IRUCAA@TDC : Low-level (gallium-aluminum-arsenide) laser irradiation of Par-C10 cells and acinar cells of rat parotid gland

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(1)Title. Author(s) Alternative Journal URL. Low-level (gallium-aluminum-arsenide) laser irradiation of Par-C10 cells and acinar cells of rat parotid gland Onizawa, K; Muramatsu, T; Matsuki, M; Ohta, K; Matsuzaka, K; Oda, Y; Shimono, M Lasers in medical science, 24(2): 155-161 http://hdl.handle.net/10130/1418. Right. Posted at the Institutional Resources for Unique Collection and Academic Archives at Tokyo Dental College, Available from http://ir.tdc.ac.jp/.

(2) Title page. Low-level (Ga-Al-As) laser irradiation to Par-C10 cells and acinar cells of rat parotid gland Katsuhiro Onizawa1), Takashi Muramatsu2), Miwako Matsuki2)3), Kazumasa Ohta4), Kenichi Matsuzaka5), Yutaka Oda6), Masaki Shimono2) 1) Department of Pathology, Tokyo Dental College 2) Oral Health Science Center, Department of Pathology, Tokyo Dental College 3) Department of Physiology, Nihon University School of Dentistry at Matsudo 4) Oral Health Science Center, Department of Biochemistry, Tokyo Dental College 5) Oral Health Science Center, Department of Clinical Pathophysiology, Tokyo Dental College 6) Oral Health Science Center, Department of Dental Materials Science, Tokyo Dental College. Tokyo Dental College 1-2-2, Masago, Mihama-ku, Chiba, 261-8502, Japan. 1) Short title: Parotid acinar cells after low-level laser irradiation 2) Key words: acinar cell, parotid gland, low-level laser irradiation, amylase, HSP25 3) Number of words in abstract: 168 4) Number of words in abstract and text: 2953 5) Number of tables and figures: 4 figures 6) Number of cited references:34. Corresponding author Takashi Muramatsu* Oral health Science Center, Department of Pathology, Tokyo Dental College 1-2-2, Masago, Mihama-ku, Chiba, 261-8502, Japan Tel: +81-43-270-3782; Fax: +81-43-270-3784 e-mail; [email protected]. 1.

(3) Abstract We investigated cell response, including cell proliferation and expression of heat stress protein to clarify the influence of low-level (Ga-Al-As diode) laser irradiation on rat parotid gland acinar cell-derived Par-C10 cells. Furthermore, we also investigated amylase release and cell death to irradiation in rat parotid gland acinar cells. Number of Par-C10 cells in the laser-irradiated groups was higher than that in the non-irradiated group at days 5 and 7, although the difference was statistically significant (p<0.01). Higher expression of HSP25 and bcl-2 was seen at days 1 and 3 in the irradiated group. Amylase release assay showed no significant difference between irradiated group and non-irradiated group statistically. Trypan blue exclusion assay revealed that there was no difference in the ratio of dead to live cells between the irradiated- and non-irradiated groups. These results suggest that low-level laser irradiation promotes cell proliferation and expression of anti-apoptosis proteins in Par-C10 cells, but it does not significantly affect amylase secretion and does not induce rapid cell death in isolated rat parotid gland acinar cells.. 2.

(4) Introduction Saliva exerts an anti-bacterial effect and plays an important role in a number of processes, including digestion, dilution, buffering, neutralization of pH and remineralization of enamel in the oral cavity. Loss of saliva secretion and function occurs with aging and due to diseases such as Sjogren’s syndrome and chronic sialadenitis. Furthermore, radiation therapy for oral cancer damages salivary gland, resulting in xerostomia, thereby disrupting homeostasis in the oral cavity. Symptomatic treatments such as artificial saliva have been tried in patients with compromised saliva secretion. However, there are limitations to such treatments, and promotion, activation and regeneration of destroyed or dysfunctional salivary gland cells would offer a better alternative. The ability to regenerate ruptured acinar cells in salivary gland would greatly improve treatment of salivary gland lesions. Laser technology has seen great advances over recent years, with much basic research and clinical application being seen in the medical field. Studies on high-level laser irradiation have shown that this technology is useful for cutting biological materials and inducing coagulation and necrosis in target tissues with subsequent reaction in surrounding tissues. However, remarkable clinical success has also been reported with low-level laser irradiation. Since the report of Mester et al [1]. on low-level laser irradiation of mouse skin to investigate wound healing, many researchers have investigated the influence of laser irradiation on wound healing [1-5], improvement of microcirculation [6], nerve activation [7], treatment of chronic pain and anti-inflammation [8]. The influence of low-level laser irradiation on activation of salivary gland cells has been also investigated [9-13]. Previous investigations showed degenerative and inflammatory changes after helium/neon (He-Ne) low-level laser. 3.

