell assay. Results:Promoter methylation was only found in ACC-Mcell line and not in ACC-2 cell line.Treatment of ACC-Mcells with 5-aza-dC partially reversed the hypermethylation status of the RECK gene and significantly enhanced the expression level of mRNA and pro-tein of RECK,suppressed ACC-Mcell invasive ability.Conclusion:5-aza-dC can inhibit ACC-Mcell invasion by reversal of hyperm-ethylation status of RECK gene.

Effects of 5-aza-2′deoxycytidine on RECK gene expression and tumor invasion in salivary adenoid cystic carcinoma

Reversion-inducing cysteine-rich protein with kazal motifs (RECK), a novel tumor suppressor gene that negatively regulates matrix metalloproteinases (MMPs), is expressed in various normal human tissues but downregulated in several types of human tumors. The molecular mechanism for this downregulation and its biological significance in salivary adenoid cystic carcinoma (SACC) are unclear. In the present study, we investigated the effects of a DNA methyltransferase (DNMT) inhibitor, 5-aza-2′deoxycytidine (5-aza-dC), on the methylation status of the RECK gene and tumor invasion in SACC cell lines. Methylation-specific PCR (MSP), Western blot analysis, and quantitative real-time PCR were used to investigate the methylation status of the RECK gene and expression of RECK mRNA and protein in SACC cell lines. The invasive ability of SACC cells was examined by the Transwell migration assay. Promoter methylation was only found in the ACC-M cell line. Treatment of ACC-M cells with 5-aza-dC partially reversed the hypermethylation status of the RECK gene and significantly enhanced the expression of mRNA and protein, and 5-aza-dC significantly suppressed ACC-M cell invasive ability. Our findings showed that 5-aza-dC inhibited cancer cell invasion through the reversal of RECK gene hypermethylation, which might be a promising chemotherapy approach in SACC treatment.

The effect and mechanism of sodium butyrate on the invasion and migration in human salivary ;adenoid cystic carcinoma cell line ACC-M / 中国癌症杂志

Background and purpose: Researches demonstrated that the butyric acid sodium salt (sodium butyrate, NaB) has effect on the inhibition of tumor cell proliferation, differentiation and apoptosis-promoting, while the mechanism on salivary adenoid cystic carcinoma(SACC) is still uncertain. This study mainly probed into the impact of different concentration of sodium butyrate on the migration and invasion of SACC cell line ACC-M, and its mechanism of action. Methods:MTT assay explored the optimal concentration of sodium butyrate on the cell ACC-M and the observation of cell growth. Transwell assay was used to detect the effects of sodium butyrate on the ACC-M cells on the aspact of invasion and migration ability. Fluorescence real-time quantitative PCR (RT-PCR) and Western blot were used to test respectively the expression of HMGB1, TLR4 mRNA and protein in ACC-M after functioned by 5 group drugs with different concentrations. Results:Compared with the control group, on the one hand, the concentration 0.625, 1.25, 2.5, 5 and 10 mmol/L of sodium butyrate could effectively inhibit cell proliferation and apparently showing concentra-tion-dependence (P<0.05);On the other hand, 5 sets concentration of sodium butyrate could also effectively inhibit invasion and migration ability of ACC-M cells in vitro (P<0.05), as well as reducing the expression of HMGB1, TLR4 mRNA and protein in ACC-M cells (P<0.05). Furthermore related analysis showed that the decline of TLR4 protein expression was positively correlated with inhibition of HMGB1 (r=0.810, P<0.05). Conclusion:Sodium butyrate has an effect on inhibiting ACC-M cell proliferation, signiifcantly reducing ACC-M cell invasion and migration capabilities, and reducing expression of HMGB1, TLR4 mRNA and protein, and both expression amount are positively correlated, Meanwhile the positively correlation suggests that sodium butyrate probably achieve the inhibition ability by lowering the expression of HMGB1, TLR4 mRNA and protein in ACC-M cell.

Significance of RhoA and Snail expression in salivary adenoid cystic carcinoma / 天津医药

Objective To investigate the relationship of RhoA and Snail expressions, and the invasion and metastasis in salivary adenoid cystic carcinoma (SACC). Methods The expressions of RhoA protein and Snail protein in 55 samples of SACC (SACC group ) and 20 samples of para-carcinoma normal tissues(control group) were detected using immunohisto?chemical method. The relationship between RhoA protein and Snail protein expressions and clinical and pathological charac?teristics were analyzed. Results The positive expressions of RhoA protein (69.1% vs 5.0%) and Snail protein (72.7% vs 10.0%) were significantly higher in SACC group than those in control group (P < 0.05). The positive expression rates of RhoA protein and Snail protein were significantly higher in patients with lymph node metastasis than those in patients with?out lymph node metastasis. The positive expression rates of RhoA protein and Snail protein were significantly higher in pa?tients atⅢ+Ⅳstage than those in patients atⅠ+Ⅱstage. The positive expression rates of RhoA protein and Snail protein were significantly higher in substantive carcinal tissues than those in screen roller type and tubular carcinal tissues. The posi?tive expression of Snail protein was significantly higher in substantive and tubular carcinal tissues than that in screen roller type carcinal tissues (P<0.05). There were no significant differences in positive expression rates of RhoA and Snail between different gender, age and different carcinal tissues. There was a positive correlation beween expression rates of RhoA and Snail protein in SACC (r=0.414, P<0.001). Conclusion RhoA and Snail may both facilitate the infiltration and metastasis of SACC through RhoA/ROCK/PKD1/NF-kappa B/Snail signaling pathways.

