Human Salivary Gland Tumor Cells Research Articles

Increased cell killing effect in neutron capture enhanced proton beam therapy

Thermal neutrons generated in the body during proton beam therapy (PBT) can be used to cause boron neutron capture reactions and have recently been proposed as neutron capture enhanced PBT (NCEPBT). However, the cell killing effect of NCEPBT remains underexplored. Here, we show an increase in the cell killing effect of NCEPBT. Using Monte Carlo simulations, we showed that neutrons generated by proton beam irradiation are uniformly spread on tissue culture plates. Human salivary gland tumor cell line (HSG), human osteosarcoma cell line (MG63), human tongue squamous cell carcinoma cell line (SAS), and human malignant melanoma cell line (G-361) were irradiated with X-rays, proton beams, and proton beams with 10B-enriched boronophenylalanine (boron concentration of 20 and 80 ppm). The relative biological effectiveness (RBE) values of proton beams alone, proton beams with 20 ppm boron, and proton beams with 80 ppm boron for HSG, MG63, SAS, and G-361 were 1.02, 1.07, and 1.23; 1.01, 1.08, and 1.44; 1.05, 1.09, and 1.46; and 1.04, 1.13, and 1.63, respectively. NCEPBT with high boron concentration showed high RBE and a high sensitizing effect. Our results confirm an increase in the cell killing effect of NCEPBT, should aid in its clinical use, and warrant its further investigation.

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.

Cytotoxicity of Propolis Extracts obtained using Dichloromethane and Hexane Solvent on Human Salivary Gland Tumor Cell Line.

This in vitro study aimed to investigate the effect of propolis extracts from two different solvents on human submandibular salivary gland (HSG) tumor cell line. Propolis was extracted by dichloromethane (DCM) and hexane (HEX). Crude extracts were prepared from 6.25 to 200 µg/mL in Dulbecco's modified eagle medium without serum. Flavonoid and total phenolic contents of crude extracts were measured using a modified colorimetric method. The cytotoxicity was evaluated by 3-[4, 5-dimethylthiazol-2-yl]-2,5 diphenyl-tetrazolium (MTT) assay and lactate dehydrogenase (LDH) release assay. The statistics were analyzed by independent sample t-test. Propolis extracts obtained using DCM and HEX exhibited comparable % yield (38.58 and 38.25) and physical characteristics and different amounts of flavonoid (0.439 ± 0.02 and 0.250 ± 0.01 mg catechin/g sample) and total phenolic compounds (3.759 ± 0.03 and 1.618 ± 0.03 mg gallic acid equivalents/g sample). The DCM group at 25, 50, 100, and 200 µg/mL as well as the HEX group at 50, 100, and 200 µg/mL significantly displayed a decrease in % cell viability and an increase in % cytotoxicity, compared with the untreated control group (P < 0.05). The DCM group showed the half-maximal inhibitory concentration (IC50) of MTT (42.93 ± 2.70) and LDH (34.94 ± 0.22). The HEX group showed the IC50 of MTT (61.30 ± 5.39) and LDH (42.32 ± 1.00). Propolis extracts obtained using both DCM and HEX are effective to inhibit HSG viability. Regarding to the cell morphological observation, MTT and LDH assays, propolis extracts obtained using DCM and HEX exhibited the cytotoxic effect on HSG tumor cell line. Based on our knowledge, this research demonstrates the first preliminary result suggesting propolis as a natural product of choice for salivary gland cancer prevention and therapy.

