Oral Diseases (2014) doi:10.1111/odi.12223 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd All rights reserved www.wiley.com
ORIGINAL ARTICLE
Hydrogen sulfide accelerates cell cycle progression in oral squamous cell carcinoma cell lines Z Ma1, Q Bi2, Y Wang3 1
Department of Special Dentistry, Peking University School and Hospital of Stomatology, Beijing; 2Department of Oral Surgery, Hospital for Oral Disease Prevention and Treatment, Harbin; 3Central Laboratory, Peking University School and Hospital of Stomatology, Beijing, China
OBJECTIVE: To investigate the cell cycle regulator role of the third gaseous transmitter hydrogen sulfide (H2S) in three oral SCC cell lines by using NaHS, a donor of H2S. METHODS: The synchronized oral squamous cell carcinoma cell lines (Cal27, GNM, and WSU-HN6) were treated with different concentrations of NaHS and then subjected to cell proliferation, cell cycle, and Western blot analyses. RESULTS: The CCK-8 assay results showed that the exogenously administered H2S donor, NaHS, induced CAL-27, and GNM cell proliferation in a concentrationdependent manner, and the cell cycle analysis indicated that NaHS accelerated cell cycle progression of the synchronized CAL-27, GNM, and WSU-HN6 cells. Western blot analysis revealed that the cell cycle regulatory genes RPA70 and RB1 were significantly down-regulated and that proliferating cell nuclear antigen (PCNA) and CDK4 were markedly up-regulated by NaHS at specific time points in the cell cycle. In addition, our results indicated that the phosphorylation of Akt and Erk1/2 was involved in exogenous H2S-induced oral SCC cell proliferation. CONCLUSIONS: H2S is a potential pro-proliferative factor of human oral SCC cells that accelerates the progression of the SCC cell cycle; thus, H2S plays a deleterious role in oral SCC cancer development. Oral Diseases (2014) doi:10.1111/odi.12223 Keywords: hydrogen sulfide; squamous cell carcinoma; cell cycle; proliferation
Correspondence: Yixiang Wang, Central Laboratory, Peking University School and Hospital of Stomatology, 22 Zhongguancun Avenue South, Haidian District, Beijing 100081, China. Tel: +86 10 82195537, Fax: +86 10 62173402, E-mail: [email protected]. Received 7 September 2013; revised 2 January 2014; accepted 8 January 2014
Introduction Halitosis is a common symptom characterized by a noticeably unpleasant odor consisting of volatile sulfur compounds exhaled in the breath from mouth (Zalewska et al, 2012; Feller and Blignaut, 2005). Studies have indicated that halitosis causes not only psychological, but also social problems, which lead to social anxiety and depression (Zalewska et al, 2012). Although the existence of this unpleasant odor in the oral cavity is unfavorable, a more important clinical facet of halitosis is hydrogen sulfide (H2S) that contributes to that odor has been proven to serve as an important physiological regulator in multiple systems (Lee et al, 2003). At present, H2S is considered the third gaseous transmitter along with nitric oxide (NO) and carbon monoxide (CO) and plays multiple physiological and pathophysiological roles in various body bioreactions, such as synaptic potentiation (Kimura et al, 2005), vasorelaxation and cardioprotection (Streeter et al, 2013), inflammation (Whiteman et al, 2010), shock (Bracht et al, 2012), and diabetes(Szabo, 2012). Oral squamous cell carcinoma (SCC) is a subtype of head and neck squamous cell carcinoma (HNSCC) located in the oral cavity. HNSCC is the sixth most common malignancy worldwide (Parkin et al, 2005). Histologically speaking, SCC is the most common histological type of head and neck cancer, and 90% of oral cancer are SCCs (Lozano et al, 2012). Although the great progression has been made in the 20th century, the 5-year survival rate of patients with HNSCC still remains at approximately 50% and has not improved significantly over the past decades (Bose et al, 2013). The main season is that oral cavity is open to the environment, and so many factors, such as smoking, alcohol, viral/bacterial infections, and halitosis, can affect the development of oral cancer. We cannot fully understand the effects of these factors on the process of oral cancer development. To date, H2S, a main contributor to halitosis, has been proven to exert different but important functions in many systems. However, little is known regarding the role of H2S in the oral cavity, particularly with respect to oral
H2S accelerates cell cycle progression in oral SCC
Z Ma et al
2
SCC. In this study, we investigated the role of the third gaseotransmitter, namely H2S, in three oral SCC cell lines through a cell counting kit-8 (CCK-8) assay, flow cytometry, and Western blot analysis. Our data indicated that exogenous H2S could accelerate SCC cell cycle progression by promoting oral SCC cell proliferation through an increase in Akt and Erk1/2 phosphorylation.
