ACC-2 Cells Research Articles

MicroRNA155 in the growth and invasion of salivary adenoid cystic carcinoma

The carcinogenesis mechanism of adenoid cystic carcinoma (ACC) of the salivary gland is poorly understood. MicroRNA155 (miRNA155) has been involved in the carcinogenesis of many malignant tumors. The present study aims to examine the role of miRNA155 in tumor growth and invasion of ACC. MiRNA155 expression was determined in ACC specimens along with normal salivary glands by quantitative PCR. Using ACC-2 cells as a model for ACC, cell proliferation was examined by MTT assay after knocking down miRNA155 expression, and cell cycle analysis was performed. Invasive capacity of ACC-2 cells was examined by a Transwell culture assay. The effect of miRNA155 on tumor growth was also examined in vivo using mouse models. The effect of miRNA155 on epidermal growth factor receptor (EGFR)/NF-κB was studied by quantitative PCR and electrophoretic mobility shift assay. MiRNA155 was over-expressed in ACC. Proliferation of ACC-2 cells was markedly inhibited by knocking down miRNA155, resulting from a blockade of cell cycle in the G1 phase. Inhibition of miRNA155 significantly suppressed the invasive capacity of ACC-2 cells. In vivo growth of ACC-2 cell-derived tumors was significantly slower by inhibition of miRNA155. Inhibition of miRNA155 also resulted in decreased expression of EGFR and RelA (NF-κB). The results suggest that miRNA155 facilitates cell cycle progression and promotes invasion in ACC and that the EGFR/NF-κB pathway might participate in mediating the effects of miRNA155. This study has provided insights into the carcinogenic mechanisms of ACC and identified new targets for intervention of salivary ACC.

Premetastatic vasculogenesis in oral squamous cell carcinoma xenograft-draining lymph nodes

To study vascular anatomy on oral cancer-draining lymph nodes before metastasis in mice. Cell lines: highly lymph metastatic oral squamous cell carcinoma SASL1m and non-metastatic human adenoid cystic carcinoma ACC2. Bone marrow transplants and xenografts: Nude mice were lethally irradiated and transplanted with bone marrow cells from EGFP(+) mice. SASL1m or ACC2 cells were implanted in the tongue. Non-xenografted mice were used as controls. In addition, we injected conditioned medium from SASL1m or ACC2 in transplanted mice. Immunohistochemistry: Primary tumors and neck lymph nodes were resected and stained with anti-mouse Podoplanin and CD31. Images were visualized in a confocal microscope. Image analysis: Areas covered by EGFP, CD31 and Podoplanin were measured and compared statistically. Expression microarrays: Transcriptomic microarray analysis compared SASL1 to ACC2 cells. Interactomes were generated to reveal altered pathways. SASL1m cells induced the assemblage of blood vessels in cancer-free, tumor-draining lymph nodes. These blood vessels incorporated bone marrow-derived EGFP(+)CD31(+) cells. Notably, SASL1m-conditioned medium induced a similar reaction. Non-metastatic cells failed to produce any change. Microarray and pathway analyses revealed the upregulation of Transforming Growth Factor-β, Lysyl Oxidase-like 2, Slit homolog 3 and Protease Serine 22. The upregulation of these genes was confirmed in xenografts. This study suggests that a blood supply for new tumors is established in lymph nodes before metastasis. It also suggests that premetastatic vasculogenesis and primary tumor angiogenesis may be mediated by different mechanisms.

Flavokawain B induces apoptosis of human oral adenoid cystic cancer ACC-2 cells via up-regulation of Bim and down-regulation of Bcl-2 expression

Novel effective drugs are still urgently needed in the prevention and treatment of oral adenoid cystic carcinoma (ACC). In this study, we have assessed the antitumor potential and molecular mechanisms of flavokawain B (FKB) as a kava chalcone on the ACC-2 cell line in vitro. The results demonstrated that FKB could significantly inhibit the cell proliferation of ACC-2 in a dose-dependent manner that was associated with induced apoptosis and cell cycle G2-M arrest, and the half maximal inhibitory concentration (IC50) of flavokawain-B treatment for 48h was estimated to be 4.69± 0.43µmol/L. Mechanistically, FKB could induce the release of cytochrome c from mitochondria into the cytosol, and activate the cleavage of caspase-3 and, eventually, the poly(ADP-ribose) polymerase (PARP), in a dose-dependent manner, leading to marked apoptotic effect of ACC-2 cells. The apoptotic action of FKB was associated with the increased expression of proapoptotic proteins: Bim, Bax, Bak and a decreased expression of antiapoptotic Bcl-2. Among them, Bim expression was significantly induced by FKB, and knockdown of Bim expression by short-hairpin RNAs attenuated the inhibitory effect induced by FKB on ACC-2 cells. These results suggest Bim may be one of the potential transcriptional targets, and suggests the potential usefulness of FKB for the prevention and treatment of ACC.

