HSG Cell Research Articles

PANoptosis induced by inflammatory cytokines promotes salivary glands radiation injury.

Salivary glands are frequently damaged in patients undergoing radiotherapy for head and neck cancer. Whether PANoptosis, which is characterized by pyroptosis, apoptosis, and necroptosis, occurs during radiation injury to the salivary glands and its role remain unclear. Radiation-induced injury models of mouse submandibular gland, as well as primary acinar cells and HSG cell lines were established to determine the presence of radiation-induced PANoptosis. Several programmed cell death inhibitors, PFTα, disulfiram, Nec-1 and zVAD, were used to compare the effects of different cell death pathway on radiation injury. The LEGENDplex™ Human Inflammation Panel was used to characterize the inflammatory landscape secreted by salivary gland cells after radiotherapy. Single 15Gy or 8Gy radiotherapy triggered PANoptosis in mouse submandibular gland or salivary gland cells. Compared to the suppression of pyroptosis, apoptosis, or necroptosis alone, the inhibition of PANoptosis is more effective in preventing radiation injury to the salivary glands (p < 0.0001). The levels of multiple inflammatory cytokines were significantly up-regulated in the supernatants of HSG cells within 48 h after IR. Neutralizing inflammatory cytokines are capable of inhibiting salivary glands PANoptosis. Inhibition of PANoptosis induced by inflammatory cytokines can effectively prevent radiation injury of salivary glands.

A methodology to abridge microdosimetric distributions without a significant loss of the spectral information needed for the RBE computation in carbon ion therapy.

In order to compute the relative biological effectiveness (RBE) of ion radiation therapy with the Mayo Clinic Florida microdosimetric kinetic model (MCF MKM), it is necessary to process entire microdosimetric distributions. Therefore, a posteriori RBE recalculations (i.e., for a different cell line or another biological endpoint) would require whole spectral information. It is currently not practical to compute and store all this data for each clinical voxel. To develop a methodology that allows to store a limited amount of physical information without losing accuracy in the RBE calculations nor the possibility of a posteriori RBE recalculations. Computer simulations for four monoenergetic 12 C ion beams and a 12 C ion spread-out Bragg peak (SOBP) were performed to assess lineal energy distributions as a function of the depth within a water phantom. These distributions were used in combination with the MCF MKM to compute the in vitro clonogenic survival RBE for human salivary gland tumor cells (HSG cell line) and human skin fibroblasts (NB1RGB cell line). The RBE values were also calculated with a new abridged microdosimetric distribution methodology (AMDM) and compared with the reference RBE calculations using the entire distributions. The maximum relative deviation between the RBE values computed using the entire distributions and the AMDM was 0.61% (monoenergetic beams) and 0.49% (SOBP) for the HSG cell line, while 0.45% (monoenergetic beams) and 0.26% (SOBP) for the NB1RGB cell line. The excellent agreement between the RBE values computed using the entire lineal energy distributions and the AMDM represents a milestone for the clinical implementation of the MCF MKM.

The Mayo Clinic Florida microdosimetric kinetic model of clonogenic survival: formalism and first benchmark against in vitro and in silico data

Objective. To develop a new model (Mayo Clinic Florida microdosimetric kinetic model, MCF MKM) capable of accurately describing the in vitro clonogenic survival at low and high linear energy transfer (LET) using single-event microdosimetric spectra in a single target. Methodology. The MCF MKM is based on the ‘post-processing average’ implementation of the non-Poisson microdosimetric kinetic model and includes a novel expression to compute the particle-specific quadratic-dependence of the cell survival with respect to dose (β of the linear-quadratic model). A new methodology to a priori calculate the mean radius of the MCF MKM subnuclear domains is also introduced. Lineal energy spectra were simulated with the Particle and Heavy Ion Transport code System (PHITS) for 1H, 4He, 12C, 20Ne, 40Ar, 56Fe, and 132Xe ions and used in combination with the MCF MKM to calculate the ion-specific LET-dependence of the relative biological effectiveness (RBE) for Chinese hamster lung fibroblasts (V79 cell line) and human salivary gland tumor cells (HSG cell line). The results were compared with in vitro data from the Particle Irradiation Data Ensemble (PIDE) and in silico results of different models. The possibility of performing experiment-specific predictions to explain the scatter in the in vitro RBE data was also investigated. Finally, a sensitivity analysis on the model parameters is also included. Main results. The RBE values predicted with the MCF MKM were found to be in good agreement with the in vitro data for all tested conditions. Though all MCF MKM model parameters were determined a priori, the accuracy of the MCF MKM was found to be comparable or superior to that of other models. The model parameters determined a priori were in good agreement with the ones obtained by fitting all available in vitro data. Significance. The MCF MKM will be considered for implementation in cancer radiotherapy treatment planning with accelerated ions.

