Human Salivary Gland Cell Line Research Articles

Cell Survival Computation via the Generalized Stochastic Microdosimetric Model (GSM2); Part II: Numerical Results.

In the present paper we numerically investigate, using Monte Carlo simulation, the theoretical results predicted by the Generalized Stochastic Microdosimetric Model (GSM2), as shown in the published companion paper. Taking advantage of the particle irradiation data ensemble (PIDE) dataset, we calculated GSM2 biological parameters of human salivary gland (HSG) and V79 cell lines. Further, exploiting the TOPAS-microdosimetric extension, we simulated the microdosimetric spectra of different radiation fields of therapeutic interest generated by four different ions (protons, helium-4, carbon-12 and oxygen-16) each at three different residual ranges. We investigated the properties of the initial damage distributions as well as the cell survival curve predicted by GSM2, focusing especially on the non-Poissonian effects naturally included in the model. GSM2 successfully computed cell survival curves, accurately describing experimental behavior even under challenging LET and dose conditions.

Formation of spread-out Bragg peak for helium-ion beam using microdosimetric kinetic model

PurposeTo evaluate the applicability of microdosimetric kinetic model (MKM) to helium-ion therapy by forming a spread-out Bragg peak (SOBP) of a helium-ion beam using the MKM developed for carbon-ion radiotherapy and confirming the predictions in biological experiments. MethodsUsing a ridge filter, a 90-mm wide SOBP for a 210 MeV/u helium-ion beam was created in a broad beam delivery system. The ridge filter was designed such that a uniform biological response was achieved with a cell survival rate of 7% over the SOBP region. Biological experiments were then performed using the SOBP beam in a human salivary gland (HSG) cell line to measure the cell survival rates. ResultsThe biological responses were uniform in the SOBP region, as expected by the MKM; however, the mean of the measured cell survival rates was (11.2 ± 0.6) % in the SOBP region, which was 60% higher than the designed rate. When investigating the biological parameters of the HSG cell line used in the experiments, we found that they were altered slightly from the MKM parameters used for carbon-ion radiotherapy. The new β parameter reproduced the measured survival rates within 6.5% in the SOBP region. ConclusionWe produced biologically uniform SOBP using MKM for carbon-ion radiotherapy. The measured survival rates in the SOBP region were higher than expected, and the survival rates were reproduced by modifying the MKM parameter. This study was limited to one SOBP, and further investigations are required to prove that MKM is generally applicable to helium-ion radiotherapy.

Relative Biological Effectiveness Values of Spot-scanning Proton Beam Therapy at Shonan Kamakura General Hospital.

This study aimed to confirm the relative biological effectiveness (RBE) values of the proton beam therapy (PBT) system installed in Shonan Kamakura General Hospital. Clonogenic cell-survival assays were performed with a human salivary gland (HSG) cell line, a human tongue squamous-cell carcinoma cell line (SAS), and a human osteosarcoma cell line (MG-63). Cells were irradiated with proton beams and X-rays with different doses (1.8, 3.6, 5.5, and 7.3 Gy for proton beams, and 2, 4, 6, and 8 Gy for X-rays). Proton beam irradiation used spot-scanning methods and three different depths (at the proximal, center, and distal sides of the spread-out Bragg peak). RBE values were obtained from a comparison of the dose that resulted in a surviving fraction of 10% (D10). D10 of proton beams at the proximal, center, and distal sides and X-rays in HSG were 4.71, 4.71, 4.51, and 5.25 Gy, respectively; those in SAS were 5.08, 5.04, 5.01, and 5.59 Gy, respectively; and those in MG-63 were 5.36, 5.42, 5.12, and 6.06 Gy, respectively. The RBE10 values at the proximal, center, and distal sides in HSG were 1.11, 1.11, and 1.16 respectively; those in SAS were 1.10, 1.11, and 1.12, respectively; and those in MG-63 were 1.13, 1.12, and 1.18, respectively. RBE10 values of 1.10-1.18 were confirmed by in vitro experiments using the PBT system. These results are considered acceptable for clinical use in terms of therapeutic efficacy and safety.