(5) irradiation, histopathologically[9, 10]. On the other hand, another study found no detectable degenerative changes in rat submandibular gland cells after gallium-arsenide (Ga-As) low-level laser irradiation [13]. However, the influence of low-level laser irradiation on acinar cells in salivary gland remains to be fully clarified. Functional evaluation of processes such as amylase release, in particular, has yet to be done in salivary gland. Furthermore, expression of stress proteins such as heat shock protein (HSP) and anti-apoptosis protein such as bcl-2 remains to be investigated, despite the fact that laser irradiation is stressful to salivary gland acinar cells. In this study, we investigated cell response, including cell proliferation and expression of heat stress protein and anti-apoptosis protein, after low-level (Ga-Al-As diode) laser irradiation in an immortalized cell, Par-C10, derived from rat parotid gland acinar cells. Furthermore, we also investigated amylase release and cell death in order to clarify the influence of the laser irradiation on isolated acinar cells in rat parotid gland.. Materials and Methods All experiments were carried out according to the Guidelines for the Treatment of Animals established by Tokyo Dental College.. Cells used in this study. We used two kinds of cells, Par-C10 derived from rat parotid acinar cell [14] (Par-C10; courtesy of Dr. David Quissell, Colorado University), and isolated rat parotid acinar cells in this study.. 4.

(6) The characteristics of Par-C10 was described in an earlier report [14] by Quissell et al. The Par-C10 cells were used to investigate cell proliferation and response to stress such as HSP. However, the cells do not produce amylase and are inappropriate for functional assay such as amylase releasing. Isolate rat parotid acinar cells were used for amylase assay and trypan blue exclusion assay. However, the cells survived within 2 h, and were inadequate for investigation of long term experiments. Therefore, we employed two kinds of the cells.. Cell line and culture. The Par-C10 cells were grown on a 24-well culture plate (Corning Life Sciences, Corning, NY, USA) using Dulbecco’s modified Eagle medium (DMEM)/F-12 (Invitrogen, Grand Island, NY, USA) containing 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) and penicillin-streptomycin (100 units/ml, Invitrogen, Grand Island, NY, USA) at 37°C in a humidified incubator containing 5% CO2. Twenty-four hours prior to laser irradiation, Par-C10 cells were trypsinized and spread on a 24-well culture plate at density of 1x105 cells / well.. Preparation of isolated rat parotid acinar cells. Isolated rat parotid acinar cells were prepared as described by Dohke et al [15] with modification. Thirty-six six-week-old male Sprague-Dawley rats were intravenously anesthetized with 100 mg/kg of 2.5 % sodium thiopental before experiments. The parotid. 5.

(7) glands were extracted and placed in a small volume of Krebs–Ringer-bicarbonate (KRB) composed of 116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO4, 1.8 mM CaCl2, 0.96 mM NaH2PO4, 25 mM NaHCO3, and 5 mM Hepes (pH 7.4); and 11.1 mM glucose was equilibrated in an atmosphere of 95% O2 / 5% CO2. The glands were minced on a sheet of dental wax with a razor and then incubated at 37°C for 10 min in 5 ml KRB containing trypsin (0.2 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) and 0.5% bovine serum albumin (BSA). The glands were separated by centrifugation at 1600 rpm for 30 sec and incubated in 5 ml Ca2+–Mg2+-free KRB containing 2 mM EGTA, trypsin inhibitor (0.2 mg/ml) and 0.5% BSA at 37°C for 5 min. The solution was removed by centrifugation as described above, and the glands were further incubated at 37°C for 5 min in 5 ml Ca2+–Mg2+-free KRB without trypsin inhibitor. The Ca2+–Mg2+-free KRB was removed by centrifugation, and then the minced glands were incubated at 37°C for 10 min in 5 ml KRB containing collagenase A (0.74 mg/ml) and 0.5% BSA. The resulting suspension was passed through 8 layers of nylon mesh, and separate dispersed cells were gently layered onto KRB containing 4% BSA. The cells were collected by centrifugation at 1200 rpm for 5 min, re-suspended in KRB containing 4% BSA, and washed by centrifugation again. The resulting cells were suspended in the appropriate amounts of KRB containing 0.5% BSA, and incubated at 37°C for 5 min. The isolated parotid acinar cells were spread on a 24-well dish at a density of 4x105 cells / well, and then used for the laser irradiation experiment.. Irradiation with Ga-Al-As diode laser The basal characteristics of the Ga-Al-As diode laser (LD15; wavelength; 810 nm, power peak 15 watts, Dentek Laser Systems Production, Vienna, Austria) regarding distance. 6.