The expression and significance of Slug,EMMPRIN and E-cadherin in salivary adenoid cystic carcinoma / 实用口腔医学杂志

Objective:To investigate the expression of Slug,EMMPRIN and E-cadherin in salivary adenoid cystic carcinoma (SACC)and its correlation with clinicopathological characteristics,and the correlation among themselves.Methods:Slug,EMMPRIN and E-cadherin expression in 1 1 5 SACC cases of SACC was examined by immunohistochemical staining.The results and clinicopatho-logical data were statistically analyzed.Results:High positive expression frequencies of Slug(76.5%)and EMMPRIN(69.6%)and low positive expression frequency of E-cadherin(51 .3%)were found in 1 1 5 SACC cases.The expression of Slug and EMMPRIN was positively associated with the histopathological types,clinical stages,perineural invasion,recurrence and distance metastasis(P <0.05).The expression of E-cadherin was negatively associated with the histopathological types,clinical stages,perineural invasion and distance metastasis(P <0.05).There was a significant correlation between Slug and EMMPRIN expression(P <0.05),negative correlation between EMMPRIN and E-cadherin expression(P <0.05)and between Slug and E-cadherin expression(P <0.05).Con-clusion:The expression of Slug,EMMPRIN and E-cadherin is closely correlated to the clinicopathological characteristics of SACC.

Research progress of signaling pathways mediating the proliferation, invasion and metastasis of salivary gland adenoid cystic carcinoma / 肿瘤研究与临床

Salivary gland adenoid cystic carcinoma is one of the most common malignant salivary gland tumors.Its invasion and metastasis are multi-stage and multifactorial processes,in which complex mechanisms and multiple signaling pathways are involved.Here,this article reviews the research progress in signaling pathways of salivary gland adenoid cystic carcinoma.

The effects of siRNA targeting Mcl-1 on biological behavior of salivary adenoid cystic carcinoma SACC-2 cells / 实用口腔医学杂志

Objective:To explore the effect of siRNA targeting myeloid cell leukemia-1(Mcl-1)on the biological behavior of salivary adenoid cystic carcinoma cells.Methods:The chemically synthesized Mcl-1-siRNA was transfected into salivary adenoid cystic carci-noma SACC-2 cells.The expression levels of Mcl-1-mRNA and Mcl-1protein were examined by Real-time PCR and western blotting respectively.MTT assay,transwell chamber and flow cytometry were used to determine the effect of Mcl-1-siRNA on SACC-2 cell pro-liferation,migration and apoptosis.Results:Compared with the control group,liposome group and NC-siRNA group,SACC-2 cell proliferation rate of Mcl-1-siRNA group was obviously slowed down.48 h after transfection,the migration of SACC-2 cells in Mcl-1-siRNA group(39 ±9.0)were lower than that in control group(69 ±6.0).The apoptosis rate of Mcl-1-siRNA group(8.6%)was sig-nificantly higher than that in control group(1.9%).Conclusion:Silence Mcl-1 can inhibit cell proliferation and migration and pro-mote apoptosis of salivary adenoid cystic carcinoma cells.

Induction effect of icotinib on apoptosis of salivary adenoid cystic carcinoma ACC-M cells through p38-MAPK pathway / 吉林大学学报(医学版)

Objective To explore the influence of icotinib in the apoptosis of the human salivary adenoid cystic carcinoma cells ACC-M, and to clarify the mechanism of icotinib for the treatment of salivary adenoid cystic carcinoma.Methods The ACC-M cells were randomly divided into control group,2,4,8μmo1·L-1 icotinib groups,p38-MAPK inhibitor SB203580 (20μmol· L-1 )group,SB203580 (20 μmol· L-1 )+4μmo1 · L-1 icotinib group;the cells were collected 4 h after treatment.The viability of ACC-M cells was measured by MTT assay.The apoptosis of ACC-M cells was assessed by caspase-3 activity kit. The expression of p-p38-MAPK protein was determined by Western blotting analysis.Results Compared with control group,the inhibitory rates of growth of the ACC-M cells in icotinib groups were significantly decreased (P<0.05 ), and the activities of caspase-3 were increased (P<0.05),and the expression levels of p-p38-MAPK were significantly increased (P<0.05).Compared with 4μmo1·L-1 icotinib group,the expression level of p-p38-MAPK in SB203580+icotinib group were decreased (P < 0.05 ), and the activity of caspase-3 was decreased dramatically (P < 0.05 ). Conclusion Icotinib may induce the apoptosis of ACC-M cells through the activation of p38-MAPK signaling pathway.