Materials and methods Reagents Sodium hydrosulfide (NaHS) was obtained from SigmaAldrich (St Louis, MO, USA). Freshly made NaHS solution was used as the H2S donor. Antibodies against replication protein A 70 kD subunit (RPA70), phosphorylatedretinoblastoma protein1 (RB1), proliferating cell nuclear antigen (PCNA), cyclin-dependent kinase 4 (CDK4), phosphorylated-Erk1/2 (p-Erk), total-Erk1/2 (t-Erk), phosphorylated-Akt (p-Akt), and total Akt (t-Akt) were purchased from Cell Signaling Technology (Danvers, MA, USA). An antibody against b-actin was purchased from Santa Cruz Biotechnology (St Louis, MO, USA). DMEM medium and fetal bovine serum (FBS) were purchased from Invitrogen (Grand Island, NY, USA). Cell culture The human oral SCC cell lines CAL-27, GNM, and WSU-HN6 were maintained in DMEM medium supplemented with 10% FBS, 100 lg ml 1 streptomycin, and 100 IU ml 1 penicillin at 37°C 5% CO2 incubator. Cell proliferation assay Cells were cultured in 96-well tissue culture plates (1 9 104 cells per well) with 10% FBS for 24 h. Then, the cells were exposed to different concentrations of NaHS (different doses of NaHS were used in different plates) and sealed with parafilm for an additional 24 h. The cell proliferation was measured by a CCK-8 assay. Briefly, 10 ll CCK-8 solution was added to each well, and the plates were incubated for additional 1 h. The absorbance was measured using a spectrometer at a wave length of 450 nm. Cell cycle analysis by flow cytometry Cells were cultured in T25-cm2 flasks (8 9 105 cells per flask) with 5 ml of 10% FBS DMEM medium overnight. The cells were then cultured in serum-free medium for an additional 30 h. After synchronization, the cells were exposed to different concentrations of NaHS for the indicated times for cell cycle analysis. The cells were harvested by trypsinization and fixed in 70% ethanol at 4°C overnight. The fixed cells were washed twice with PBS, treated with RNase A (50 lg ml 1) for 30 min at room temperature, and then stained with propidium iodide. The stained cells were analyzed for cell cycle using a Beckman Coulter XL instrument (Beckman Coulter, Brea, CA, USA). Western blot As described above, the synchronized cells were treated with 500 and 1000 lM NaHS or with the indicated Oral Diseases
concentrations of NaHS for 5 h and were then lysed with RIPA lysis buffer supplemented with protease inhibitors. The protein concentration was determined by the bicinchoninic acid (BCA) method. Equal amounts of cell lysates were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in 5% non-fat dry milk for 1 h and probed with antibodies against RPA70, RB1, PCNA, CDK4, pErk1/2, t-Erk1/2, p-Akt, t-Akt, and b-actin separately at 4°C overnight. After incubation with the corresponding horseradish peroxidase-linked secondary antibody, the immunoreactive proteins were visualized using the ECL reagent (Applygen Technology, Beijing, China). Statistical analysis The results are expressed as means s.d. Differences between groups were analyzed by one-way ANOVA. Significance was established at the P < 0.05 level.