Protein kinase D1 induced epithelial–mesenchymal transition and invasion in salivary adenoid cystic carcinoma via E‐cadherin/Snail regulation

Salivary adenoid cystic carcinoma (SACC) is a malignant tumor, which is characterized by a higher incidence of distant metastasis. The aim of this study was to investigate the role and mechanism of protein kinase D1 (PKD1) in regulating the epithelial-mesenchymal transition (EMT) and promotes the metastasis in SACC. We analyzed the expression of PKD1 in 40 SACC patients and different metastatic potential cell lines. Then, we investigated whether the migration and growth of SACC were regulated by PKD1 using shRNA interference or inhibition of kinase active in vitro cell. Moreover, the mechanism by which PKD1 regulates the stability of Snail protein was determined. Finally, nude mice were used to testify the function of PKD1 via tail vein injection. PKD1 was correlated with metastasis and poor prognosis of SACC patients. PKD1 inhibition attenuated proliferation, migration, invasion, and EMT of SACC cells. Conversely, kinase active PKD1 could induce EMT and promoted cell migration in human HSG cell. Furthermore, downregulation of PKD1 regulated Snail via phosphorylation at Ser-11 on Snail protein and promotion of proteasome-mediated degradation, and reduced lung metastasis in vivo. Our results suggest that PKD1 induces the EMT and promotes the metastasis, which illustrate that PKD1 may be a potential prognostic biomarker and serve as a potential therapeutic target for SACC patients.

Nerve growth factor protects salivary glands from irradiation-induced damage

AimsRadiotherapy has become a basic treatment modality for head and neck cancer. However, radiotherapy results in inevitable side effects, particularly radiation sialadenitis, that significantly impairs quality of life. A previous study indicated that nerve growth factor (NGF) has a radio-protective effect, but the mechanism was not determined in salivary glands. In this study, we explored the functional role and mechanism regarding how NGF protects salivary glands against IR-induced damage. Main methodsHuman salivary gland (HSG) cells and C57BL/6 mice were selected to establish an IR-induced salivary gland damage model in vitro and in vivo. Recombinant NGF protein and NGF siRNA and over-expression plasmids were applied to manipulate NGF expression in vitro. AAV-NGF was retrogradely perfused into the submandibular gland (SMG) through the SMG duct to manipulate NGF expression in vitro. Small-molecule inhibitors and siRNAs were applied to inhibit AKT and JNK. Western blotting, quantitative PCR, flow cytometry and histology assays were performed to analyse the functional role and mechanism of NGF. Key findingsOur study demonstrated that NGF expression was upregulated following radiotherapy both in human HSG cells and mouse SMG tissues. NGF could reduce IR-induced HSG cell apoptosis, and AAV-mediated gene therapy could restore the salivary flow rate and protect the salivary gland against IR-induced apoptosis in vivo. Mechanistically, NGF protects salivary glands from IR-induced apoptosis by de-phosphorylating JNK kinase rather than promoting AKT phosphorylation. SignificanceThe current study findings indicated that the modulation of the NGF pathway might prevent IR-induced salivary hypo-function.

Abstract 1353: Role of TLK1B/NEK1 axis in DNA DSB repair: Implications for prostate cancer progression