(8) between source, target and effective irradiation energy have been reported in earlier studies [16, 17]. To provide a sufficiently wide area (30 mm in diameter), the distance from the bottom of the culture plate to the tip of the probe was set at 50 mm. Par-C10 and isolated parotid acinar cells were spread in 24-well culture plates and irradiated at an output power of 10 watts with a continuous wave under an atmosphere set at 37°C. Effective irradiation energy was equivalent to about 61% of the source energy. We employed 100 J/cm2 (25.21 sec), 300 J/cm2 (65.63 sec) and 500 J/cm2 (126.05 sec) as the irradiation energy. Laser irradiation was applied once to the cells, and the cells were harvested at each time point. In the controls, no irradiation was carried out.. Cell growth The Par-C10 cells were detached using 0.25% trypsin (WAKO, Tokyo, Japan) at 1, 3, 5 and 7 days after irradiation, and counted using a Coulter Counter (Beckman Coulter, Fullerton, CA, USA). Results and growth curves were plotted.. Western blot In order to determine HSP25 expression, Western blot was carried out according to the method of Amano et al [18]. Cells were lysed in a radio-immunoprecipitation assay (RIPA) buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris [pH=7.4]) containing inhibitors at 1, 3, 5, and 7 days after irradiation. Twenty-five micrograms of total lysate was subjected to 7.5% SDS-PAGE, and then transferred onto a PVDF membrane (BioRad, Melvilla, NY, USA). The membranes were then incubated with anti-HSP25 antibody (StressGen Biotechnologies, Victoria, BC, Canada, dilution 1:10 000), anti-bcl-2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, dilution 1:1000) and. 7.

(9) anti-actin antibody (Sigma-Aldrich, St Louis, Mo, USA, dilution 1:2500) at 4°C overnight. After washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated rabbit IgG (1:1 000, Amersham, Rochester, MI, USA) at room temperature for 1 h. Immunoreactive bands were detected using the ECL Western blot analysis system (Amersham). Fold change relative to the control (Fold-Delta) was determined from densitometric data after normalization to actin for HSP25 and bcl-2. Value of control at each day was arbitrarily set at 1.0.. Amylase release assay The isolated parotid acinar cells in the KRB were treated with 10–6 M isoproterenol (IPR, Sigma-Aldrich, St. Louis, MO, USA) immediately after laser irradiation. Samples for amylase assay were obtained from the KRB at 0, 10, 20 and 30 min. The samples were diluted (1:10) in Dulbecco’s PBS (Invitrogen, Grand Island, NY, USA) and used for assay of released amylase. The retained cells were lysed in homogenizing buffer (20 mM phosphate buffer pH 7.4, 0.1% Triton X-100). The lysate was diluted (1:50) in homogenizing buffer and used for assay of total amylase. The activity of released amylase was measured with a spectrometer (Amersham Biosciences, wave length = 530 nm) according to the method by Bernfeld [19] (1955) and Fujita-Yoshigaki [20](2005). Released amylase activity was expressed as a percentage of the total amylase activity of the cells.. 8.

(10) Trypan blue exclusion assay In order to determine whether laser irradiation affected isolated parotid acinar cells, trypan blue exclusion assay was carried out. In brief, the isolated parotid acinar cells were stained with 0.5 % trypan blue solution for 30 s at immediately after, at 10 min after and at 30 min after irradiation. The number of both stained and total cells was determined with a hemocytometer, and the ratio of stained cells per total cells was calculated.. Statistical analysis Statistical significance of multiple comparisons was evaluated using the Steel test. Statistical significance was set at p<0.01.. Results Growth of Par-C10 after low-level laser irradiation To determine the relationship between laser irradiation and cell growth, we prepared Par-C10 cells. While both the laser-irradiated cells and control cells showed daily growth, laser-irradiated Par-C10 cells showed an increase in rate of growth in all groups (100, 300 and 500 J/cm2) (Fig. 1). The number of cells in the laser-irradiated groups was higher than that in the non-irradiated group at days 5 and 7, and this difference was statistically significant (p<0.01). Furthermore, at day 7, statistical significance between 100 J/cm2 and 500 J/cm2 (p<0.01) was confirmed, while no significance between 100 J/cm2 and 300 J/cm2, and between 300 J/cm2 and 500 J/cm2. However, no statistically significant differences were observed among the irradiated. 9.