HCG11 inhibits salivary adenoid cystic carcinoma by upregulating EphA2 via binding to miR-1297

Ephrin receptor A2 (EphA2) was reported to be related to the tumorigenesis of salivary adenoid cystic carcinoma (SACC), which is a rare malignancy accounting for less than 1% of all oral and maxillofacial tumors. This research aimed to assess the molecular mechanisms of EphA2 in SACC. The expression of long non-coding RNA human leukocyte antigen complex group 11 (HCG11), microRNA-1297 (miR-1297), and EphA2 in SACC cell lines compared with normal human salivary gland (HSG) cell line was measured by reverse transcription-quantitative polymerase chain reaction. EphA2 protein level was detected by western blot. 5-ethynyl-2'-deoxyuridine (EdU), colony formation, Transwell, and wounding healing experiments were applied to evaluate SACC cell proliferation, migration, and invasion. The relationship among HCG11, miR-1297, and EphA2 was confirmed by luciferase reporter, RNA pulldown, and RNA immunoprecipitation experiments. HCG11 and EphA2 were downregulated while miR-1297 was upregulated in SACC cells. EphA2 overexpression suppressed SACC cell proliferation, migration, and invasion. HCG11 bound to miR-1297 to reduce the inhibition of miR-1297 on EphA2 expression. EphA2 knockdown reversed the suppression of HCG11 overexpression on SACC cell phenotypes. This study identified the HCG11/miR-1297/EphA2 regulatory axis in SACC, which might provide novel therapeutic targets for SACC.

Estimate of the Biological Dose in Hadrontherapy Using GATE.

Simple SummaryThis study presents the implementation of a biological dose module using the Monte Carlo software, GATE. Both mMKM and NanOx biophysics models of cell survival predictions were used as input. The code was validated in terms of biological dose, relative biological effectiveness and cell survival against experimental data from the HIMBC (Hyogo, Japan) ion beam line.For the evaluation of the biological effects, Monte Carlo toolkits were used to provide an RBE-weighted dose using databases of survival fraction coefficients predicted through biophysical models. Biophysics models, such as the mMKM and NanOx models, have previously been developed to estimate a biological dose. Using the mMKM model, we calculated the saturation corrected dose mean specific energy (Gy) and the dose at 10% D10 for human salivary gland (HSG) cells using Monte Carlo Track Structure codes LPCHEM and Geant4-DNA, and compared these with data from the literature for monoenergetic ions. These two models were used to create databases of survival fraction coefficients for several ion types (hydrogen, carbon, helium and oxygen) and for energies ranging from 0.1 to 400 MeV/n. We calculated α values as a function of LET with the mMKM and the NanOx models, and compared these with the literature. In order to estimate the biological dose for SOBPs, these databases were used with a Monte Carlo toolkit. We considered GATE, an open-source software based on the GEANT4 Monte Carlo toolkit. We implemented a tool, the BioDoseActor, in GATE, using the mMKM and NanOx databases of cell survival predictions as input, to estimate, at a voxel scale, biological outcomes when treating a patient. We modeled the HIBMC 320 MeV/u carbon-ion beam line. We then tested the BioDoseActor for the estimation of biological dose, the relative biological effectiveness (RBE) and the cell survival fraction for the irradiation of the HSG cell line. We then tested the implementation for the prediction of cell survival fraction, RBE and biological dose for the HIBMC 320 MeV/u carbon-ion beamline. For the cell survival fraction, we obtained satisfying results. Concerning the prediction of the biological dose, a 10% relative difference between mMKM and NanOx was reported.

Apigenin, a Single Active Component of Herbal Extract, Alleviates Xerostomia via ERα-Mediated Upregulation of AQP5 Activation.

