Proliferation Of Adenoid Cystic Carcinoma Cells Research Articles

Wnt/β-catenin-C-kit axis may play a role in adenoid cystic carcinoma prognostication

Adenoid cystic carcinoma (ACC) is one of the most common malignant salivary gland tumors. ACC is composed of myoepithelial and epithelial neoplastic cells which grow slowly and have a tendency for neural invasion. The long term prognosis is still relatively poor. Although several gene abnormalities, such as fusions involving MYB or MYBL1 oncogenes and the transcription factor gene NFIB, and overexpression of KIT have been reported in ACC, their precise functions in the pathogenesis of ACC remain unclear. We recently demonstrated that the elevated expression of Semaphorin 3A (SEMA3A), specifically expressed in myoepithelial neoplastic cells, might function as a novel oncogene-related molecule to enhance cell proliferation through activated AKT signaling in 9/10 (90%) ACC cases. In the current study, the patient with ACC whose tumor was negative for SEMA3A in the previous study, revisited our hospital with late metastasis of ACC to the cervical lymph node eight years after surgical resection of the primary tumor. We characterized this recurrent ACC, and compared it with the primary ACC using immunohistochemical methods. In the recurrent ACC, the duct lining epithelial cells, not myoepithelial neoplastic cells, showed an elevated Ki-67 index and increased cell membrane expression of C-kit, along with the expression of phosphorylated ERK. Late metastasis ACC specimens were not positive for β-catenin and lymphocyte enhancer binding factor 1 (LEF1), which were detected in the nuclei of perineural infiltrating cells in primary ACC cells. In addition, experiments with the GSK-3 inhibitor revealed that β-catenin pathway suppressed not only KIT expression but also proliferation of ACC cells. Moreover, stem cell factor (SCF; also known as KIT ligand, KITL) induced ERK activation in ACC cells. These results suggest that inactivation of Wnt/β-catenin signaling may promote C-kit-ERK signaling and cell proliferation of in metastatic ACC.

Adenoid cystic carcinoma cells produce exosomes that promote tumor cell proliferation

To observe the effect of exosomes released by adenoid cystic carcinoma (ACC) cell line SACC-83 on the proliferation of ACC cells. Exosomes were isolated from SACC-83 cell culture supernatants using total exosome isolation reagents. The whole-mount exosomes were characterized using transmission electron microscope and Western blotting. The exosomes were labeled with green fluorescent dye PKH67 and co-cultured with SACC-83 cells for 48 h, followed by staining with Alexa Fluor 594 phalloidin and DAPI to observe exosome uptake by the cells using laser scanning confocal microscopy (LSCM). The cell proliferation was assessed using MTT assay and wound healing assay, and the expressions of ERK and P-ERK in the co-cultured SACC-83 cells were detected using Western blotting. The exosomes isolated from SACC-83 cells showed a size range of 30-100 nm and expressed the exosomal markers CD9, CD63 and TSG101. LSCM showed exosome uptake by SACC-83 cells, which exhibited accelerated proliferation and significantly enhanced P-ERK expression (P < 0.05) without significant changes in ERK expression. SACC-83 cells produce exosomes that promote the tumor cell proliferation and enhances the cellular expression of P-ERK, suggesting a potential role of MAPK/ERK pathway activation in exosome-mediated acceleration of ACC cell proliferation.

Inhibitory effect of chidamide on the growth of human adenoid cystic carcinoma cells

Adenoid cystic carcinoma (ACC) is a malignant epithelial neoplasm that limitedly responses to chemotherapy at the cost of significant toxicity. There is no single targeted drug approved by Food and Drug Administration (FDA) for ACC. Genomic landscape studies have revealed that frequently mutated pathways in ACC often involve in chromatin remodeling, which interfere multiple histone related proteins. Chidamide is a novel histone deacetylase inhibitor (HDACi) approved in clinical practice that was designed to increase the acetylation level of histone H3. It demonstrated anticancer effects in various cancers in preclinical study, but not in ACC. In this study, we aimed to investigate the anticancer effects of chidamide alone or in combination with cisplatin (cDDP) on ACC in vitro and in vivo. The results showed that chidamide alone or in combination with cDDP effectively inhibited the growth and proliferation of ACC cells in a dose- and time-dependent manner. Chidamide arrested cell cycle in G2/M phase by up-regulating the acetylation of histone H3 and interfering phosphorylation of AKT protein. Chidamide alone or in combination with cDDP did not induce distinct apoptosis in ACC cells. In vivo experiments showed that chidamide combining cDDP exerted significant inhibitory effects on ACC. These suggest that chidamide may be a promising candidate drug for the treatment of patients with ACC.