Mammalian target of rapamycin regulates isoliquiritigenin-induced autophagic and apoptotic cell death in adenoid cystic carcinoma cells

Previous studies, including those from our laboratory, have demonstrated that isoliquiritigenin (ISL), a flavonoid isolated from licorice, is a promising cancer chemotherapeutic agent. However the mechanisms underlying its anticancer effects are still far from clear. We now show, for the first time, that ISL triggers the mammalian target of rapamycin (mTOR)-dependent autophagic and apoptotic cell death in adenoid cystic carcinoma (ACC). Exposure of both ACC-2 and ACC-M cells to ISL resulted in several specific features for autophagy, including the appearance of membranous vacuoles, formation of acidic vesicular organelles, punctate pattern of LC3 immunostaining, and an increase in autophagic flux. Moreover, ISL treatment also resulted in significantly increased apoptosis in ACC cells. The ISL-mediated autophagic and apoptotic cell death were obviously attenuated by transfection with dominant negative Atg5 (DN-Atg5(K130R)) plasmids or treatment with 3-methyladenine(3-MA). In additon, the data also revealed that the autophagic and apoptotic cell death induced by ISL occurred through a mTOR-dependent pathway. More importantly, the xenograft model using ACC-M cells provided further evidence of the occurrence of ISL-induced autophagy and apoptosis in vivo, correlating with the suppresson of mTOR activation as well as up-regulation of Atg5 expression. Taken together, these findings in our study suggest that induction of mTOR-dependent autophagic and apoptotic cell death may be an important mechanism in cancer chemotherapy by ISL.

Effects of 125 I seeds interstitial brachytherapy on orbital adenoid cystic carcinoma

Background Adenoid cystic carcinoma is a malignant tumor originating from lacrimal gland epithelium with high recurrence rate and mortality because of its invasiveness. Although surgery, radiotherapy and chemotherapy are used clinically,the curative effect is not enough satisfied. Objective This study was to provide an experimental basis for clinical application of 125 Ⅰ seeds interstitial brachytherapy. Methods Human ACC-2 cell links were transplanted subcutaneously in the back of 30 SPF BALB/C nude mice to establish the ACC model,and 25 of these mice with suitable sizes of tumor were selected and randomly divided into the G1 (0. 4 mCi 125 Ⅰ ), G2 (0. 6 mCi 125 Ⅰ ) ,G3(0. 8mCi 125 Ⅰ ) ,and G4( 1.0mCi 125 Ⅰ ) groups according to the different therapeutic radioactivity treatments,with 5 nude mice for each group,and a 125 Ⅰ seed without radioactivity was used in 5 mice as the control group. The dimensions of the tumors were measured at 2-day intervals and the inhibition rates of tumors were calculated. Nude mice were killed 14 days later by the broken neck method,and the amount of apoptosis and necrosis as well as the maximum effective radius of tumor to 125Ⅰ seed were detected under the transmission electron microscope and routine pathological examination. Results Fourteen day after operation, the dimension of tumors was (3713.19±243.23)mm3 in the G0 group;while in the G1 ,G2 ,G3 and G4 groups,the dimensions of tumors were (3113.35±316.54) mm3,(2635.85±261.21) mm3, (2538.37±312.16) mm3 and(1686.28±231.65) mm3,respectively, showing a significant decrease in comparison with the Go group( P＜0. 05 ). The tumor inhibitory rates in the G1 ,G2,G3 and G4 group were(20. 11±3.09)％, (36. 18±2.54)％ ,(40. 83±4. 17)％ ,and(66. 63±5.34)％ ,with an obvious elevation with the increase in the dose of 125 Ⅰ ( F=120. 240,P=0. 000). Correlative analysis showed that the intensity of radioactivity from 125Ⅰ had a positive correlation with tumor inhibitory rate (r =0. 653,P =0. 008 ). The maximum effective radius were ( 5.2 ±0.5 ) mm, ( 6.4 ±0. 7 ) mm, ( 7.4 ±0.4 ) mm, and ( 8.2 ±0. 5 ) mm in the G1 ,G2, G3 and G4 groups, with the considerable differences among them (F=29. 22, P=0. 000). Radioactivity of 125 Ⅰ exhibited positive correlation with the maximum effective radius ( r =0. 609, P =0. 004). Conclusions 125 Ⅰseed implantation brachytherapy can inhibit the growth of the transplanted ACC in BALB/C nude mice by suppressing the proliferation of tumor cells. It is a safe ,feasible and effective method to treat adenoid cystic carcinoma. Key words: 125 Ⅰ seeds; Adenoid cystic carcinoma; Brachytherapy