Abstract Defects in DNA Damage Response and Repair are linked to the majority of cancers, including Prostate Cancer (PCa). Common genotoxic stresses that cause damage to the DNA, or replication fork collapse are generated when PCa cells face Androgen Deprivation Therapy (ADT), and must be bypassed to allow continuous replication and division. Tousled like kinase (TLK1) splice variant TLK1B is implicated in DNA damage repair pathways and is translationally increased following various stresses, including the DDR. We found that the expression of TLK1B is rapidly increased following a shift of LNCaP cells to charcoal-stripped serum (ADT), via an activation of mTOR and phosphorylation of 4EBP1 (inhibitor of the translation factor eIF4E). We recently uncovered the existence of the important DDR axis TLK1B&amp;gt; NEK1&amp;gt; ATR&amp;gt;Chk1.(1). TLK1 phosphorylates NEK1 at T141 and activates its autophosphorylation of Y315. A defect in DNA repair in NEK1-deficient cells is suggested by persistence of DNA double strand breaks (DSB) after low dose ionizing radiation (IR).(2). Cells repair DSB either via HR or NHEJ. We investigated whether TLK1 phosphorylation is essential for NEK1 activity in HR repair. siRNA-mediated NEK1 deficient cells have decreased kinetics of Rad54-S572 phosphorylation and persistent Rad51 foci when induced with Doxorubicin that creates DSB.(3). We tested a NEK1-T141A overexpressing (Hek293) mutant for its effect on Rad54-S572 phosphorylation (pRad54) during a time course of recovery from Doxorubicin. There was no significant delay in pRad54 kinetics compared to the control, unless endogenous (WT) NEK1 was knocked down, in which case we observed a significant delay in pRad54 in NEK1 mutant cells. When DSBs occur during DNA damage, Rad51 becomes localized at DSB repair foci. Delay in Rad54 phosphorylation results in persistent Rad51 foci since aspects of HR subsequent to Rad51 filament formation and strand invasion are impaired. We observed increased Rad51 foci that persisted in NEK1-T141A mutants following two hours of Doxorubicin treatment and 14 hours of recovery. A quantitative approach to monitor HR in NEK1-T141A mutants was obtained with the generation of a stable DR-GFP recombination reporter substrate (4) in HSG cell line. Transfection with the megabase cutter endonuclease I-SceI, creates a DSB in a defective double GFP cassette which would generate a functional GFP gene conversion product. Results indicate that approximately 14-17 % cells from parent population undergoes HR in WT, NEK1 overexpressing, and NEK1-T141A mutants. We would expect statistically significant conclusion from improvising GFP reporter experiments with cells in which endogenous Nek1 and Rad54 is knocked down, consistent with the results obtained with pRad54. Another approach would be inhibiting TLK1B interaction with NEK1 to block HR repair which is thought to be critical in Prostate Cancer progression to Castration Resistant Prostate Cancer. Citation Format: Ishita Ghosh, Arrigo DeBenedetti, Gulshan Sunavala. Role of TLK1B/NEK1 axis in DNA DSB repair: Implications for prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1353.

Small Integrin-Binding Ligand N-Linked Glycoproteins (Siblings): A Study On Human Salivary Gland Cancer

Salivary gland carcinomas constitute a rare but deadly group of head and neck cancers, but timely diagnosis is often delayed due to inherent variability in etiology, heterogeneity and histopathological characterization. SIBLINGs are a family of secreted glycophosphoproteins that include osteopontin (OPN), bone sialoprotein (BSP), dentin sialophosphoprotein (DSPP), dentin matrix protein-1 (DMP-1), and matrix extracellular phosphoglycoprotein (MEPE). SIBLINGs were first discovered in bone and teeth, and were considered to be exclusively expressed in mineralized tissue. In addition to mineralized tissue, SIBLINGs have now been shown to have variable expression in normal, non-mineralized tissue and in cancers. However, there have been no studies evaluating SIBLING expression in human salivary gland cancers. Our study tested the hypothesis that SIBLINGs, specifically, BSP, DSPP and OPN, would be significantly overexpressed in human salivary gland cancer. We also hypothesized that the cancer secretome would influence SIBLING expression in normal salivary gland cells. Methods: Normal and cancerous human salivary gland tissue obtained from the NDRI were processed using routine immunohistochemistry techniques to evaluate expression of BSP, DSP, and OPN. In addition normal HSG cell line and cancer HTB-41 cell line were evaluated using immunofluorescence techniques to localize expression of BSP, DSP and OPN. Normal HSG, cancer HTB-41 and HSG* cells (normal HSG cells exposed to a cancer HTB-41 secretome) were propagated using routine cell culture techniques for 24, 48, and 72 hours. Western blotting techniques were utilized ii to quantify and compare SIBLING protein expression levels in HSG, HTB-41 and HSG* cells. Normal HSG, cancer HTB-41, and HSG* cells were processed via immunoflourescence in order to observe localization of SIBLINGs. Results: Immunohistochemistry and western blot showed increased expression of SIBLINGs in human salivary gland cancers. Furthermore, immunoflourescence revealed distinct localization of SIBLING proteins in HSG and HTB-41 cell lines. In terms of HSG*, it was found that cells exposed to cancer secretome exhibited similar SIBLING expression to HTB-41. Conclusion: Our studies confirm that SIBLING proteins are selectively expressed in human salivary gland cancer. Also, the cancer secretome is found to affect SIBLING expression in normal cells, similar to HTB-41 cancer cell lines.