Xerostomia is a common symptom in menopausal women, suggesting the role of sex steroids in disease development. Shreds of literature had reported the potential use of herbal extracts to relieve xerostomia. However, a cocktail of multiple components in herbal extract makes it difficult to understand the exact mechanism of action. Aquaporin5 (AQP5), the specific aquaporin expressed in salivary glands, plays an important role in salivary secretion as a downstream of estrogen signaling. In this study, we aimed to unravel a single active herbal component as a therapeutic for xerostomia and investigate its mechanism of action. The effects of apigenin (flavonoid), dauricine (alkaloids), protopine (alkaloids), and lentinan (polysaccharides) on AQP5 transcription were screened in vitro. Only apigenin robustly induced AQP5 transcription and expression, and this effect was even robust compared to the effect of estradiol (E2, a positive control). Overexpression of estrogen receptor α (ERα) in the human salivary gland cell line (HSG) upregulated the AQP5 transcription and expression and the knockdown ERα reversed this effect, suggesting the role of ERα signaling on AQP5 activation in HSG cells. Docking results showed apigenin-specific binding sites in ERα. We further analyzed the therapeutic effect of apigenin on ovariectomized mice as a xerostomia model. The saliva secretion in the xerostomia group was reduced to one-third of the sham group, whereas the apigenin or E2 treatment for 12 weeks reversed this effect. Meanwhile, the water consumption in the xerostomia group was augmented obviously compared to the sham group, whereas the water consumption in the apigenin and E2 group was declined to the level of the sham group. Immunohistochemistry of submandibular glands revealed the downregulation of AQP5 expression in xerostomia mice compared to control. Apigenin, or E2 treatment, upregulated AQP5 expression in xerostomia mice. In conclusion, apigenin, a single active component of herbal extract, upregulated AQP5 expression in HSG cells via activation of ERα signaling and restored saliva flow rates in OVX mice. These results revealed apigenin as a single active component of herbal extract with the potential to treat xerostomia.

Non-Lethal Concentration of MeHg Causes Marked Responses in the DNA Repair, Integrity, and Replication Pathways in the Exposed Human Salivary Gland Cell Line.

In Brazilian northern Amazon, communities are potentially exposed and vulnerable to methylmercury (MeHg) toxicity through the vast ingestion of fish. In vivo and in vitro studies demonstrated that the salivary glands as a susceptible organ to this potent environmental pollutant, reporting alterations on physiological, biochemical, and proteomic parameters. However, the alterations caused by MeHg on the gene expression of the exposed human salivary gland cells are still unknown. Therefore, the goal was to perform the transcriptome profile of the human salivary gland cell line after exposure to MeHg, using the microarray technique and posterior bioinformatics analysis. The cell exposure was performed using 2.5 µM MeHg. A previously published study demonstrated that this concentration belongs to a range of concentrations that caused biochemical and metabolic alterations in this linage. As a result, the MeHg exposure did not cause lethality in the human salivary gland cells line but was able to alter the expression of 155 genes. Downregulated genes (15) are entirety relating to the cell metabolism impairment, and according to KEGG analysis, they belong to the glycosphingolipid (GSL) biosynthesis pathway. On the other hand, most of the 140 upregulated genes were related to cell-cycle progression, DNA repair, and replication pathway, or cellular defenses through the GSH basal metabolism. These genomic changes revealed the effort to the cell to maintain physiological and genomic stability to avoid cell death, being in accordance with the nonlethality in the toxicity test. Last, the results support in-depth studies on nonlethal MeHg concentrations for biomarkers identification that interpret transcriptomics data in toxicological tests serving as an early alert of physiological changes in vitro biological models.

The Alteration of Salivary Immunoglobulin A in Autism Spectrum Disorders.