Adaptation of stochastic microdosimetric kinetic model to hypoxia for hypo-fractionated multi-ion therapy treatment planning

For hypo-fractionated multi-ion therapy (HFMIT), the stochastic microdosimetric kinetic (SMK) model had been developed to estimate the biological effectiveness of radiation beams with wide linear energy transfer (LET) and dose ranges. The HFMIT will be applied to radioresistant tumors with oxygen-deficient regions. The response of cells to radiation is strongly dependent on the oxygen condition in addition to radiation type, LET and absorbed dose. This study presents an adaptation of the SMK model to account for oxygen-pressure dependent cell responses, and develops the oxygen-effect-incorporated stochastic microdosimetric kinetic (OSMK) model. In the model, following assumptions were made: the numbers of radiation-induced sublethal lesions (double-strand breaks) are reduced due to lack of oxygen, and the numbers of oxygen-mediated lesions are reduced for radiation with high LET. The model parameters were determined by fitting survival data under aerobic and anoxic conditions for human salivary gland tumor cells and V79 cells exposed to helium-, carbon-, and neon-ion beams over the LET range of 18.5–654.0 keV μm−1. The OSMK model provided good agreement with the experimental survival data of the cells with determination coefficients >0.9. In terms of oxygen enhancement ratio, the OSMK model reproduced the experimental data behavior, including slight dependence on particle type at the same LET. The OSMK model was then implemented into the in-house treatment planning software for the HFMIT to validate its applicability in clinical practice. A treatment plan with helium- and neon-ion beams was made for a pancreatic cancer case assuming an oxygen-deficient region within the tumor. The biological optimization based on the OSMK model preferentially placed the neon-ion beam to the hypoxic region, while it placed both helium- and neon-ion beams to the surrounding normoxic region. The OSMK model offered the accuracy and usability required for hypoxia-based biological optimization in HFMIT treatment planning.

Microdosimetric measurements as a tool to assess potential in-field and out-of-field toxicity regions in proton therapy

Relative biological effectiveness (RBE) variations are thought to be one of the primary causes of unexpected normal-tissue toxicities during tumor treatments with charged particles. Unlike carbon therapy, where treatment planning is optimized on the basis of the RBE-weighted dose, a constant RBE value of 1.1 is currently used in proton therapy. Assuming a uniform value can lead to under- or over-dosage, not just to the tumor but also to surrounding normal tissue. RBE changes have been linked with dose/fraction, the biological endpoint and beam properties. Understanding radiation quality and the associated RBE can improve the prediction of normal-tissue toxicities.In this study, we exploited microdosimetry for characterizing radiation quality in proton therapy in-field, and off-beam at 20 (beam edge), 50 (close out-of-field) and 100 (far out-of-field) mm from the beam center. We measured the lineal energy y spectra in a water phantom irradiated with 152 MeV protons, from which beam quality as well as the physical dose could be obtained. Taking advantage of the linear quadratic model and a modified version of the microdosimetric kinetic model, the microdosimetric data were combined with radiobiological parameters (α and β) of human salivary gland tumor cells for assessing cell survival RBE and RBE-weighted dose.The results indicate that if a dose of 60 Gy is delivered to the peak, the beam edge receives up to 6 Gy while the close and far out-of-field regions receive doses on the order of 10−3 Gy and 10−4 Gy, respectively. The RBE estimate in-beam shows large variations, ranging from 1.0 ± 0.2 at the entrance channel to 2.51 ± 0.15 at the tail. The beam edge follows a similar trend but the RBE calculated at the Bragg peak depth is 2.27 ± 0.17, i.e. twice the RBE in-beam (1.05 ± 0.15). Out-of-field, the estimated RBE is always significantly higher than 1.1 and increases with increasing lateral distance, reaching the overall highest value of 3.4 ± 0.3 at a depth of 206 mm and a lateral distance of 10 mm. The combination of RBE and dose into the biological dose points to the beam edge and the end-of-range in-beam as the areas with the highest risk of potential toxicities.

Adaptation of stochastic microdosimetric kinetic model for charged-particle therapy treatment planning