Targeting the Oncogenic Transcriptional Regulator MYB in Adenoid Cystic Carcinoma by Inhibition of IGF1R/AKT Signaling.

Adenoid cystic carcinoma (ACC) is an aggressive cancer with no curative treatment for patients with recurrent/metastatic disease. The MYB-NFIB gene fusion is the main genomic hallmark and a potential therapeutic target. Oncogenic signaling pathways were studied in cultured cells and/or tumors from 15 ACC patients. Phospho-receptor tyrosine kinase (RTK) arrays were used to study the activity of RTKs. Effects of RTK inhibition on cell proliferation were analyzed with AlamarBlue, sphere assays, and two ACC xenograft models (n = 4-9 mice per group). The molecular effects of MYB-NFIB knockdown and IGF1R inhibition were studied with quantitative polymerase chain reaction, immunoblot, and gene expression microarrays. All statistical tests were two-sided. The MYB-NFIB fusion drives proliferation of ACC cells and is crucial for spherogenesis. Intriguingly, the fusion is regulated through AKT-dependent signaling induced by IGF1R overexpression and is downregulated upon IGF1R-inhibition (% expression of control ± SD = 27.2 ± 1.3, P < .001). MYB-NFIB regulates genes involved in cell cycle control, DNA replication/repair, and RNA processing. The transcriptional program induced by MYB-NFIB affects critical oncogenic mediators normally controlled by MYC and is reversed by pharmacological inhibition of IGF1R. Co-activation of epidermal growth factor receptor (EGFR) and MET promoted proliferation of ACC cells, and combined targeting of IGFR1/EGFR/MET induced differentiation and synergistically inhibited the growth of patient-derived xenografted ACCs (ACCX5M1, % growth of control ± SD = 34.9 ± 20.3, P = .006; ACCX6, % growth of control ± SD = 24.1 ± 17.5, P = .04). MYB-NFIB is an oncogenic driver and a key therapeutic target in ACC that is regulated by AKT-dependent IGF1R signaling. Our studies uncover a new strategy to target an oncogenic transcriptional master regulator and provide new important insights into the biology and treatment of ACC.

Apigenin inhibits the proliferation of adenoid cystic carcinoma via suppression of glucose transporter-1.

Apigenin is a natural phyto-oestrogen flavonoid, which exerts various biological effects, including anti-oxidative, anti-inflammatory and anticancer activities. In addition, apigenin has recently been reported to target hypoxic markers; however, there are currently no studies regarding the association between apigenin and glucose transporter-1 (GLUT-1) in adenoid cystic carcinoma (ACC). The present study investigated whether apigenin inhibits the proliferation of ACC cells or suppresses the expression of GLUT-1 in ACC cells. The results of the present study demonstrated that apigenin inhibits ACC-2 cell growth in a dose- and time-dependent manner. Treatment with apigenin also induced apoptosis and G2/M-phase arrest in a dose- and time-dependent manner. Corresponding with the above results, the expression levels of GLUT-1 were significantly decreased following treatment in a dose- and time-dependent manner. These results suggest that the inhibition of ACC-2 cell growth by apigenin may be due to the decreased expression of GLUT-1.

Expression of survivin in adenoid cystic carcinoma of the lacrimal gland and the effect of intervention with arsenic trioxide in vitro.