(11) groups at days 1 and 3 (Fig. 1).. Expression of HSP25 and bcl-2 Western blot analysis revealed that HSP25, bcl-2 and actin were expressed in Par-C10 cells at all time points (Fig. 2). Actin was used as an internal control and density of the band was confirmed equally in all the samples. While no change was detected in the control group through the time points, a change was seen in the irradiated group. Higher expression of HSP25 was seen at days 1 and 3 in the irradiated group. When the irradiation at 300 J/cm2 was carried out, the stronger expression was seen compare to that at 100 and 500 J/cm2. However, HSP25-expression in the irradiated groups at days 5 and 7 became to be similar to that in the control group (Fig. 2). Higher expression of bcl-2 was also detected in the 300 and 500 J/cm2 irradiated group at days 1 and 3. At days 5 and 7, the expression in the irradiation group became same or lower than that in control group (Fig. 2).. Released amylase in isolated rat parotid acinar cells after low-level laser irradiation The results of amylase activity are shown in Fig. 3. Released amylase was increased time-dependently. No significant differences were observed between the irradiated groups and the control group at any irradiation density and at all time points statistically.. Trypan blue exclusion assay The results of the trypan blue exclusion assay are shown in Fig. 4. The ratio of trypan blue-positive cells per total cells was about 15% at immediately after laser. 10.

(12) irradiation (100, 300 and 500 J/cm2), the same as in the control group. At 10 min after laser irradiation, this ratio showed a slight increase (range from16.5 to 20 %), although it was still similar to that in the control group. At 30 min after laser irradiation, the ratio increased to 20%, similar to that in the control group. No statistically significant differences were observed between the irradiated groups and the control group at any irradiation density.. Discussion Cell growth Whether low-level-laser irradiation of salivary gland cells accelerates cell growth remains to be determined. Samar et al. and Plavnik et al. reported degenerative and necrotic changes using He-Ne laser irradiation [9, 10], while Takeda showed frequent mitotic findings in granular duct cells following gallium-arsenide (Ga-As) low-level laser irradiation at 24 h, suggesting a mitogenic effect with low-level laser irradiation [13]. In the present study, we found that the number of Par-C10 cells in the irradiated groups was higher than that in the non-irradiated group. These data are compatible with the results by earlier studies that have reported promotion of cellular proliferation with low-level laser irradiation [17, 21-23]. Furthermore, our results also showed that number of the irradiated cell at 500 J/cm2 was higher than that at 100 J/cm2 at days 7. The low-level laser irradiation may induce cell proliferation in Par-C10 cells irradiation-dose-dependently as well as earlier studies [17, 21-23].. HSP25 and bcl-2 expression Laser irradiation can be considered a stress signal for cells [24]. The most. 11.

(13) obvious feature of response to stress is that it leads to an increase in synthesis of HSPs [25, 26], which are believed to function in the protection and recovery of cells from environmental or pathological stress [25, 26]. HSP25 and 70 have been found to exert an anti-apoptotic effect [25, 27] and promote cell survival [28]. In the present study, higher HSP25 expression was observed at days 1 and 3 in the laser-irradiated group. The higher expression of HSP25 may explain this suppression of apoptotic activity. The response of HSP25 to stimuli is important in cell growth, repair and regeneration, since HSP25 plays an anti-apoptotic role in the inhibition of the JNK pathway early in the healing process [29]. Miyata et al. reported that low-level laser irradiation did not appear to promote JNK pathway in dental pulp cells [30]. Furthermore, Shefer et al. reported that low-energy laser irradiation promoted the survival and cell cycle entry of skeletal muscle satellite cells [31]. Moreover, bcl-2 proto-oncogene is a member of a gene family that includes cell death suppressors and cell death promoters. Its gene product, the protein bcl-2, is a 26 kDa putative membrane-associated protein which acts as a cell death suppressor that facilitates cell survival by regulating apoptosis [32]. As bcl-2 is an apoptosis inhibiting protein, our results that bcl-2 was detected exclusively in the laser-irradiated group, are reasonable. Taken together with the earlier reports and the findings of this study, low-level laser irradiation induces expression of HSP25 and cell survival.. Amylase release after laser irradiation Many studies have investigated low-level laser irradiation in various types of cell, including fibroblasts [23], dental pulp cells[33], periodontal ligament cells [22] and ectoblastic cells [17]. A few studies have also investigated the influence of low-level laser irradiation on salivary gland cells [9-13]. Although these earlier reports demonstrated. 12.