Expression of the membrane-cytoskeletal linker Ezrin in salivary gland adenoid cystic carcinoma

The aim of this study was to investigate the relationship between membrane cytoskeleton linker protein Ezrin and CD44v6, iNOS, Ki-67, and clinicopathologic characteristics, and the prognostic significance of Ezrin expression in salivary gland adenoid cystic carcinoma (SACC). Immunohistochemistry and reverse-transcription polymerase chain reaction were used to quantify the expression of Ezrin, CD44v6, inducible nitric oxide synthase (iNOS), and Ki-67 in 75 primary SACCs, 25 tumor-free salivary tissues, and 2 SACC cell lines (ACC-M and ACC-2). Survival analysis was performed to find the prognostic significance of Ezrin expression. Expressions of Ezrin, CD44v6, iNOS, and Ki-67 in SACC tissues, especially with distant metastasis, were significantly higher than in tumor-free tissues. Ezrin mRNA and protein levels in ACC-M cells were significantly higher than in ACC-2 cells. Ezrin, CD44v6, iNOS, and Ki-67 expressions were significantly higher in solid pattern than in cribriform and tubular patterns. Ezrin and its partners, CD44v6, iNOS, and Ki-67, were significantly related to tumor size, clinical stage, perineural and vascular invasion, and recurrence. Furthermore, Ezrin had an independent prognostic effect on overall survival. The increased expression of Ezrin and its partners, CD44v6, iNOS, and Ki-67, in SACC correlated with histologic patterns, may play a role in distant metastasis, and might indicate poor clinical outcome.

Maslinic acid induces apoptosis in salivary gland adenoid cystic carcinoma cells by Ca2+-evoked p38 signaling pathway

Maslinic acid (MA) is a triterpenoid with a high concentration that exists in olives. This natural compound, which has shown multiple biological activities, was proved to be an anti-tumoral agent more recently. We have investigated the mechanisms of MA with regard to its inhibitory effects on the growth of salivary gland adenoid cystic carcinoma (ACC). We demonstrated that MA at 10-100 μM reduced cell viability in a dose-dependent manner, IC(50) of 43.68 μM, and 45.76 μM, respectively in cultured ACC-2 and ACC-M cells. Fifty micromolars of MA efficiently induced apoptosis as indicated by AO/EB staining, electronic microscopy, flow cytometry, and activation of caspase-3 activity. MA induced an elevation of [Ca(2+)](i) in a dose-dependent manner, and cell viability inhibition and cell apoptosis induced by MA were blocked by an intracellular Ca(2+) chelator, BAPTA-AM. The elevation of [Ca(2+)](i) induced by MA was blocked by EGTA or TRPV channel inhibitor suggesting TRPV channel involved in calcium influx induced by MA. MA also activated ERK and p38 MAPK in a time-dependent manner. MA induced cell apoptosis and activation of caspase-3 activity were reversed by SB203580, but not by PD98059, suggesting that the apoptosis induction of MA was via p38 MAPK, but not via ERK. Chelation of intracellular Ca(2+) with BAPTA reversed MA induced p38 MAPK phosphorylation, but SB203580 did not block MA-evoked elevation of [Ca(2+)](i), suggesting a Ca(2+)-evoked p38 MAPK signaling involved in MA-induced apoptosis in ACC cells. Taken together, in ACC cells, maslinic acid induced an increase in [Ca(2+)](i), which evoked p38 MAPK phosphorylation, subsequently activated caspase-3 leading to apoptosis.