Objectives: Autism spectrum disorders (ASD) are neurodevelopmental disorders with changes in the gut and oral microbiota. Based on the intimate relationship between the oral microbiota and oral mucosal immunity, this study aimed to investigate changes in salivary immunoglobulin A (IgA) level in ASD and the underlying mechanism for any such changes.Methods: We recruited 36 children diagnosed with ASD and 35 normally developing children and measured their salivary IgA content using enzyme-linked immunosorbent assay (ELISA). The valproate (VPA) -treated ASD mouse model was established by prenatal exposure to valproate and mouse salivary IgA content was also quantified by ELISA. The submandibular glands of VPA and control mice were isolated and analyzed using qRT-PCR, immunofluorescence staining, and flow cytometry. ASD-related Streptococci were co-incubated with the human salivary gland (HSG) cell line, and western blotting was used to detect the levels of relevant proteins.Results: We found that salivary IgA content was significantly decreased in patients with ASD and had a significant ASD diagnostic value. The salivary IgA content also decreased in VPA mice and was significantly correlated with autistic-like behaviors among them. The mRNA and protein levels of the polymeric immunoglobulin receptor (Pigr) were downregulated in the submandibular glands of VPA mice and the Pigr mRNA level was positively correlated with mouse salivary IgA content. HSG cells treated with ASD-related Streptococci had reduced PIGR protein level.Conclusion: Therefore, protective IgA levels were reduced in the saliva of individuals with ASD, which correlated with the bacteria-induced downregulation of Pigr in salivary glands. This study suggests a new direction for ASD diagnosis and prevention of oral diseases in ASD cohorts and provides evidence for the ASD mucosal immunophenotype in the oral cavity.

Association Between N-Desmethylclozapine and Clozapine-Induced Sialorrhea: Involvement of Increased Nocturnal Salivary Secretion via Muscarinic Receptors by N-Desmethylclozapine.

Clozapine-induced sialorrhea (CIS) is a common side effect of clozapine. There is no established standard treatment of CIS since the underlying mechanism remains unknown. This study aimed to elucidate the mechanisms involved in CIS. In our clinical study, a prospective observational study evaluated the association between serum and saliva concentrations of clozapine or its metabolites and Drooling Severity and Frequency Scale (DSFS) score. In our in vivo study, we first developed a new CIS animal model; subsequently, we measured salivary secretion and concentrations of clozapine or its metabolites in the animal model. In our in vitro study, we measured the calcium ion (Ca2+) response to evaluate the effect of clozapine or its metabolites on human salivary gland cell line (HSY cells) and then examined whether their effect was inhibited by atropine. In our clinical study, serum and saliva N-desmethylclozapine concentrations were significantly correlated with nocturnal DSFS score. In our in vivo study, daily single oral administration of 100 mg/kg clozapine for 7 days significantly increased salivary secretion in rats. Furthermore, N-desmethylclozapine concentrations in serum and submandibular glands of the rats were higher than clozapine concentrations. In our in vitro study, N-desmethylclozapine only elicited an increase in the intracellular Ca2+ in HSY cells. N-desmethylclozapine-induced Ca2+ responses were inhibited by atropine. These results suggest that N-desmethylclozapine is implicated in CIS by increasing nocturnal salivation via the muscarinic receptors. Moreover, our developed animal model that reflects CIS in clinical condition plays a key role as a bridge between basic and clinical research. SIGNIFICANCE STATEMENT: Clozapine-induced sialorrhea (CIS) is a severe and frequent adverse reaction, but the mechanism underlying CIS is less well understood. This paper reports that N-desmethylclozapine, a metabolite of clozapine, is implicated in CIS by increasing nocturnal salivation via the muscarinic receptors and that oral administration of clozapine at 100 mg/kg once daily for 7 days to rat is the optimum method for establishing the new animal model reflecting the clinical scenario of CIS.

JAK Inhibitors and Oxidative Stress Control.