Epigenetic regulation of CFTR in salivary gland

Cystic fibrosis transmembrane conductance regulator (CFTR) plays a key role in exocrine secretion, including salivary glands. However, its functional expression in salivary glands has not been rigorously studied. In this study, we investigated the expression pattern and regulatory mechanism of CFTR in salivary glands using immunohistochemistry, western blot analysis, Ussing chamber study, methylation-specific PCR, and bisulfite sequencing. Using an organ culture technique, we found that CFTR expression was first detected on the 15th day at the embryonic stage (E15) and was observed in ducts but not in acini. CFTR expression was confirmed in HSG and SIMS cell lines, which both originated from ducts, but not in the SMG C-6 cell line, which originated from acinar cells. Treatment of SMG C-6 cells with 5-aza-2'-deoxycytidine (5-Aza-CdR) restored the expression level of CFTR mRNA in a time-dependent manner. Restoration of CFTR was further confirmed by a functional study. In the Ussing chamber study, 10 μM Cact-A1, a CFTR activator, did not evoke any currents in SMG C-6 cells. In contrast, in SMG C-6 cells pretreated with 5-Aza-CdR, Cact-A1 evoked a robust increase of currents, which were inhibited by the CFTR inhibitor CFTRinh-172. Furthermore, forskolin mimicked the currents activated by Cact-A1. In our epigenetic study, SMG C-6 cells showed highly methylated CG pairs in the CFTR CpG island and most of the methylated CG pairs were demethylated by 5-Aza-CdR. Our results suggest that epigenetic regulation is involved in the development of salivary glands by silencing the CFTR gene in a tissue-specific manner.

Synthesis of functionalized gold nanoparticles capped with 3-mercapto-1-propansulfonate and 1-thioglucose mixed thiols and “in vitro” bioresponse

The synthesis, characterization and assessment of biological behavior of innovative negatively charged functionalized gold nanoparticles is herein reported, for potential applications in the field of radiotherapy and drug delivery. Gold nanoparticles (AuNPs) functionalized with two capping agents, i.e., the 3-mercapto-1-propansulfonate (3-MPS) and 1-β-thio-D-glucose (TG), have been on purpose synthesized and fully characterized. Advanced characterization techniques including X-Ray Photoelectron Spectroscopy (XPS) were applied to probe the chemical structure of the synthesized nanomaterials. Z-potential and Dynamic Light Scattering measurements allowed assessing the nanodimension, dispersity, surface charge and stability of AuNPs. Transmission Electron Microscopy (TEM) and Flame Atomic Absorption Spectroscopy (FAAS) were applied to the “in vitro” HSG cell model, to investigate the nanoparticles-cells interaction and to evaluate the internalization efficiency, whereas short term cytotoxicity and long term cell killing were evaluated by means of MTT and SRB assays, respectively. In conclusion, in order to increase the amount of gold atoms inside the cell we have optimized the synthesis for a new kind of biocompatible and very stable negatively charged TG-functionalized nanoparticles, with diameters in a range that maximize the uptake in cells (i.e., ∼15nm). Such particles are very promising for radiotherapy and drug delivery application

MTA1 regulation of ERβ pathway in salivary gland carcinoma cells

Although Metastatic-tumor antigen 1 (MTA1) is differentially expressed in metastatic cancer and coregulates the status and activity of nuclear receptors, its role upon estrogen receptor β (ERβ) - a potent tumor suppressor, remains poorly understood. Here we investigated whether MTA1 regulates the expression and functions of ERβ, an ER isoform predominantly expressed in salivary gland cancer cells. We found that the depletion of the endogenous MTA1 in the HSG and HSY salivary duct carcinoma cell lines enhances the expression of ERβ while MTA1 overexpression augmented the expression of ERβ in salivary duct carcinoma cells. Furthermore, MTA1 knockdown inhibited the proliferations and invasion of HSG and HSY cells. The noted ERβ downregulation by MTA1 overexpression involves the process of proteasomal degradation, as a proteasome inhibitor could block it. In addition, both MTA1 knockdown and ERβ overexpression attenuated the cell migration and inhibited the ERK1/2 signaling in the both cell lines. These findings imply that MTA1 dysregulation in a subset of salivary gland cancer might promote aggressive phenotypes by compromising the tumor suppressor activity of ERβ, and hence, MTA1-ERβ axis might serve a new therapeutic target for the salivary gland cancer.