RUNX3 expression and its methylation of 5'-CpG island in salivary gland adenoid cystic carcinoma cell lines ACC-2, ACC-3 and ACC-M

To investigate the methylation of 5'-CpG island and expression of RUNX3 in salivary gland adenoid cystic carcinoma cell lines. RT-PCR (reverse transcription-polymerase chain reaction), laser scanning confocal microscope (LSCM) and Western blot were used to detect the expression of RUNX3 gene and protein in salivary gland adenoid cystic carcinoma cell lines, ACC-2, ACC-3, and ACC-M, before/after a treatment of 5-Aza-dc respectively. A weak expression of RUNX3 was found in ACC-2 and ACC-3. And no expression of RUNX3 was found in ACC-3 cell line. After a treatment of 300 nmol/L 5-Aza-dc for 72 hours, the expression of RUNX3 in ACC-2 and ACC-3 cells was enhanced, and in ACC-M was restored. LSCM results showed that the RUNX3 protein was located mainly in the cytoplasm of ACC cell lines. After a treatment of 300 nmol/L 5-Aza-dc for 72 h, both nuclear and cytoplasmic location of RUNX3 positive signals were found in the ACC-2 and ACC-3 cells. However, a weak positive signal was still only found in the cytoplasm of ACC-M cells. Partial methylation in promoter 5'-CpG island of RUNX3 gene was found in all three cell lines. And the methylation degree of CpG island was 50%, 75% and 33% in ACC-2, ACC-M and ACC-3 respectively. After a treatment of 5-Aza-dc, the RUNX3 gene showed unmethylated status in all three cell lines. The methylation of RUNX3 plays an important role in the silencing of RUNX3 expression in ACC cell lines. The cytoplasmic mislocalization of RUNX3 may be correlated with the inhibition of its function in ACC cells.

VCR-loaded nanoparticles as targeted delivery system for the treatment of orbital adenoid cystic carcinoma

To prepare and observe the properties of Folate Receptor-mediated VCR-loaded nanoparticles, which is abbreviated as FA-PLGA (VCR)-NP and to study the inhibitory effect of FA-PLGA (VCR)-NP in ACC-2 cells in vitro and in ACC in BALB/c-nu mice. The mAified W/O/W extraction-evaporation technique was chosen to prepare FA-PLGA (VCR)-NP. Tumor cells were divided into three groups: VCR, PLGA (VCR)-NP and FA-PLGA (VCR)-NP. Seven doses (0.05, 0.25, 0.50, 1.00, 5.00, 10.00, and 30.00 mg/L) of VCR were tested in the cell culture mAel. After 1, 2, 3, 4 and 5 days, the cell growth inhibition ratio was evaluated by MTT colorimetry. Nude mice mAel of orbital ACC was built by injecting ACC cell suspension and divided into four groups: VCR, PLGA (VCR)-NP, FA-PLGA (VCR)-NP, and control group. After 1 day, 7 days and 14 days, the inhibition ratio of gross tumor volume was observed. Residual concentrations of VCR in tumors were evaluated by HPLC. The feature of histopathology was observed by electron microscopy. The effect of empty nanoparticles on ACC-2 cells was compared with normal control group using t-test to analyze. On account of different drugs, concentration, time and the interaction of them, multivariate analysis of variance was used to analyze their relationship. Inhibition rate and residual volume of drug concentrations were compared using one-factor analysis of variance and LSD method. FA-PLGA (VCR)-NP were smooth and spherical with a mean particle size of 249.2 nm. The drug loading efficiency was 4.53%. The release of VCR from PLGA nanoparticles can persist for 14 d. After blank particles PLGA-NP and ACC-2 cells were co-cultured for 5 days, cell viability had remained at more than 80 percent (t = 1.952 ∼ 3.285, P = 0.081 ∼ 0.190). The inhibitory effect of FA-PLGA (VCR)-NP was more effective than VCR alone after a period of time (F = 4.798 ∼ 563.479, P = 0.000 ∼ 0.006). The effects of the treatment were both in dose-dependent and time-dependent manner. Targeting particles could attach to tumor surface, via folate receptor. FA was competitive inhibitor of this recolonization. The volume inhibition ratios of FA-PLGA (VCR)-NP and PLGA (VCR)-NP were significant higher than VCR (P = 0.016, P = 0.029). The inhibition ratio of FA-PLGA (VCR)-NP was higher than that of PLGA (VCR)-NP, but there was no statistical difference (P = 0.376). There was significant different between residual concentrations of VCR on the 1(st), 7(th) and 14(th) days. TEM pictures showed a mass of electron-dense microspheres in tumor cells on the 14(th) day. Tumor necrosis was obvious, while surrounding tissues were normal. FA-PLGA (VCR)-NP are stable and have high drug entrapment efficiency and high effect of growth inhibition in vitro. It can be proposed as a potentially controlled and targeted delivery system for the treatment of ACC.