The microdosimetric kinetic (MK) model underestimates the cell-survival fractions for high linear energy transfer (LET) and high dose irradiations. To address the issue, some researchers previously extended the MK model to the stochastic microdosimetric kinetic (SMK) model. In the SMK model, the radiation induced cell-survival fractions were estimated from the specific energies zd and zn absorbed by a microscopic subnuclear structure domain and a cell nucleus, respectively. By taking the stochastic nature of zn as well as that of zd into account, the SMK model could reproduce the measured cell-survival fractions for radiations with wide LET and dose ranges. However, treatment planning based on the SMK model was unrealistic in clinical practice due to its long computation time and huge memory space required for the computation. In this study, we modified the SMK model to shorten the computation time and to reduce the memory space required for the computation. By using the dose-averaged cell-nucleus specific energy per event in the SMK formalism, the stochastic nature of zn was reflected onto the estimated cell-survival fractions. The accuracy of the modified SMK model was examined through the comparison between the estimated and the measured survival fractions of human salivary gland tumor cells and V79 cells. We then implemented the modified SMK model into the in-house treatment planning software for scanned charged-particle therapy to validate its applicability in clinical practice. As examples, treatment plans of helium-, carbon-, and neon-ion beams were made for an orbital tumor case. The modified SMK model could reproduce the measured cell-survival fractions more accurately compared to the MK model especially for high-LET and high-dose irradiations. In summary, the modified SMK model offers the accuracy and simplicity required in treatment planning of scanned charged-particle therapy for wide LET and dose ranges.

The motogenic effect of EGF and TGF-α on the migration of tumor cells from the oral region

Aim: Epithelial-to-mesenchymal transition (EMT) is recognized as a hallmark of cancer. The change in phenotype of epithelial cells enables them to migrate and the cancer therefore, to become more aggressive. The presence of the epidermal growth factor receptor (EGFR) has been implicated in the spread and aggressive nature of oral tumors. The aims of this present study were to investigate the role of two different growth factors, both ligands of the receptor, and their influence on EMT and cellular motility. Methods: A number of assays were used to investigate the response of the cells to the two growth factors (EGF and transforming growth factor (TGF-α)). These techniques included the 2-D scratch assay, a 2-D-cell scatter assay, and immunocytochemistry. Normal keratinocytes (HaCaT), oral adenoid squamous cell carcinoma (TYS), and human salivary gland tumor cells were used in this study. Results: The results of the scratch and scatter assays indicated that EGF and TGF-α exerted differential effects upon the cells. EGF and TGF-α stimulated the migration of cancer cells in scratch assays. Scatter assays have revealed that both EGF and TGF-α induce EMT. EGF- and TGF-α-induced scattering is EGFR dependent and localization of EMT markers (E-cadherin and vimentin) might play an important role in scattering. These responses were also found to be dependent upon cell type, indicating that the assay could also be used as a simple screen for active growth factor. The observed effects were also dose dependant (data not shown). Conclusion and implication for translation to clinic: We have previously reported that although EGF and TGF-α have ultimately similar effects on motility, it would appear that different mechanisms are responsible. This data extends our knowledge of the action of these growth factors to epithelial cell lines and will inform future testing of the effects of EGF and TGF-α inhibitors.

Enhanced radiobiological effects at the distal end of a clinical proton beam: in vitro study

In the clinic, the relative biological effectiveness (RBE) value of 1.1 has usually been used in relation to the whole depth of the spread-out Bragg-peak (SOBP) of proton beams. The aim of this study was to confirm the actual biological effect in the SOBP at the very distal end of clinical proton beams using an in vitro cell system. A human salivary gland tumor cell line, HSG, was irradiated with clinical proton beams (accelerated by 190 MeV/u) and examined at different depths in the distal part and the center of the SOBP. Surviving fractions were analyzed with the colony formation assay. Cell survival curves and the survival parameters were obtained by fitting with the linear–quadratic (LQ) model. The RBE at each depth of the proton SOBP compared with that for X-rays was calculated by the biological equivalent dose, and the biological dose distribution was calculated from the RBE and the absorbed dose at each position. Although the physical dose distribution was flat in the SOBP, the RBE values calculated by the equivalent dose were significantly higher (up to 1.56 times) at the distal end than at the center of the SOBP. Additionally, the range of the isoeffective dose was extended beyond the range of the SOBP (up to 4.1 mm). From a clinical point of view, this may cause unexpected side effects to normal tissues at the distal position of the beam. It is important that the beam design and treatment planning take into consideration the biological dose distribution.

Clinical oxygen enhancement ratio of tumors in carbon ion radiotherapy: the influence of local oxygenation changes.