Remodeling of the Cytoskeleton and Regulation of Store-Operated Calcium Entry

Store-operated calcium entry (SOCE) is a critical Ca2+ entry and is activated by ER-Ca2+ store depletion while refilling the stores leads inactivation of the process. STIM1 is the main Ca2+ sensor protein in the ER that responds to store depletion, aggregates and translocates to ER-PM junctional domains, where it interacts with, and activates, the channels mediating SOCE, such as Orai1. However, little is known about other proteins that interact with STIM1 to facilitate the regulation of SOCE. In this study, we identified 155 specific STIM1 binding partners using a shotgun proteomic approach on STIM1 immunoprecipitated complex from HSG cell lysates. In order to determine quantitative changes in the STIM1 proteome during stimulation status, mixtures of lysates from control cells and stimulated cells with Tg were analyzed using a SILAC (Stable Isotope Labeling by Amino acids in Cell culture) approach. Our analysis reveal several interesting changes in the STIM1 proteome upon stimulation. As a result of the SILAC findings, we found that cytoskeletal interacting/remodeling proteins such as CDC42, ARP2, N-WASP, and zyxin interact with STIM1 and Orai1. In addition to inducing changes in cell morphology and actin cytoskeleton, some of these also significantly impact SOCE. Together, our data suggest that cytoskeletal remodeling has an important role in the assembly of the Orai1-STIM1 complex and regulation of SOCE.

SU‐E‐T‐26: A Study On the Influence of Photonuclear Reactions On the Biological Effectiveness of Therapeutic High Energy X‐Ray Beam

Purpose:Photons from a modern high‐energy therapeutic linear accelerator used in X‐ray radiotherapy causes photonuclear reactions in an accelerator or patient's body. The aim of this study is to evaluate the biological effectiveness including these particles by Microdosimetric Kinetic Model (MKM) based on microdosimetry.Methods:A linear accelerator operating at 15 MV was used. CR‐39 was used to obtain LET spectra of secondary ions selectively, as CR‐39 is regarded insensitive to photons. CR‐39 was put on the central axis of the X‐ray beam at depths of 0, 5 and 10 cm in plastic phantom at a source to detector distance of 100 cm. Pits formed by the traversal of ions were etched then analyzed to obtain restricted LET distribution. Frequency‐mean and dose‐mean lineal energy was evaluated from the relationship between the restricted LET and the lineal energy required to evaluate the biological effectiveness by MKM. The relationship was calculated by Monte Carlo simulations with GEANT4.Results:Restricted LET distributions of secondary particles showed broad distributions that decreases exponentially with increasing LET. Frequency‐mean and dose‐mean lineal energy were determined uniquely within the scope of the energies of secondary particles generated from photons of 15 MeV. The frequency‐mean lineal energies at the depth of 0, 5 and 10 cm were 15.1, 16.0 and 19.7 keV/μm respectively, and the dose‐mean lineal energies were 18.6, 20.5 and 19.6 keV/μm, respectively. RBE of secondary particles for HSG cell evaluated by MKM was about 2.0 at all depths, and RBE of all particles including photons was evaluated 1.0.Conclusion:We investigated the biological effectiveness of secondary particles by photonuclear reactions. The method to evaluate RBE by MKM was established with measurements and simulations. However, the influence of these secondary ions on RBE was found negligible in the entire biological effectiveness of the high‐energy X‐ray.This study has been supported by JSPS KAKENHI Grant Number 25861144.