(14) morphological changes, the influence of low-level laser irradiation on salivary gland acinar cells remains to be fully clarified. Functional evaluation of processes such as amylase release, in particular, has yet to be done. Our results showed no statistical differences of amylase release between the irradiation groups and the control groups. Amylase releasing is associated with activation of beta-adrenergic receptor [20]. In the present study, IPR (10–6 M) was used in both irradiated and control groups for promotion of amylase secretion, but no statistical differences. In general, cells do not function when they are in proliferating phase. Our earlier report using the same laser device and condition showed that MC3T3-E1 (osteoblastic cells) did not well express non-collagenous proteins such as osteopontin and osteocalcin when the cells were in proliferating phase [17]. Therefore, in this study, we summarized low-level laser irradiation was associated with cell proliferation, but not amylase releasing in the rat parotid acinar cells.. Irradiation energy density Many investigations using low-level laser irradiation have been reported in various cells and tissues[5, 17, 22, 23, 33], with irradiation energy ranging from 0.1 to10 J/cm2. The Ga-Al-As diode laser device used in this study is capable of a higher output power than other similar devices. However, the effect of high-output laser irradiation on cells remains to be fully investigated. Matsuzaka et al. carried out laser irradiation of osteoblastic cells MC3T3 cells at about 200 J/cm2, and investigated cell growth and differentiation using the same device as in this study [17]. However, no studies have investigated the effect of high-output laser irradiation on salivary gland cells. The results of the trypan blue exclusion assay in this study showed that there were no differences. 13.

(15) between the irradiated and non-irradiated acinar cells, suggesting that the output condition (parameter) of the present study did not lead to acute cell death, regardless of irradiation energy. Furthermore, anti-apoptosis proteins were detected after laser irradiation. The results of this study suggest that high-power output irradiation of cells using the Ga-Al-As diode laser does not cause the kind of severe degenerative change shown by Samar et al. and Plavnik et al. who used He-Ne laser irradiation [9, 10], and it would be associated with apoptosis-inhibiting proteins such as HSP25 and bcl-2.. Conclusion In conclusion, the results of the present study suggest that low-level (Ga-Al-As) laser irradiation at 100, 300 and 500 J/cm2 induces cell proliferation, HSP25 and bcl-2 expression in Par-C10 cells, but is not effective for amylase releasing.. Acknowledgments We would like to thank Professor Hiroshi Sugiya (Department of Oral Physiology, Nihon University, School of Dentistry at Matsudo) for his helpful advice, and Associate Professor Jeremy Williams, Tokyo Dental College, for his assistance with the English of the manuscript. This work was supported by a grant from the Oral Health Science Center (HRC5A03) at Tokyo Dental College.. 14.

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(20) Figure legends Fig. 1: Cell growth in Par-C10 after low-level laser irradiation. Number of Par C10 cells in laser irradiated groups was higher than that in control group at days 5 and 7. The difference was statistically significant (p<0.01). Furthermore, at day 7, statistical significance between 100 J/cm2 and 500 J/cm2 (p<0.01) was confirmed, while no significance between 100 J/cm2 and 300 J/cm2, and between 300 J/cm2 and 500 J/cm2. However, there were no statistically significant differences among irradiated groups at days 1 and day 3. *: statistical significant (p<0.01). n: number of experiments. Fig. 2: HSP25 and bcl-2 expression in Par-C10 cell after low-level laser irradiation. While no change was seen in control (non-irradiated) group, a drastic change was seen in irradiated group. Higher expression of HSP25 was seen at days 1 and 3 in irradiated groups than in control group. However, HSP25-expression in the irradiated groups at days 5 and 7 became to be similar to that in the control group. Higher expression of bcl-2 was also detected in the 300 and 500 J/cm2 irradiated group at days 1 and 3. At days 5 and 7, the expression in the irradiation group became same or lower than that in control group. Fold change relative to the control (Fold-Delta) was determined from densitometric data after normalization to actin for HSP25 and bcl-2. Value of control at each day was arbitrarily set at 1.0.. 19.

(21) Fig. 3: Amylase release after low-level laser irradiation in isolated rat parotid acinar cells. Released amylase increased gradually in both irradiated and control (non-irradiated) groups. However, amylase release in irradiated groups was not statistically significant compare to that in control group at each time point. n: number of experiments. Fig. 4: Trypan blue exclusion assay after low-level laser irradiation in isolated rat parotid acinar cells. Ratio of trypan blue-positive cells per total cells was about 15% immediately after laser irradiation (100, 300, and 500 J/cm2) and about 20% at 30 min after irradiation. This ratio showed no difference between irradiated and control groups. n: number of experiments. 20.

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