The effect of carbon ion radiotherapy on hypoxic tumors has recently been questioned because of low linear energy transfer (LET) values in the spread-out Bragg peak (SOBP). The aim of this study was to investigate the role of hypoxia and local oxygenation changes (LOCs) in fractionated carbon ion radiotherapy. Three-dimensional tumors with hypoxic subvolumes were simulated assuming interfraction LOCs. Different fractionations were applied using a clinically relevant treatment plan with a known LET distribution. The surviving fraction was calculated, taking oxygen tension, dose and LET into account, using the repairable–conditionally repairable (RCR) damage model with parameters for human salivary gland tumor cells. The clinical oxygen enhancement ratio (OER) was defined as the ratio of doses required for a tumor control probability of 50% for hypoxic and well-oxygenated tumors. The resulting OER was well above unity for all fractionations. For the hypoxic tumor, the tumor control probability was considerably higher if LOCs were assumed, rather than static oxygenation. The beneficial effect of LOCs increased with the number of fractions. However, for very low fraction doses, the improvement related to LOCs did not compensate for the increase in total dose required for tumor control. In conclusion, our results suggest that hypoxia can influence the outcome of carbon ion radiotherapy because of the non-negligible oxygen effect at the low LETs in the SOBP. However, if LOCs occur, a relatively high level of tumor control probability is achievable with a large range of fractionation schedules for tumors with hypoxic subvolumes, but both hyperfractionation and hypofractionation should be pursued with caution.

Abstract 5625: Wnt inhibitory factor 1 is a potent growth inhibitory agent for salivary gland tumor cells

Abstract Background: Aberrant activation of the Wingless-type (Wnt)/β-catenin pathway plays an important role in many human cancers. We have shown that Wnt inhibitory factor 1 (WIF1), a Wnt antagonist, is rearranged in salivary gland pleomorphic adenomas and down-regulated in carcinoma ex-pleomorphic adenomas. Here, we studied the mechanisms of WIF1 down-regulation and also the potential therapeutic implications of WIF1 in human salivary gland tumor cells. Aims: To characterize the mechanisms of WIF1 down-regulation in salivary gland tumor cells and to determine the effects of restoration of WIF1 expression on salivary gland tumor cell growth. Methods: Salivary gland tumor cell lines were treated with the demethylating agent 5-aza-2′-deoxycytidine (50 µM) for 4 days. Then we isolated total RNA and performed real-time RT-PCR to determine WIF1 mRNA expression. To determine the growth suppressive effects of WIF1, salivary gland pleomorphic adenoma and carcinoma ex-pleomorphic adenoma cells were stably or transiently transfected with either empty vector or pCI blast-WIF1. The growth inhibitory role of WIF1 was also assessed by exposure of salivary gland tumor cells to pure WIF1 protein. Cell proliferation was assessed at different time points by MTT assay. We performed cell cycle analysis after 72 h of WIF1 transfection by flow cytometry using FACSCalibur analyzer. Results: Treatment with DAC caused about 30-fold and 6-fold increase in WIF1 mRNA expression in salivary gland carcinoma ex-pleomorphic adenoma cells and pleomorphic adenoma cells, respectively compared with vehicle treatment. Stable transfection of salivary gland tumor cells with WIF1 vector induced dramatic nuclear fragmentation and cell death. No viable colonies were observed. Transient transfection with pCI blast-WIF1 or exposure to WIF1 protein significantly decreased the proliferation of salivary gland tumor cells. Cell cycle analysis showed a significant accumulation of cells in G1 phase suggesting that WIF1 re-expression induces G1 arrest in salivary gland tumor cells. Conclusions: Our findings demonstrate that WIF1 is epigenetically silenced by promoter hypermethylation in carcinoma ex-pleomorphic adenoma cells and pleomorphic adenoma cells. Importantly, salivary gland tumor cells stably transfected with WIF1 are non-viable, and exposure to high levels of WIF1 protein resulted in significant growth inhibition. These data show that WIF1 is a potent growth inhibitory agent for salivary gland tumor cells and may serve as a potential therapeutic drug for salivary gland cancer. Grant support: This work was supported by the Oklahoma Center for the Advancement of Science &amp; Technology (LQ) (HR08-018). LQ holds a Presbyterian Health Foundation Endowed Chair in Otorhinolaryngology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5625. doi:1538-7445.AM2012-5625