Primary Sjögren's syndrome (SjS) is a complex autoimmune epithelitis, with few treatment options, but the use of Janus kinase (JAK) inhibitors is promising because suppression of the JAK/signal transducer and activator of transcription (STAT) pathway improves sicca manifestations. Playing a primary and pathogenic role in disease development, the oxidative stress response is upregulated in activated salivary gland epithelial cells (SGECs) from patients with SjS. Therefore, the aim of this study was to investigate whether JAK inhibitors would suppress SGEC activation in response to an oxidative stress. For this purpose, the human salivary gland (HSG) cell line was used, and cells were treated with the reactive oxygen species (ROS) inducer hydrogen peroxide (H2O2) or with interferons (IFN Type I and Type II), used as positive controls, to mimic activated SGECs as observed in SjS patients. Afterward, the levels of the intracellular adhesion molecule-1 (ICAM-1) and the regulatory programmed-death ligand-1 (PD-L1) were measured by real-time PCR and flow cytometry, and the STAT1/3 phosphorylation status was assessed by Western blotting. Using the HSG cell line, our results showed that both ICAM-1 and PD-L1 are induced by ROS through pSTAT3, and that this activation pathway is reversed by the use of JAK inhibitors, AG490 and ruxolitinib, as well as by N-acetylcysteine, which is a direct inhibitor of ROS. These findings open new perspectives regarding the pathogenesis and therapeutic possibilities for SjS.

Dysregulated interleukin 11 in primary Sjögren's syndrome contributes to apoptosis of glandular epithelial cells.

The purpose of this study was to explore the potential function of interleukin-11 (IL-11) in the pathogenesis of primary Sjögren's syndrome (pSS) patients. Real-time polymerase chain reaction was performed to examine IL-11 expression in the labial glands of 30 pSS patients and 30 healthy controls. Immunohistochemistry was conducted to assess the distribution of IL-ll-positive cells in labial glands. The human salivary gland (HSG) cell line was used to study the effects of IL-11 on gland epithelial cells in vitro. Cell viability and cell proliferation were examined by CCK-8 kit and EdU assay, respectively. The population of apoptotic cells was detected in flow cytometry followed by Annexin V/PI and Hoechst staining. We found that the expression levels of IL-11 were remarkably decreased in pSS labial glands and were positively correlated with C-reactive protein levels and negatively correlated with rheumatoid factor levels. Fewer numbers of glandular epithelial cells were observed to be positively stained with IL-11 antibody in labial glands from pSS patients than those in healthy control patients. After IL-11 treatment, the viability and proliferation of HSG cells were significantly higher than those in the control group. The total apoptotic and necrotic rates of HSG cells in the group after IL-11 treatment were significantly lower. In conclusion,the results indicated that IL-11 promoted viability and proliferation and inhibited apoptotic and necrotic rates of glandular epithelial cells. In pSS, downregulated IL-11 might contribute to the apoptosis of salivary gland epithelial cells. However, it might be a potential target to alleviate the pathological atrophy of glandular epithelial cells in pSS patients.

MMP-9 Inhibition Suppresses Interferon-γ-Induced CXCL10 Production in Human Salivary Gland Ductal Cells.

Gene expression profiling of lip salivary gland (LSG) has shown that C-X-C motif chemokine 10 (CXCL10) and matrix metalloproteinase 9 (MMP9) expression is upregulated in primary Sjögren's syndrome (pSS) patients. Although CXCL10 and MMP-9 are both associated with pSS pathogenesis, the potential relationship between these two factors has not been investigated. In this study, we used LSG sections from pSS patients and human salivary gland cell lines to investigate the relationship between CXCL10 and MMP-9. Immunofluorescence analyses revealed that CXCL10 and MMP-9 were co-expressed in the LSG of pSS patients, particularly in expanded ductal cells. Furthermore, RT-qPCR analyses on human salivary gland ductal NS-SV-DC cells confirmed that CXCL10 expression was induced by interferon (IFN)-γ, whereas that of MMP9 was stimulated by IFN-α, tumor necrosis factor-α, and interleukin-1β. Remarkably, MMP-9 inhibition in IFN-γ-stimulated NS-SV-DC cells significantly decreased CXCL10 mRNA and secreted protein levels. Further analyses established that MMP-9 inhibition in IFN-γ-stimulated NS-SV-DC cells decreased STAT1 phosphorylation and hence suppressed IFN-γ signaling. Collectively, these results suggest that in addition to its reported role in the destruction of acinar structures, MMP-9 is involved in the IFN-γ-induced production of CXCL10 in pSS lesions. We believe that our findings open the door to the development of novel treatments for pSS, based on the modulation of MMP-9 activity.