WE-A-BRA-05: Proton Ultra High Dose-Rate Effect on HSG Cell Survival Curve

Purpose: One of the important issues that we are facing in the current radiation process is the long treatment time for irradiating protons to the tumor moving with respiration. In order to improve this problem, we are currently developing the highly precise and very short time protonIGRT using the high intensity beam from cyclotron and the real‐time images acquired by two flat panel detectors attached to the gantry. The dose‐rate by using this method will reach 10 to 100 times of the present one. The purpose of this study is to investigate the relative biological effectiveness (RBE) of HSG cell in such an ultra high dose‐rate regime and its LET dependence by using the colony assay method. Material and method: We attached the HSG cells at the bottom of the plastic chamber, and irradiated the spatially and temporally homogeneous proton beam. We used 235MeV proton beams with the different beam current of 10nA and 300nA in order to study the dose‐rate effect. The chamber was molded in a Polyethylene block with a hole which fits tightly to the chamber. It was placed at plateau(1.75, 114Gy/min, yD=0.56keV/ m), then at Bragg‐peak(8, 325Gy/min, yD=3.19keV/ m) to see the LET dependence of RBE at high dose‐rate.Result: There were no significant splits observed in survival curves of HSG cell over the protondose‐rate. The ratio of RBE at lower dose‐rate to that at higher dose‐rate was 0.98−+0.08 at Bragg‐peak and was 0.96−+0.11 at plateau. On the other hand, the RBE ratio at Bragg‐peak to plateau was 1.13–1.20, which suggests that the position dependence of RBE cannot be neglected. Conclusion: We conclude that in the therapeutic planning of high dose‐rateradiation, the present RBE can be consistently used. Instead, the RBE enhancement toward the Bragg‐peak and beyond should be reconsidered.

Functional expression of the alpha 2-macroglobulin receptor CD91 in salivary gland epithelial cells

CD91 molecule is a multifunctional receptor of alpha 2-macroglobulin, heat-shock proteins and calreticulin. CD91 has been implicated in cross-presentation of peptides chaperoned by these proteins to MHC molecules, thus eliciting antigen-specific immune responses. Hence, CD91 is considered as a major regulator of innate and acquired immune responses. Herein, we show that CD91 molecules are expressed by human salivary gland epithelial cells (SGEC), as indicated by immunohistochemical studies in minor salivary gland biopsy tissues ( n = 21) as well as by the analyses of human long-term cultured non-neoplastic SGEC lines ( n = 11) and the neoplastic HSG cell line. In these cell lines CD91 expression was evaluated by RT-PCR, flow cytometry and confocal microscopy. Standard internalization assays revealed that HSG and SGECs are capable to bind and internalize the CD91 ligand alpha 2-macroglobulin. This internalization is specific, as attested by inhibition studies using unlabeled alpha 2-macroglobulin and a blocking antibody against human CD91 receptor. Conclusively, our findings indicate that SGEC functionally express CD91 receptor, suggesting that this pathway might be involved in the presentation of exogenous antigens in SGEC.

Histamine H1 Receptor Induces Cytosolic Calcium Increase and Aquaporin Translocation in Human Salivary Gland Cells

One of the common side effects of antihistamine medicines is xerostomia (dry mouth). The current consensus is that antihistamine-induced xerostomia comes from an antimuscarinic effect. Although the effect of antihistamines on salivary secretion is both obvious and significant, the cellular mechanism whereby this happens is still unclear because of the lack of knowledge of histamine signaling in human salivary glands. Here, we have studied histamine receptors and the effect of antihistamines on human submandibular acinar cells. In primary cultured human submandibular gland and a HSG cell line, histamine increased the intracellular Ca(2+) concentration. The histamine-induced cytosolic free Ca(2+) concentration ([Ca(2+)](i)) increase was inhibited by histamine H1 receptor-specific antagonists, and the expression of the functional histamine H1 receptor was confirmed by reverse transcription-polymerase chain reaction. Interestingly, histamine pretreatment did not inhibit a subsequent carbachol-induced [Ca(2+)](i) rise without "heterologous desensitization." Chlorpheniramine inhibited a carbachol-induced [Ca(2+)](i) increase at a 100-fold greater concentration than histamine receptor antagonism, whereas astemizole and cetrizine showed more than 1000-fold difference, which in part explains the xerostomia-inducing potency among the antihistamines. Notably, histamine resulted in translocation of aquaporin-5 to the plasma membrane in human submandibular gland cells and green fluorescent protein-tagged aquaporin-5 expressing HSG cells. We found that histidine decarboxylase and the histamine H1 receptor are broadly distributed in submandibular gland cells, whereas choline acetyltransferase is localized only at the parasympathetic terminals. Our results suggest that human salivary gland cells express histamine H1 receptors and histamine-synthesizing enzymes, revealing the cellular mechanism of antihistamine-induced xerostomia.