Treatment planning for a scanned carbon beam with a modified microdosimetric kinetic model

We describe a method to calculate the relative biological effectiveness in mixed radiation fields of therapeutic ion beams based on the modified microdosimetric kinetic model (modified MKM). In addition, we show the procedure for integrating the modified MKM into a treatment planning system for a scanned carbon beam. With this procedure, the model is fully integrated into our research version of the treatment planning system. To account for the change in radiosensitivity of a cell line, we measured one of the three MKM parameters from a single survival curve of the current cells and used the parameter in biological optimization. Irradiation of human salivary gland tumor cells was performed with a scanned carbon beam in the Heavy Ion Medical Accelerator in Chiba (HIMAC), and we then compared the measured depth-survival curve with the modified MKM predicted survival curve. Good agreement between the two curves proves that the proposed method is a candidate for calculating the biological effects in treatment planning for ion irradiation.

High yields of isochromatid breaks and successive formation of chromosome exchanges may lead to reproductive cell death following high-LET irradiation

Abstract To clarify the relationship between cell death and chromosomal aberrations following exposure to heavy-charged ion particles beams, exponentially growing Human Salivary Gland Tumor cells (HSG cells) were irradiated with various kinds of high energy heavy ions; 13 keV/μm carbon ions as a low-LET charged particle radiation source, 120 keV/μm carbon ions and 440 keV/μm iron ions as high-LET charged particle radiation sources. X-rays (200 kVp) were used as a reference. Reproductive cell death was evaluated by clonogenic assays, and the chromatid aberrations in G2/M phase and their repairing kinetics were analyzed by the calyculin A induced premature chromosome condensation (PCC) method. High-LET heavy-ion beams introduced much more severe and un-repairable chromatid breaks and isochromatid breaks in HSG cells than low-LET irradiation. In addition, the continuous increase of exchange aberrations after irradiation occurred in the high-LET irradiated cells. The cell death, initial production of isochromatid breaks and subsequent formation of chromosome exchange seemed to be depend similarly on LET with a maximum RBE peak around 100–200 keV/μm of LET value. Conversely, un-rejoined isochromatid breaks or chromatid breaks/gaps seemed to be less effective in reproductive cell death. These results suggest that the continuous yield of chromosome exchange aberrations induced by high-LET ionizing particles is a possible reason for the high RBE for cell death following high-LET irradiation, alongside other chromosomal aberrations additively or synergistically.

Growth inhibition of human salivary gland tumor cells by introduction of progesterone (Pg) receptor and Pg treatment

Cancer of the salivary gland is one of the common cancers in the head and the neck regions. This type of cancer develops in the minor and the major salivary glands, and it sometimes metastasizes to other organs, particularly the lung. Morphologic mimicry and similarity in the expression of steroid hormone receptors between salivary gland tumors (SGTs) and breast tumors are well-known phenomena and are occasionally debated in the field of surgical pathology. Progesterone (Pg), one of the female sex steroid hormone, is intimately involved in the development of the mammary gland. Further, it is believed that Pg plays a role in breast cancer progression. However, little is known regarding its role in SGTs. In this study, we used ACCM, a human adenoid cystic carcinoma cell line established from the salivary gland, in order to clarify the role of the Pg receptor (PR) on cell proliferation. No effect of Pg on cell proliferation was observed in the PR-deficient aggressive ACCM cells. However, after introducing PR into the ACCM cells, Pg markedly inhibited the proliferative activity of the cells. This inhibitory effect on cell proliferation was accompanied by p21 upregulation, and Id1 and c-myc downregulation. Moreover, Pg-treated PR transfectants showed significant morphological change; they appeared more flattened and spread out when compared with the ethanol-treated control cells. Our results provided significant insights into the mechanism of suppression of the proliferative property of the cells via the function of PR, and suggested that PR reintroduction therapy might be a viable method of inhibiting human SGT progression.