Methylation status of the PTEN gene in adenoid cystic carcinoma cells

The tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is deficient in various types of human tumors due to mutations or epigenetic alterations. PTEN promoter hypermethylation is a major epigenetic silencing mechanism leading to self-repression in these tumors. The present study aimed to investigate whether PTEN promoter methylation is involved in the regulation of the PTEN gene in adenoid cystic carcinoma (ACC) cells. The expression of PTEN in ACC-2 cells was found to be significantly lower than that in normal salivary gland epithelial cells using RT-PCR analysis. The existence of CpG island methylation in the PETN promoter region in ACC-2 cells was demonstrated by methylation-specific PCR (MSP) analysis and direct sequencing of MSP product. RT-PCR, Western blot analysis and luciferase assay showed that mRNA and protein expression and the promoter activity of PTEN in ACC-2 cells treated with the DNA methylation inhibitor 5-aza-2-deoxycytidine were significantly up-regulated in a time-dependent manner. These results indicate that the hypermethylation of the PTEN promoter region leads to lower expression of PTEN gene in ACC cells, which aids in the development of PTEN as a molecular marker for the early diagnosis of this carcinoma.

Activation of PI3K/Akt/IKK-α/NF-κB signaling pathway is required for the apoptosis-evasion in human salivary adenoid cystic carcinoma: its inhibition by quercetin

Quercetin, one of the most common natural flavonoids, has been reported to possess significant anti-tumor activities both in vitro and in vivo. The present study was to investigate the effects of quercetin on growth and apoptosis in human salivary adenoid cystic carcinoma (ACC). The result from MTT assay showed that quercetin decreased cell viability of both low metastatic cell line ACC-2 and high metastatic cell line ACC-M in a concentration- and time-dependent manner. Moreover, treatment with quercetin resulted in significantly increased apoptosis in ACC cells. Our data also revealed that the apoptosis induced by quercetin treatment was through a mitochondria-dependent pathway which showed close correlation with the down-regulation of the PI3K/Akt/IKK-alpha/NF-kappaB pathway. Most importantly, quercetin significantly prevented in vivo growth of ACC xenografts in nude mice, accompanied by induction of tumor cell apoptosis, suppression of NF-kappaB nuclear translocation, as well as down-regulation of Akt and IKK-alpha activation. In addition, we explored the clinical significance of the PI3K/Akt/IKK-alpha/NF-kappaB signaling axis in ACC by immunohistochemical analysis of tissue specimens followed by the clustering analyses. We determined that the PI3K/Akt/IKK-alpha/NF-kappaB pathway is ubiquitously activated in ACC and plays an essential role in the evasion of apoptosis. Taken together, the results from our study implicated that quercetin would be a promising chemotherapeutic agent against ACC through its function of down-regulating the PI3K/Akt/IKK-alpha/NF-kappaB signaling pathway.

Expression of c-kit and Slug correlates with invasion and metastasis of salivary adenoid cystic carcinoma

The overexpression of c-kit seems to be frequent and specific in salivary adenoid cystic carcinoma (ACC), however, there is little information on correlation between c-kit expression and the invasion and metastasis. Recently, the data showed that Slug, a transcription factor of epithelial-mesenchymal transitions (EMT), is a molecular target that contributes to the biological specificity of c-kit signaling pathway. In this study, the expression of c-kit and Slug was evaluated in two ACC cell lines and 121 patients with ACC. The results of real-time RT-PCR and Western blot showed that ACC-2 and ACC-M cell lines expressed c-kit and Slug mRNA and protein. The immunohistochemical assay in patients demonstrated that positive expression of c-kit and Slug was observed in 108/121 (89.26%) and 87/121 (71.90%) of cases, respectively, and that c-kit and Slug expression was significantly associated with tumor site, TNM stage, histological pattern, perineural invasion, local regional recurrence and distant metastasis of patients with ACC (P<0.05). Furthermore, there was a significant association between the positive expression of c-kit and that of Slug (P=0.046). These findings indicated that c-kit/Slug pathway might participate in the invasion and metastasis of salivary ACC.