The aim of the present study was to investigate the role of survivin in adenoid cystic carcinoma of the lacrimal gland (LGACC) and the effect of arsenic trioxide (As2O3) intervention in vitro. In total, 13 patients with LGACC were recruited from Renmin Hospital of Wuhan University between 2007 and 2012. The patients were divided into different groups, namely tumor-node-metastasis (TNM)1-2 and TNM3-4, according to the TNM classification. A total of eight samples were included in the TNM1-2 group, while five samples were included in the TNM3-4 group. In addition, six patients that suffered from other diseases (no tumor), but underwent eye surgery, were selected as the controls. The mRNA and protein expression levels of survivin were analyzed. In addition, primary adenoid cystic carcinoma (ACC) cells were cultured and the expression of survivin was silenced using short interfering RNA (siRNA), after which the cell proliferation was determined. Furthermore, the primary cultured ACC cells were treated with different doses of As2O3 to observe the effect on the apoptosis rate. The mRNA and protein expression levels of survivin in the LGACC groups were higher when compared with the controls (P<0.01). In addition, the expression levels were higher in the TNM3-4 group when compared with the TNM1-2 group (P<0.01). After silencing survivin expression using siRNA, the rate of ACC cell proliferation was shown to decrease at days 2, 3, 4 and 5 following culture, particularly in the TNM3-4 group at days 2 and 3 (P<0.05), day 4 (P<0.01 vs. TNM1-2 + siRNA); and days 3 and 5 (P<0.05), and day 2 (P<0.01 vs. TNM3-4 + siRNA). The mRNA expression levels of survivin in the TNM1-2 and TNM3-4 groups were significantly decreased following treatment with the various doses of As2O3 (2, 4 and 6 µΜ) for 48 h, and the apoptosis rate of the ACC cells was markedly increased (TNM1-2 and 3-4: As2O3 6 µM vs. As2O3 2 µM, As2O3 6 µM vs. As2O3 4 Μm, P<0.01; TNF3-4, As2O3 4 µM VS. As2O3 2 µM, P<0.01; TNM1-2, As2O3 4 µM vs. As2O3 2 µM, P<0.05). Therefore, survivin was demonstrated to be highly expressed in ACC cells of the lacrimal gland, and inhibition of survivin gene expression was found to suppress the proliferation of the ACC cells. Furthermore, As2O3 treatment was demonstrated to inhibit the mRNA expression of survivin in the ACC cells, while significantly increasing the apoptosis rate.

Abstract 5274: Wnt inhibitory factor 1 reduces the growth and migration of human adenoid cystic carcinoma cells

Abstract Background: Adenoid cystic carcinoma (ACC) is a common malignancy that arises in major and minor salivary glands. It is well characterized by relatively high rate of local recurrence, and late onset of distant metastases, which cause high morbidity and mortality. Dysregulation in the Wingless-type (Wnt)/β-catenin pathway contributes to many human tumors. Here, we report that downregulation of an important Wnt antagonist, Wnt inhibitory factor 1 (WIF1), promotes ACC cell proliferation and migration. Aims: To determine the status of WIF1 and study the effects of WIF1 on human adenoid cystic carcinoma cell growth, migration, spheroid formation and senescence. Methods: Genomic DNA isolated from human adenoid cystic carcinoma cell lines (ACC52 and ACC112) was used for methylation-specific PCR after bisulfite modification. ACC52 and ACC112 cells were treated with a demethylating agent, 5-aza-2′-deoxycytidine (DAC) [50 µM for 4 days], and used for total RNA isolation and real-time RT-PCR to determine WIF1 mRNA expression. To determine the growth suppressive effects of WIF1, ACC52 and ACC112 cells were transiently transfected with either pCI blast (vector) or pCI blast-WIF1 and assessed for cell proliferation, migration, spheroid formation and senescence. Cell proliferation was determined at different time intervals (24, 48, 72 h) by hexosaminidase assay. Cell migration and spheroid formation were assessed by scratch assay and soft agar assay, respectively. Cellular senescence was determined using senescence β-galactosidase staining kit. Results: Methylation-specific PCR demonstrate that WIF1 is silenced by promoter methylation in ACC cells. DAC treatment increased the WIF1 mRNA expression by 2.3-fold and 3.8-fold in ACC52 and ACC112 cells, respectively compared with vehicle treatment. WIF1 significantly decreased the proliferation and migration of adenoid cystic carcinoma cells. Additionally, WIF1 significantly decreased the number of spheroids formed at 7 and 17 days. Furthermore, WIF1 induced a remarkable increase in cellular senescence in both the cell lines. Conclusions: We demonstrate that WIF1 is downregulated by promoter hypermethylation in human adenoid cystic carcinoma cells. Importantly, WIF1 inhibited the cell growth, migration and cancer stemness, while inducing cellular senescence. Our findings suggest that WIF1 is a potent growth inhibitor of adenoid cystic carcinoma cells and therefore could be a potential therapeutic agent for ACC. Grant support: This work was supported by the Adenoid Cystic Carcinoma Research Foundation (LQ). LQ holds a Presbyterian Health Foundation Endowed Chair in Otorhinolaryngology. Citation Format: Ilangovan Ramachandran, Vengatesh Ganapathy, Lurdes Queimado. Wnt inhibitory factor 1 reduces the growth and migration of human adenoid cystic carcinoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5274. doi:10.1158/1538-7445.AM2014-5274