Expression of EZH2 and Ki-67 in salivary adenoid cystic carcinoma and their clinical significance / 中国肿瘤临床

Objective:This study aimed to investigate the expressions of EZH2 and Ki-67 in the salivary adenoid cystic carcino-ma (SACC) of humans and their correlation. Methods:A total of 42 cases of SACC tumor tissues and 5 cases of normal tissues were considered to determine the expressions of EZH2 and Ki-67 by immunohistochemistry. The relationship and correlation of such expres-sions with the clinicopathological characteristics were also analyzed. Results:The expression of EZH2 was notably higher in SACC than in normal tissues (P<0.05). EZH2 expression was detected in 66.67%(28/42) of the tumor tissues. This expression was correlated with pathological grade and clinical stage. By contrast, EZH2 expression did not correlate with gender, age, and localization. EZH2 was not expressed in normal tissues. The incidence of EZH2 expression in the Ki-67 positive group was 75.76%(25/33) and the incidence in the Ki-67 negative group was 33.33%(3/9). The difference between the two groups was statistically significant (P<0.05). Conclu-sion:The increased expression of EZH2 in SACC was related to tumor proliferation. EZH2 may participate in tumor cell proliferation via cell cycle management.

The effects of sulforaphane on the proliferation and apoptosis of salivary adenoid cystic carcinoma ACC-M cells / 实用口腔医学杂志

Objective:To study the effects of sulforaphane(SFN)on the proliferation and apoptosis of salivary adenoid cystic carci-noma ACC-Mcells in vitro.Methods:ACC-Mcells were treated with SFN at the doses(μmol/L)of 5,10,20,30,40,60,80 and 100 for 24,48 and 72 hours,respectively.The growth inhibition was examined with MTT assay and typan blue exclusion assay.Morpholo-gy of ACC-M cells was observed with phase contrast microscope,giemsa staining and transmission electron microscope.Flow cytome-try with Annexin-V-FITC/PI double staining was used to detect the apoptosis of ACC-M cells.Results:SFN inhibited the prolifera-tion of ACC-Mcells,the IC50 values(μmol/L)after 24,48 and 72 h treatment were 75.6,21.3 and 16.5 respectively.The highest inhibition rate was 89.2%.The growth inhibition rate of SFN on ACC-Mcells was positively correlated with concentrations of SFN and treatment time.SFN induced the apoptosis of ACC-Mcells in a dose and time dependent manner(P<0.01).Conclusion:SFN can inhibit proliferation and induce apoptosis of salivary adenoid cystic carcinoma ACC-M cells time and dose-dependently.

Immunohistochemical assays for the expression of angiogenic signaling molecules and microvessel density in adenoid cystic carcinomas of human salivary glands

Adeonoid cystic carcinoma (ACC) is one of the most common malignant tumors of salivary glands. It is characterized by a relentless regrowth especially around nerve tissues and a high rate of hematogenous distant metastasis. Clinically most deaths from salivary ACC are caused by delayed lung metastases that are resistant to conventional chemotherapy. So, knowledge of cellular and molecular properties that influence the dissemination of metastatic tumor cells, is important for new treatment strategies of metastatic lesions. We determined expressions of angiogenic signaling molecules microvessel density (MVD) using surgical specimens of human salivary ACC. Protein expressions of vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR)-2, activated VEGFR-2, and human CD31 were assessed in 20 cases of salivary ACC by immunohistochemical staining. Most of the tumors, especially ACC with a tubulocribriform pattern, were positive for antibodies of VEGF, VEGFR-2, and activated VEGFR-2. The overall percentages of the 20 specimens expressing VEGF, VEGFR-2, activated VEGFR-2 were 90, 95, and 95%, respectively. Immunoreactivities of the biomarkers in salivary ACC were higher than those in normal salivary gland. Furthermore, immune-related cells as well as tumor cells expressed VEGF/VEGFR-2. Microvessel density of salivary ACC was higher than that of normal salivary gland (P<0.05). Taken together, angiogenic signaling molecules are actively expressed in salivary ACC. And we suggest that these molecules may have critical role in the hematogenous spread of salivay ACC, which has a propensity for delayed lung metastasis. Therefore, these biomarkers can be molecular targets for therapy of metastasis of salivary ACC.

Cellular and molecular characterization of adenoid cystic carcinoma of the salivary glands