NF‐κB promotes iNOS and VEGF expression in salivary gland adenoid cystic carcinoma cells and enhances endothelial cell motility in vitro

Tumour cell-derived angiogenic-related factors and endothelial cell mobility are essential for neovascularization and haematogenous metastasis of adenoid cystic carcinoma (ACC) of the human salivary glands. Our previous study demonstrated that vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), and nuclear factor kappaB (NF-kappaB) staining correlated with ACC microvessel density. However, there still remains a lack of direct evidence to clarify the function of liposaccharide-induced NF-kappaB activity in ACC angiogenesis. This study aimed to demonstrate the function of liposaccharide-induced NF-kappaB signalling pathway (which would be relevant to angiogenesis) of ACC cell lines. A PCMV-IkappaBalphaM vector transfection assay was performed to inhibit NF-kappaB activity. Constitutive and liposaccharide-induced NF-kappaB activity, along with VEGF and iNOS expression, was detected by electrophoretic mobility shift assays, reverse transcription-polymerase chain reaction and Western blot assay. Tumour cell-derived NO(2) (-) was evaluated by a nitrite determination assay, and endothelial cell mobility was investigated by endothelial cell proliferation, migration and tube formation assays. We demonstrated that regulation of VEGF and iNOS expression utilized the NF-kappaB signalling pathway in ACC cell lines. In the constitutive and liposaccharide-induced condition, NF-kappaB activity was blocked by phosphorylation-defective IkappaBalpha. Accordingly, mRNA and protein expression of VEGF and iNOS were significantly decreased, with a great drop in endogenous NO(2) (-). Furthermore, supernatant from ACC-2/IkBaM and ACC-M/IkBaM cells resulted in much lower endothelial cell mobility than that from ACC-2 and ACC-M cells. Our results suggest that NF-kappaB promotes iNOS and VEGF expression in salivary gland ACC cells and enhances endothelial cell motility in vitro.

Radiosensitization and Anti-tumour Effects of Cytosine Deaminase and Thymidine Kinase Fusion Suicide Gene in Human Adenoid Cystic Carcinoma Cells

Herpes simplex virus thymidine kinase (HSV-TK) and Escherichia coli cytosine deaminase (CD) can convert innocuous prodrugs into cytotoxic metabolites and are being investigated for use in gene therapy for cancer. Human adenoid cystic carcinoma (ACC-2) cells transduced with a CD/HSV-TK fusion gene (ACC-2/CD-TK cells) were found to be more sensitive to radiation than ACC-2 cells when exposed to 5-fluorocytosine (5-FC; 40 microg/ml) plus ganciclovir (0.1 microg/ml) for 48 h before irradiation. Analysis of radiation survival curves for cells exposed to 5-FC plus ganciclovir before irradiation showed that ACC-2 cells had a higher capacity for sublethal damage repair (D(q) value) and greater cellular radiosensitivity (D(0) value) than ACC-2/CD-TK cells. Colony formation rate after 2 Gy of irradiation was significantly greater for ACC-2 than for ACC-2/CD-TK cells when cells were treated with 5-FC plus ganciclovir before irradiation. This study, therefore, indicates that addition of radiation might substantially improve the therapeutic potential of CD-TK fusion gene therapy of human adenoid cystic carcinomas.

Synergistic inhibitory effect of sulforaphane and 5‐fluorouracil in high and low metastasis cell lines of salivary gland adenoid cystic carcinoma

The present study aimed to evaluate the growth-inhibitory effect of sulforaphane (SFN) and a traditional chemotherapy agent, 5-fluorouracil (5-Fu), against the proliferation of salivary gland adenoid cystic carcinoma high metastatic cell line (ACC-M) and low metastasis cell line (ACC-2). Furthermore, the expression of nuclear factor kappa B (NF-kappaB) which induces resistance to anticancer chemotherapeutic agents was also detected. The combination effect of SFN and 5-Fu was quantitatively determined using the method of median effect principle and the combination index. The nuclear NF-kappaB p65 expression after treatment with the SFN-5-Fu combination was also evaluated by western blot analysis. The ACC-M and ACC-2 cells exhibited relative resistant to 5-Fu. Treatment ACCs cells with SFN and 5-Fu in combination, led to synergistic inhibition on cell growth and a decreased expression in nuclear NF-kappaB p65 protein. This synergistic inhibitory effect was more significant in ACC-M cells, which is associated with the greatly decreased expression of NF-kappaB p65 (almost 5-fold) after the combination treatment. Our results demonstrate synergism between SFN and 5-Fu at higher doses against the ACC-M and ACC-2 cells, which was associated with the decreased expression of nuclear NF-kappaB p65 protein.