Comparison of Biological Effectiveness of Carbon-Ion Beams in Japan and Germany

To compare the biological effectiveness of 290 MeV/amu carbon-ion beams in Chiba, Japan and in Darmstadt, Germany, given that different methods for beam delivery are used for each. Murine small intestine and human salivary gland tumor (HSG) cells exponentially growing in vitro were irradiated with 6-cm width of spread-out Bragg peaks (SOBPs) adjusted to achieve nearly identical beam depth-dose profiles at the Heavy-Ion Medical Accelerator in Chiba, and the SchwerIonen Synchrotron in Darmstadt. Cell kill efficiencies of carbon ions were measured by colony formation for HSG cells and jejunum crypts survival in mice. Cobalt-60 gamma rays were used as the reference radiation. Isoeffective doses at given survivals were used for relative biological effectiveness (RBE) calculations and interinstitutional comparisons. Isoeffective D(10) doses (mean +/- standard deviation) of HSG cells ranged from 2.37 +/- 0.14 Gy to 3.47 +/- 0.19 Gy for Chiba and from 2.31 +/- 0.11 Gy to 3.66 +/- 0.17 Gy for Darmstadt. Isoeffective D(10) doses of gut crypts after single doses ranged from 8.25 +/- 0.17 Gy to 10.32 +/- 0.14 Gy for Chiba and from 8.27 +/- 0.10 Gy to 10.27 +/- 0.27 Gy for Darmstadt, whereas isoeffective D(30) doses after three fractionated doses were 9.89 +/- 0.17 Gy through 13.70 +/- 0.54 Gy and 10.14 +/- 0.20 Gy through 13.30 +/- 0.41 Gy for Chiba and Darmstadt, respectively. Overall difference of RBE between the two facilities was 0-5% or 3-7% for gut crypt survival or HSG cell kill, respectively. The carbon-ion beams at the National Institute of Radiological Sciences in Chiba, Japan and the Gesellschaft für Schwerionenforschung in Darmstadt, Germany are biologically identical after single and daily fractionated irradiation.

Cimetidine inhibits salivary gland tumor cell adhesion to neural cells and induces apoptosis by blocking NCAM expression

BackgroundCimetidine, a histamine type-2 receptor antagonist, has been reported to inhibit the growth of glandular tumors such as colorectal cancer, however the mechanism of action underlying this effect is unknown. Adenoid cystic carcinoma is well known as a malignant salivary gland tumor which preferentially invades neural tissues. We demonstrated previously that human salivary gland tumor (HSG) cells spontaneously express neural cell adhesion molecule (NCAM), that HSG cell proliferation may be controlled via a homophilic (NCAM-NCAM) binding mechanism and that NCAM may be associated with perineural invasion by malignant salivary gland tumors. We further demonstrated that cimetidine inhibited NCAM expression and induced apoptosis in HSG cells. Here, we investigated the effects of cimetidine on growth and perineural/neural invasion of salivary gland tumor cells.MethodsIn this study, we have examined the effect of cimetidine on cancer cell adhesion to neural cells in vitro, one of the critical steps of cancer invasion and metastasis. We have also used an in vivo carcinogenesis model to confirm the effect of cimetidine.ResultsWe have demonstrated for the first time that cimetidine can block the adhesion of HSG cells to neural cell monolayers and that it can also induce significant apoptosis in the tumor mass in a nude mouse model. We also demonstrated that these apoptotic effects of cimetidine might occur through down-regulation of the cell surface expression of NCAM on HSG cells. Cimetidine-mediated down-regulation of NCAM involved suppression of the nuclear translocation of NF-κB, a transcriptional activator of NCAM gene expression.ConclusionThese findings suggest that growth and perineural/neural invasion of salivary gland tumors can be blocked by administration of cimetidine via induction of apoptosis and in which NCAM plays a role.