Induction of apoptosis in human salivary gland tumor cells by anti-NCAM antibody

Neural cell adhesion molecule (NCAM) is a type of cell surface glycoprotein and a member of the immunoglobulin superfamily. It has been reported that NCAM may be associated with perineural invasion by malignant salivary gland tumors such as adenoid cystic carcinoma. We have previously demonstrated that NCAM is constitutively expressed in the human salivary gland tumor cell line HSG, in vitro. In the present study, we have aimed to clarify the hypothesis that NCAM-mediated inhibition of salivary gland tumor proliferation is caused by homophilic binding and involves the prevention of signal transduction for perineural invasion using HSG cells. NCAM mRNA and protein expression was found to decrease in a dose-dependent manner upon treatment with the anti-NCAM antibody (MAb NCAM) for 24 h. The MTT assay showed a significant reduction in the number of viable HSG cells. Confocal laser microscopy showed that HSG cells underwent apoptosis after treatment with MAb NCAM. The activation of caspases 3, 7 and 9 was observed in HSG cells after treatment with MAb NCAM, thus confirming that apoptosis was induced by the activated caspases. Apaf-1 activity was also detected in HSG cells in a dose-dependent manner after treatment with MAb NCAM. The up-regulation of TGF-beta1-mediated NCAM expression appeared to lead to the activation of homophilic NCAM binding, further accelerating HSG cell proliferation. In addition, the localization of NCAM in adenoid cystic carcinomas (ACCs) was examined using an immunohistochemical method. NCAM was slightly to moderately positive in 9 of 13 cases (69.2%) of ACC. These findings suggest that NCAM is associated not only with a cell-to-cell adhesion mechanism, but also with tumorigenesis, including growth, development and perineural invasion in human salivary gland tumors.

Detection of photodynamic therapy-induced early apoptosis in human salivary gland tumor cells in vitro and in a mouse tumor model

We studied the detection of apoptosis of malignant human salivary gland tumor cells induced by photodynamic therapy (PDT) using the photosensitizer mono-L-aspartyl chlorin e6 (NPe6) in vitro and in vivo in mice receiving transplanted human salivary gland tumor (HSG) cells. An immunohistocytochemical method using a monoclonal antibody (MoAb), M30, which reacts with the product resulting from the cleavage of cytokeratin (CK) 18 by activated caspase, was applied to detect the apoptosis of HSG cells induced by PDT. Significant amounts of immunoreactive products were observed in the cytoplasm of HSG cells after PDT. In vitro, M30-positive cells increased from 2 h after PDT, increased rapidly from 8 h and reached a peak 24 h after PDT. In vivo, a peak of early apoptosis was confirmed two hours after PDT. In comparison with DNA fragmentation detected by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method, the destroyed tumor cells were observed sporadically 24 h after PDT. These results suggest that immunohistocytochemical staining with the MoAb M30 may be useful for detecting early apoptosis induced by PDT. Futhermore, PDT using NPe6 is effective in inducing apoptosis of HSG cells at an early stage, which suggests the possibility of the therapy being ideal for treatment of human malignant neoplasms.