Measurement of annexin V uptake and lactadherin labeling for the quantification of apoptosis in adherent Tca8113 and ACC-2 cells

Phosphatidylserine (PS) exposure occurs during the cell death program and fluorescein-labeled lactadherin permits the detection of PS exposure earlier than annexin V in suspended cell lines. Adherent cell lines were studied for this apoptosis-associated phenomenon to determine if PS probing methods are reliable because specific membrane damage may occur during harvesting. Apoptosis was induced in the human tongue squamous carcinoma cell line (Tca8113) and the adenoid cystic carcinoma cell line (ACC-2) by arsenic trioxide. Cells were harvested with a modified procedure and labeled with lactadherin and/or annexin V. PS exposure was localized by confocal microscopy and apoptosis was quantified by flow cytometry. The detachment procedure without trypsinization did not induce cell damage. In competition binding experiments, phospholipid vesicles competed for more than 95 and 90% of lactadherin but only about 75 and 70% of annexin V binding to Tca8113 and ACC-2 cells. These data indicate that PS exposure occurs in three stages during the cell death program and that fluorescein-labeled lactadherin permitted the detection of early PS exposure. A similar pattern of PS exposure has been observed in two malignant cell lines with different adherence, suggesting that this pattern of PS exposure is common in adherent cells. Both lactadherin and annexin V could be used in adherent Tca8113 and ACC-2 cell lines when an appropriate harvesting procedure was used. Lactadherin is more sensitive than annexin V for the detection of PS exposure as the physical structure of PS in these blebs and condensed apoptotic cell surface may be more conducive to binding lactadherin than annexin V.

Effect of exogenous bFGF on the proliferation of human adenoid cystic carcinoma ACC-2 cells

To observe the effects of basic fibroblast growth factor (bFGF) on human adenoid cystic carcinoma ACC-2 cell line proliferation and ERK, cyclin D1/p21(waf/cip1) signaling pathways, human adenoid cystic carcinoma cells (ACC-2) were cultured and the influence of bFGF of different concentrations on cell proliferation was determined by MTT. Protein was detected by immuno-precipitation and ERK activity by using ERK agent kit. p-ERK(1/2) and down-stream cyclin D1, p21(waf/cip1) expression were detected by Western blotting and the interfering role of mitogen protein-activated kinase (MEK) suppressor U0126 in the afore-mentioned indicators was examined. MTT demonstrated ACC-2 cell proliferation was substantially enhanced by bFGF, immuno-precipitation displayed ERK activity was up-regulated by bFGF, and immuno-imprinting also showed p-ERK(1/2), cyclin D1 expression was greatly enhanced and p21(waf/cip1) expression was inhibited by bFGF. U0126 suppressed the effect of bFGF. It is concluded that bFGF can promote the proliferation of human adenoid cystic carcinoma ACC-2 cells, and its pathways are associated with the up-regulated activity and expression of p-ERK(1/2), inhibited p21waf/cip1 expression and enhanced cyclin D1 expression.

A20 inhibits human salivary adenoid cystic carcinoma cells invasion via blocking nuclear factor-κB activation

A20, also known as tumor necrosis factor alpha induced protein 3 (TNFaip3), is a cytoplasmic zinc finger protein that inhibits nuclear factor kappa-B (NF-kappaB) activity and prevents tumor necrosis factor (TNF)-mediated programmed cell death. NF-kappaB is a transcription factor that regulates expression of genes involved in cell proliferation, cell survival and anti-apoptosis. Several studies have implicated that the NF-kappaB signal pathway is associated with angiogenesis and clinico-pathological process of adenoid cystic carcinoma (ACC) of the salivary glands. The ability of overexpression of A20 to influence the biological behavior and invasion of ACC cells was examined. The cells were stably transfected with full-length A20 cDNA. Stable gene transfer was verified by realtime-polymerase chain reaction (PCR) and Western blot analysis. The change of cell biological behavior was examined by methyl thiazolyl tetrazolium (MTT) and NF-kappaB luciferase reporter assay and the invasion of the cells was examined by a Matrigel invasion chamber. pEGPFN3-A20 gene was stably transferred into ACC-2 cells and overexpressed. When cells were treated with TNFalpha, the NF-kappaB activity of ACC-2-A20 cells could be down-regulated about 46.32% in contrast to ACC-2-GFP cells (P < 0.05). A20 potently inhibited growth of A20 transfectant ACC-2-A20 compared with control vector transfected groups and the ACC-2 empty control group (P < 0.05). The ACC-2-A20 cells showed significantly reduced ability to invade through Matrigei-coated filters compared to ACC-2-GFP and ACC-2 cells. The inhibition rate was up to 71.05% (P < 0.05). A20 gene transfer is associated with decreased tumor invasion, in part via the down-regulation of NF-kappaB expression, providing evidence for a potential application of A20 in designing a treatment modality for salivary gland cancers such as ACC.