Lipid Rafts Determine Clustering of STIM1 in Endoplasmic Reticulum-Plasma Membrane Junctions and Regulation of Store-operated Ca2+ Entry (SOCE)

Store depletion induces STIM1 to aggregate and relocate into clusters at ER-plasma membrane junctions where it functionally interacts with and activates plasma membrane channels that mediate store-operated Ca(2+) entry (SOCE). Thus, the site of peripheral STIM1 clusters is critical for the regulation of SOCE. However, what determines the location of the STIM1 clusters in the ER-PM junctional regions, and whether these represent specific sites in the cell is not yet known. Here we report that clustering of STIM1 in the subplasma membrane region of the cell and activation of TRPC1-dependent SOCE are determined by lipid raft domains (LRD). We show that store depletion increased partitioning of TRPC1 and STIM1 into plasma membrane LRD. TRPC1 and STIM1 associated with each other within the LRD, and this association was dynamically regulated by the status of the ER Ca(2+) store. Peripheral STIM1 clustering was independent of TRPC1. However, sequestration of membrane cholesterol attenuated thapsigargin-induced clustering of STIM1 as well as SOCE in HSG and HEK293 cells. Recruitment and association of STIM1 and TRPC1 in LRD was also decreased. Additionally STIM1(D76A), which is peripherally localized and constitutively activates SOCE in unstimulated cells, displayed a relatively higher partitioning into LRD and interaction with TRPC1, as compared with STIM1. Disruption of membrane rafts decreased peripheral STIM1(D76A) puncta, its association with TRPC1 and the constitutive SOCE. Together, these data demonstrate that intact LRD determine targeting of STIM1 clusters to ER-plasma membrane junctions following store depletion. This facilitates the functional interaction of STIM1 with TRPC1 and activation of SOCE.

Cimetidine induces apoptosis of human salivary gland tumor cells

It has been reported that cimetidine, a histamine type-2 receptor (H2R) antagonist, inhibits the growth of glandular tumors such as colorectal cancer. However, its effects against salivary gland tumors are still unknown. We demonstrated previously that human salivary gland tumor (HSG) cells spontaneously express the neural cell adhesion molecule (NCAM) and also that HSG cell proliferation could be controlled via a homophilic (NCAM-NCAM) binding mechanism and that NCAM may be associated with perineural invasion by malignant salivary gland tumors. In the present study, we investigated the effects of cimetidine via the expression of NCAM on tumor growth and perineural/neural invasion in salivary gland tumor cells. Expression of both NCAM mRNA and protein was found to decrease in a dose-dependent manner upon treatment with cimetidine for 24 h. The MTT assay and confocal laser microscopy clearly showed that HSG cells underwent apoptosis after treatment with cimetidine. Activation of caspases 3, 7, 8 and 9 was observed in HSG cells after cimetidine treatment, thus confirming that the apoptosis was induced by the activated caspases. Apaf-1 activity was also detected in HSG cells in a dose-dependent manner after treatment with cimetidine. We also found that the cimetidine-mediated down-regulation of NCAM expression in HSG cells did not occur via blocking of the histamine receptor, even though H2R expression was observed on HSG cells, as two other H2R antagonists, famotidine and ranitidine, did not show similar effects. We demonstrated for the first time that cimetidine can induce significant apoptosis of salivary gland tumor cells, which express NCAM, at least in part by down-regulation of NCAM expression on the cells. These findings suggest that the growth, development and perineural/neural invasion of salivary gland tumor cells can be blocked by cimetidine administration through down-regulation of NCAM expression, as well as induction of apoptosis.

Interferon-gamma sensitizes the human salivary gland cell line, HSG, to tumor necrosis factor-alpha induced activation of dual apoptotic pathways

Activated immune cells secrete proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha), interferon-gamma (IFN-gamma) and Fas ligand (FasL) and these cytokines have been reported to induce apoptosis in numerous cell types. Apoptotic cell death has been associated with the progression of numerous autoimmune diseases. Proinflammatory cytokines are reportedly involved in apoptosis in the salivary glands of patients with Sjögren's syndrome (SS); an autoimmune disorder characterized by the destruction of salivary and lachrymal glands. In this study, we used the HSG cell line to determine if exposure to proinflammatory cytokines induces apoptosis in human salivary gland cells. In addition, we identified the mediators controlling the apoptotic process in response to TNF alpha and IFN gamma. TNF-alpha and IFN-gamma induced apoptosis in HSG cells and resulted in the activation of caspase 8 and the "death receptor" pathway. We further determined that caspase 9 and the "mitochondrial" pathway was also activated. Induction of the intrinsic and extrinsic pathways in HSG cells resulted in substrate cleavage by effector caspases, in particular the cleavage of alpha II spectrin, an autoantigen in Sjögren's syndrome. Our results suggest that HSG cells provide a model system to study processes regulating proinflammatory cytokine-induced apoptotic cell death.