唾液腺腺様嚢胞癌の増殖・進展メカニズムに関する研究―ＮＣＡＭの発現とその機能について―

Neural cell adhesion molecule (NCAM) is one cell surface glycoprotein and a member of the immunoglobulin superfamily. It has been reported that NCAM may be associated with perineural invasion by malignant glandular tumors, such as bile duct carcinoma or gallbladder carcinoma. However, it remains unclear whether NCAM is really associated with the development of salivary gland tumors. Previous studies have demonstrated that NCAM is constitutively expressed in the human salivary gland tumor cell line HSG in vitro. In this study, molecular mechanisms of perineural invasion were examined using HSG cells in which NCAM-mediated inhibition of salivary gland tumor proliferation is caused by homophilic binding. In addition, the localization of NCAM in adenoid cystic carcinomas (ACCs) was examined immunohistochemically. The levels of NCAM mRNA and protein expression were found to decrease in a dose-dependent manner on treatment with anti-NCAM antibody. The MTT assay revealed a significant reduction in the number of viable HSG cells. Confocal laser microscopy showed that HSG cells undergo apoptosis in response to treatment with anti-NCAM antibody. The activation of caspases 3, 7, and 9 was observed in HSG cells after treatment with anti-NCAM antibody, thus confirming the induction of activated caspase-mediated apoptosis. The up-regulation of TGF-β1-mediated NCAM expression seemed to lead to the activation of homophilic NCAM binding, further accelerating HSG cell proliferation. Immunohistochemical examinations revealed that NCAM was slightly to moderately positive in 9 of 13 cases (69.2%) of ACC.These findings suggest that NCAM is associated not only with a cell-to-cell adhesion mechanism but also with tumorigenesis, including growth, development, and perineural invasion in human ACCs.

Hypoxia-induced angiogenesis of cultured human salivary gland carcinoma cells enhances vascular endothelial growth factor production and basic fibroblast growth factor release.

The angiogenic activity of two human salivary gland tumor cell lines, ACCS from adenoid cystic carcinoma and IT-2 from mucoepidermoid carcinoma, was examined by stimulating tube formation by bovine capillary endothelial cells (BCE). ACCS and IT-2 were cultured in 20 or 3% oxygen, representing normoxic and hypoxic conditions, respectively, and conditioned medium (CM) was obtained from each culture. The BCE tubes stimulated by hypoxic CM were 1.59 (ACCS) and 1.42 (IT-2) times longer than those stimulated by normoxic CM. The tube-forming activity of CM was inhibited by preincubation with either anti-vascular endothelial growth factor (VEGF) IgG or anti-basic fibroblast growth factor (bFGF) IgG, suggesting that both VEGF and bFGF with angiogenic activity were present in the CM. This was confirmed by ELISA, which also demonstrated increased concentrations of both proteins in the hypoxic CM. Northern blot analysis showed an increased VEGF mRNA level in both carcinoma cells with hypoxia, while hypoxia did not affect the bFGF mRNA level in either cell line. The results suggest that both VEGF and bFGF are major angiogenesis factors in salivary gland tumors, and hypoxia-induced angiogenesis results from upregulation of VEGF and increased release of bFGF.

Role of plasminogen activators, metalloproteinases and the tissue inhibitor of metalloproteinase-1 in the metastatic process of human salivary-gland adenocarcinoma cells.

An in vitro system has been established in which conversion from non-metastasizing to metastasizing adenocarcinoma cells can be induced, and subsequently subjected to analysis of the expression of proteases and tissue inhibitor of metalloproteinases-1 (TIMP-1). A human salivary-gland adenocarcinoma cell clone HSGc, with no metastatic ability, was exposed to N-methyl-N-nitrosourea (MNU). Following exposure to MNU, cells with altered morphology were cloned. Upon s.c. inoculation into nude mice, MNU-treated HSGc clones formed metastatic foci in various organs, and then 5 metastasizing clones were isolated. Evaluation of expression of tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), metalloproteinases and TIMP-1 was performed by means of enzyme immunoassay, zymogram, or immunoblot. MNU-treated HSGc and metastasizing clones were found to secrete high levels of tPA, while HSGc produced undetectable levels of this enzyme. Expression of uPA was not observed in any of the cell clones. When the secretion of gelatinolytic enzymes was examined, metastasizing clones produced higher levels of 57- and 32-kDa, but not of 92- or 72-kDa gelatinases, as compared to HSGc cells. Although TIMP-1 was detected in all cell clones, metastasizing clones secreted less TIMP-1 than HSGc cells; in addition, one metastasizing clone produced TIMP-1 with a molecular weight distinct from that of 28-kDa TIMP-1. Our results suggest that the acquisition of metastatic ability by human salivary-gland tumor cells is closely associated with increased secretion of several metalloproteinases as well as decreased or altered TIMP-1 expression.