Promoter methylation as a common mechanism for inactivating E‐cadherin in human salivary gland adenoid cystic carcinoma

The role of promoter methylation in the inactivation of E-cadherin (E-cad) in salivary gland adenoid cystic carcinoma (ACC) is unknown. The objective of this study was to determine the role and potential clinical implications of promoter methylation of E-cad in salivary gland ACC. The promoter methylation status of E-cad was determined by using methylation-specific polymerase chain reaction (PCR) analysis in 60 primary salivary gland ACC tissues and 3 ACC cell lines. The level of E-cad protein expression was determined by immunohistochemical analysis of each tumor. E-cad protein and messenger RNA (mRNA) expression levels were examined by immunohistochemical analysis and reverse transcriptase-PCR in 3 ACC cell lines. Associations between molecular alterations and patients' clinicopathologic characteristics were analyzed statistically. E-cad mRNA expression was examined in a 5-azacytidine-treated ACC-2 cell line. Promoter methylation of E-cad was detected in 34 of 60 tumors (57%). Of those 34 tumors, 18 tumors (53%) showed no E-cad protein expression, whereas only 5 of the remaining 26 tumors (19%) without E-cad promoter methylation showed no E-cad protein expression (P = .01). Tumors that had E-cad promoter methylation had a significantly higher histologic grade (P = .01) and more perineural invasion (P = .02) compared with tumors that did not have methylation. All 3 ACC cell lines exhibited E-cad promoter methylation and a lack of E-cad mRNA and protein expression, whereas 5-azacytidine restoredE-cad mRNA expression in the ACC-2 cell line. E-cad frequently is inactivated in salivary gland ACC through promoter methylation, and E-cad promoter methylation may play a role in tumor cell differentiation and perineural invasion.

In vitro angiogenesis and expression of nuclear factor κB and VEGF in high and low metastasis cell lines of salivary gland Adenoid Cystic Carcinoma

BackgroundAdenoid cystic carcinoma is a high malignant carcinoma characterized by intensive local invasion and high incidence of distant metastasis. Although many reports have demonstrated that angiogenesis has played an important role in tumor metastasis, the relationship between metastasis characters and angiogenesis ability in high and low metastasis cell lines of Adenoid cystic carcinoma has rarely been reported. The present study aimed to compare the angiogenesis ability of ACC-M (high metastasis) and ACC-2 (low metastasis) cell lines in vitro. Furthermore, the activity of nuclear factor κappa B and the expression of vascular endothelial growth factor (VEGF) in ACC-2 and ACC-M were also detected.MethodsElectrophoretic mobility shift assay was used to detect nuclear factor κappa B activity. Semi-quantitative RT-PCR was used to quantify the mRNA level of VEGF. Immuofluorescence double staining and semi-quantitative confocal laser scanning analysis was carried out to detect nuclear factor κappa B nuclear localization and staining intensity of VEGF. The angiogenesis ability of ACC-M and ACC-2 was compared by an in vitro three-dimensional angiogenic model assay. The vector transfection assay was performed to transfect the PCMV-IκBαM vector into ACCs cell lines expressing the phosphorylation defective IκBαM.ResultsNuclear factor κappa B activity and the rate of nuclear factor κappa B nuclear localization in ACC-M was significantly higher than that in ACC-2. Moreover, ACC-M exhibited higher mRNA and protein levels of vascular endothelial growth factor than ACC-2. VEGF mRNA expression was effectively decreased by inhibition of nuclear factor κappa B activity. Furthermore, ACC-M could remarkably stimulate the migration and tube formation of endothelial cells and induce The umbilical vein endothelial cells sprouting into the gel matrix.ConclusionThese results implicated that ACCs cells with higher metastasis feature might present greater angiogenesis ability.