JAK Inhibitors Suppress Innate Epigenetic Reprogramming: a Promise for Patients with Sjögren's Syndrome.

Pathogenesis of primary Sjögren's syndrome (SjS) remains obscure. However, recent data demonstrate the implication of epigenetic alterations in the DNA methylation/hydroxymethylation process in SjS mostly affecting genes regulated by two innate cytokines, interferon α (IFNα) and IFNγ as well as the oxidative stress pathways. The Janus kinase (JAK) signal transducer and activator of transcription (STAT) pathway is known to be activated by IFN and reactive oxygen species (ROS). This prompts us to test the potential implication of JAK/STAT signaling on DNA methylation/hydroxymethylation alterations in SjS. For this purpose, the human salivary gland (HSG) cell line was used and cells were treated with both types of IFNs and H2O2 to mimic activated salivary gland epithelial cells (SGEC) as observed in SjS patients. Afterwards, the global DNA level of methylcytosine and hydroxymethylcytosine, the expression of the DNA methylating enzymes (DNMTs) and ten-eleven translocation (TETs) methyl cytosine dioxygenase that controls DNA hydroxymethylation, both at transcriptional and at protein level, as well as STAT phosphorylation and ROS status were determined. Our results showed that expression of TET3 and in turn global DNA hydroxymethylation is controlled through the induction of STAT3 mediated by IFNα, IFNγ, and H2O2. On the other hand, treatment with JAK inhibitors (AG490 and ruxolitinib) reverses this process, suggesting a novel treatment pathway for patients with autoimmune diseases and Sjögren's syndrome.

Upregulation of IL‐6 expression in human salivary gland cell line by IL‐17 via activation of p38 MAPK, ERK, PI3K/Akt, and NF‐κB pathways

The human salivary gland (HSG) cell line has so far been used as invitro models for study of the influence of cytokines and pharmacologic agents on salivary glands, as well as a model system for inflammation in Sjögren's syndrome (SS). This study aimed to determine the effect of IL-17 on IL-6 production and the underlying molecular mechanism regulated by the HSG cell line. Immunofluorescence analyses, RT-PCR, and Western blot were conducted to evaluate the IL-17 receptor (IL-17R) expression in cultured HSG cells. Real-time PCR and ELISA were then utilized to establish the mRNA and protein levels of IL-6 in IL-17-stimulated HSG cells. Western blot, flow cytometry, immunofluorescence, and inhibitor analyses were conducted to elucidate the involved signaling pathways. The HSG cells reliably expressed the IL-17R mRNA and its encoded surface-bound protein. The expression of IL-6 mRNA and protein was upregulated by stimulation of HSG cells with IL-17; this effect was impeded by IL-17- or IL-17R-neutralizing antibodies. IL-17 stimulation ended up with the fast phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), Akt, and translocation of nuclear factor-kappaB (NF-κB) in the HSG cells. p38 MAPK, Akt, and NF-κB inhibitors significantly subdued IL-6 generation in HSG cells stimulated by IL-17. PD98059, an ERK inhibitor, decreased IL-6 generation under low dose of IL-17 but not with high dose. The HSG cells expressed IL-17R and reacted to IL-17 to generate IL-6 via the stimulation of ERK, p38 MAPK, Akt, and NF-κB signaling pathways.

Cross-contamination of the human salivary gland HSG cell line with HeLa cells: A STR analysis study.