Sulforaphane induces G 2–M arrest and apoptosis in high metastasis cell line of salivary gland adenoid cystic carcinoma

New chemotherapeutic strategy should be investigated to enhance clinical management in salivary gland adenoid cystic carcinoma (ACC). Recently, sulforaphane (SFN), as a natural compound from cruciferous vegetables exhibits a potent anti-cancer activity in various tumor cells, but remains uncertain in ACC cells. The present study examined whether SFN suppresses proliferation and in ACC cells, if so, the possible molecular targets would be further investigated. Cell survives, apoptosis, cell cycle progression and molecular targets were identified by multiple detecting techniques, including trypan blue dye exclusion assay, electron microscopy, AO/EB staining, flow cytometry and immunoblotting in human lung high metastasis cell line of salivary gland adenoid cystic carcinoma (ACC-M). The results showed that 5-20 microM SFN suppressed proliferation and induced apoptosis of ACC-M cells in dose- and time-dependent manners. Cell cycle analysis demonstrated treatment of ACC-M cells with 20 microM SFN resulted in G(2)/M cell cycle arrest, which was associated with a marked decline in protein levels of G(2)/M regulatory proteins including cyclin B1 and cyclin-dependent kinase 1 (Cdk1). In terms of apoptosis, SFN increased the expression of Bax and decreased the level of Bcl-2 and subsequently triggered release of cytochrome c from mitochondria and activation of caspase-3, but Fas level and caspase-8 activity remained unchanged at all time points. Furthermore, levels of nuclear factor-kappaB (NF-kappaB) p65 in both of the cytoplasm and the nucleus have also been markedly suppressed by SFN in a time-dependent manner. Taken together, these results suggest SFN inhibits cell growth via inducing G(2)/M cell arrest and apoptosis in ACC-M cells. These events have been associated with SFN-regulated multiple targets involved in ACC-M cell proliferation. The present study provides an evidence for testing SFN efficacy in vivo and warranting future investigations to exam the clinical potential of SFN in ACC chemotherapy.

Effect of all-trans-retinoic acid on inhibition of human salivary adenoid cystic carcinoma cell proliferation in vitro

Retinoids have proved to prevent carcinogenic transformation in head and neck premalignant lesions and to decrease the rate of secondary primary carcinoma in patients with head and neck cancer. Recent studies have demonstrated the efficacy of retinoic acid in inducing differentiation therapy of carcinoma. In the present study, all-trans-retinoic acid (ATRA) was tested for its anti-proliferative activity against human salivary adenoid cystic carcinoma cell (ACC-M) by means of clonogenic assay and MTT assay, as well as cell cycle analysis in vitro. The results showed that there was a decrease in the ratio of S phase cells after 72 hours of treatment, together with an increase in the ratio of G0 and G1 phase, indicating that ATRA could inhibit the proliferation of ACC-M cell in a dose- and time-dependent manner. Steve G. F. Shen