The human salivary gland (HSG) cell line, labeled as a submandibular ductal cell line, is commonly used as in vitro models to study radiation therapy, Sjögren's syndrome, pleomorphic adenoma, mucocele, epithelial-to-mesenchymal transition, and epigenetics. However, the American Type Culture Collection (ATCC) has recently released a list of cross-contaminated cell lines that included HSG. Despite this notice, some research laboratories still use HSG as a salivary cell model. Therefore, this study examined the authenticity of HSG sampled from three different laboratories. DNA was extracted from HSG and additional salivary cell lines (NS-SV-AC, NS-SV-DC, A253, HSY) and submitted for cell line authentication with short tandem repeat (STR) analysis. All HSG samples had STR profiles indicating >80% match with HeLa in both the ATCC and Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) databases. This confirmed that HSG sampled from three different laboratories and HSY shared a common ancestry (host) with HeLa, whereas NS-SV-AC, NS-SV-DC, and A253 had unique STR profiles. Short tandem repeat analysis revealed that HSG was contaminated by the HeLa cell line. Furthermore, because genotyping of the original HSG cell line was not performed during its establishment, it will be difficult to authenticate an uncontaminated sample of HSG.

Cepharanthine Inhibits IFN-γ-Induced CXCL10 by Suppressing the JAK2/STAT1 Signal Pathway in Human Salivary Gland Ductal Cells.

Cepharanthine, a biscolaurine alkaloid isolated from the plant Stephania cephalantha Hayata, has been reported to have potent anti-inflammatory properties. Here, we investigated the effects of cepharanthine on the expression of CXCL10 (a CXC chemokine induced by interferon-gamma [IFN-γ] that has been observed in a wide variety of chronic inflammatory disorders and autoimmune conditions) in IFN-γ-treated human salivary gland cell lines. We observed that IFN-γ-induced CXCL10 production in NS-SV-DC cells (a human salivary gland ductal cell line), but not in NS-SV-AC cells (a human salivary gland acinar cell line). Cepharanthine inhibited the IFN-γ-induced CXCL10 production in NS-SV-DC cells. A Western blot analysis showed that cepharanthine prevented the phosphorylation of JAK2 and STAT1, but did not interfere with the NF-κB pathway. Moreover, cepharanthine inhibited the IFN-γ-mediated chemotaxis of Jurkat T cells. These results suggest that cepharanthine suppresses IFN-γ-induced CXCL10 production via the inhibition of the JAK2/STAT1 signaling pathway in human salivary gland ductal cells. Our findings also indicate that cepharanthine could inhibit the chemotaxis of Jurkat T cells by reducing CXCL10 production.

P2X7 receptor and NLRP3 inflammasome activation in head and neck cancer.

In this study, we investigated purinergic receptor P2X7 and NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome expressions, and their role in head and neck cancer. We found upregulation of purinergic receptor P2X7 and all NLRP3 inflammasome components in biopsied head and neck squamous cell carcinoma tissues. Similarly, the expression of purinergic receptor P2X7, apoptosis-associated speck-like protein containing CARD, and pro-form caspase 1 in A253 cells derived from epidermoid carcinoma were highly upregulated in comparison to normal Human Salivary Gland cell line. Active caspase-1 and its final product, active interleukin-1β, both increased in primed A253 cells stimulated with purinergic receptor P2X7 agonists, while this elevated NLRP3 inflammasome activity was suppressed by purinergic receptor P2X7 antagonists. However, we observed none of these effects in Human Salivary Gland cells. Inhibition of both NLRP3 inflammasome and purinergic receptor P2X7 led to the significant cell death of primed A253 cells, but had no effect on the viability of primed HSG cells or the primary cultured human fibroblast cells. Furthermore, inhibition of either purinergic receptor P2X7 or NLRP3 inflammasome decreased invasiveness of A253, and this effect became more evident when both purinergic receptor P2X7 and NLRP3 inflammasome were simultaneously blocked. Therefore, it is concluded that the purinergic receptor P2X7 and the activation of NLRP3 inflammasome play important roles in the survival and invasiveness of head and neck squamous cell carcinoma in humans.

The In Vitro Response of Tissue Stem Cells to Irradiation With Different Linear Energy Transfers

A reduction in the dose, irradiated volume, and sensitivity of, in particular, normal tissue stem cells is needed to advance radiation therapy. This could be obtained with the use of particles for radiation therapy. However, the radiation response of normal tissue stem cells is still an enigma. Therefore, in the present study, we developed a model to investigate the in vitro response of stem cells to particle irradiation. We used the immortalized human salivary gland (HSG) cell line resembling salivary gland (SG) cells to translate the radiation response in 2-dimensional (2D) to 3-dimensional (3D) conditions. This response was subsequently translated to the response of SG stem cells (SGSCs). Dispersed single cells were irradiated with photons or carbon ions at different linear energy transfers (LETs; 48.76 ± 2.16, 149.9 ± 10.8, and 189 ± 15 keV/μm). Subsequently, 2D or 3D clonogenicity was determined by counting the colonies or secondary stem cell-derived spheres in Matrigel. γH2AX immunostaining was used to assess DNA double strand break repair. The 2D response of HSG cells showed a similar increase in dose response to increasing higher LET irradiation as other cell lines. The 3D response of HSG cells to increasing LET irradiation was reduced compared with the 2D response. Finally, the response of mouse SGSCs to photons was similar to the 3D response of HSG cells. The response to higher LET irradiation was reduced in the stem cells. Mouse SGSC radiosensitivity seems reduced at higher LET radiation compared with transformed HSG cells. The developed model to assess the radiation response of SGSCs offers novel possibilities to study the radiation response of normal tissue in vitro.

Characterization of Long-Term Cultured Murine Submandibular Gland Epithelial Cells

PurposeHuman and rat salivary gland cell lines derived from tumors or genetic modification are currently available for research. Here, we attempted to culture and characterize long-term cultured cells spontaneously derived from wild type murine submandibular glands (SGs).MethodsSGs were removed from 3-week-old C57B/6J female mice and dissociated by collagenase type 1 and hyaluronidase digestion. Isolated SG epithelial cells were cultured in low calcium, serum-free growth media in the presence of cholera toxin (CT) during early passages. Single-cell colonies were isolated by limiting dilution culture after 25 passages. Early- and late-stage cell cultures were characterized for keratin 14, keratin 18, α-smooth muscle actin, and p63 by immunostaining and quantitative real-time PCR analysis.ResultsSG epithelial cells cultured in optimized media maintained their proliferative ability and morphology for over 80 passages. Long-term cultured cells expressed keratin 14, keratin 18, and p63, indicative of an epithelial phenotype.ConclusionsEpithelial cells originating from wild type murine SGs could be cultured for longer periods of time and remain phenotypically similar to ductal basal epithelium.

Defective DNA methylation in salivary gland epithelial acini from patients with Sjögren's syndrome is associated with SSB gene expression, anti-SSB/LA detection, and lymphocyte infiltration

The pathogenesis of primary Sjögren's syndrome (pSS) is complex, in part due to DNA methylation abnormalities. This study was undertaken to evaluate the importance of global DNA methylation (5mC) as determined in minor salivary glands (MSG) from well characterized pSS patients. Twenty-two pSS patients and ten controls were selected, and MSG were stained with anti-5mC, anti-5mC/anti-cytokeratin (KRT)19, or with anti-SSB/La antibodies (Ab). The DNA methylation status at the SSB gene promoter P1 and P1′ was evaluated by methylation-sensitive restriction enzymes (MSRE) coupled with PCR. The effect of the DNA demethylating drug 5 azacytidine (5-Aza) was tested in the human salivary gland (HSG) cell line. In pSS, the reduction of global DNA methylation (5mC) was associated with lymphocyte infiltration, the emergence of 5mClow and KRT19high acini, and the detection of circulating anti-SSB/La Ab, but not with disease activity (ESSDAI). Next, treating HSG cells with 5-Aza was effective in inducing SSB expression. Finally in pSS patients positive for anti-SSB/La Ab, we further observed DNA demethylation at the SSB gene promoter P1 with consequent SSB overexpression at both the transcriptional and protein levels in salivary gland epithelial cells. In conclusion, our results highlight the importance of DNA methylation in the pathophysiology of pSS and to the emergence of anti-SSB/La Ab.