Results Exogenous H2S promotes oral SCC cell proliferation To investigate the effect of H2S on the oral SCC cell lines GNM and CAL-27, the cell viability was determined using CCK-8 after the cells had been treated with increasing concentrations (0–1000 lM) of NaHS for 24 h. The results showed that H2S enhanced cell proliferation in a dose-dependent manner. Compared with the untreated control, the cells treated with NaHS at 200, 500 and 1000 lM showed significantly increased cell proliferation by 22.1 3.8%, 27.0 4.2%, and 16.1 4.5% in GNM cells (P < 0.01) and 12.4 1.3%, 17.3 1.1%, and 18.5 3.4% in CAL-27 cells (P < 0.05), respectively (Figure 1). Exogenous H2S accelerates cell cycle progression in oral SCC cell lines To verify the effect of H2S on the proliferation of GNM, CAL-27, and WSU-HN6 cells, cell cycle entry was
Figure 1 Effect of NaHS on cell proliferation. Cell proliferation was induced by NaHS treatment for 24 h using CCK-8 kit in oral SCC CAL-27 and GNM cells. NaHS-induced cell proliferation was in a dose-dependent manner. Data represent the means s.d. of two independent experiments analyzed using one-way ANOVA. *P < 0.05, **P < 0.01 compared with corresponding untreated controls
H2S accelerates cell cycle progression in oral SCC
Z Ma et al
analyzed using flow cytometry. The results showed that the cell cycle process was markedly changed when the cells were treated with NaHS. For GNM cells, compared with the corresponding untreated controls, 500 and 1000 lM NaHS exposure for 2.5 h significantly decreased the G0/G1-phase population [from 49.3 1.7% to 40.9 0.2% (P < 0.05) in 500 lM NaHS-treated cells and to 22.1 0.6% (P < 0.01) in 1000 lM NaHS-treated cells] and increased the G2/Mphase population [from 9.7 0.8% to 24.7 0.4% (P < 0.01) in 500 lM NaHS-treated GNM cells and to 40.5 3.5% (P < 0.01) in 1000 lM NaHS-treated cells]. Meanwhile, the S population was markedly decreased from 41.0 2.5% to 34.4 0.2% (P < 0.05) in 500 lM and to 36.4 2.7% (P < 0.05) in 1000 lM NaHS-treated cells. When the cells were treated with 500 and 1000 lM NaHS for 5 h, the cells in the G0/G1-phase changed [from 32.5 1.1% to 51.6 3.3% (P < 0.01) in 500 lM and to 26.9 0.6% (P < 0.01) in 1000 lM NaHS-treated cells] and in the G2/M-phase decreased [from 36.4 3.1% to 11.5 0.6% (P < 0.01) in 500 lM and to 2.4 1.9% (P < 0.01) in 1000 lM NaHS-treated cells], and the cells in the S-phase significantly increased [from 31.4 0.4% to 36.8 2.1% (P < 0.05) in 500 lM and to 70.6 0.7% (P < 0.01) in 1000 lM NaHS-treated cells]. When the cells were treated with NaHS for 7.5 h, the G0/G1-phase population changed [from 45.8 1.1% to 47.8 3.3% in 500 lM and to 15.1 0.6% (P < 0.01) in 1000 lM NaHS-treated cells], while the S-phase population significantly decreased [from 34.4 0.4% to 38.4 2.1% in 500 lM and to 40.8 0.7% (P < 0.01) in 1000 lM NaHS-treated cells]. In the cells treated with 500 lM NaHS for 7.5 h, the G2/M-phase population decreased slightly (from 19.8 0.6% to.13.8 1.1%, P < 0.05), while after treatment with 1000 lM NaHS for 7.5 h, the G2/M-phase population clearly increased (from 19.8 0.6% to 44.1 0.2%, P < 0.01) in comparison with the untreated control cells (Figure 2a). To compare the experimental results more intuitively, we drew the following histogram. The relative percentage of cells in the different cell cycle phases after NaHS exposure was normalized to the corresponding untreated controls at the same time point, and the results clearly showed the following dose-dependent trends: along with NaHS concentration increases, there was a decreasing trend in G0/G1-phase cells at the 2.5 h time point, an increasing trend in S-phase cells at the 5 h time point, and an increasing trend at the 2.5 h time point, and a decreasing trend at the 7.5 h time in the G2/ M phase (Figure 2b). Similar results were found in NaHS-treated CAL-27 and WSU-HN6 cells. For the CAL-27 cells, with the 5-h treatment with 1000 lM NaHS, the percentage of cells in S-phase was significantly increased (19.6 0.2%) compared with the untreated control (13.5 0.6%, P < 0.05). For WSU-HN6 cells, the percentage of G2/M-phase cells markedly increased from zero to 15.8 1.1% (P < 0.01), while the percentage of S-phase cells significantly decreased from 39.8 0.6% to 15.0 1.3% (P < 0.05) (Figure 2c).
H2S accelerates cell cycle progression via the downregulation of RB1 and RPA70 and the up-regulation of PCNA and CDK4 To understand the underlying mechanism of the H2Sinduced SCC cell proliferation, cell cycle-related genes, including RB1, RPA70, CDK4, and PCNA, were investigated by Western blot after oral SCC cells were treated with NaHS for 5 h. The results showed that in these three SCC cell lines, compared with their corresponding controls, the expression of RB1 and RPA70 was clearly down-regulated by 1000 lM NaHS. Meanwhile, PCNA was markedly up-regulated by 1000 lM NaHS. CDK4 was clearly up-regulated by 500 lM NaHS in the GNM and CAL-27 cells and by 1000 lM NaHS in the WSUHN6 cells (Figure 3a).
3
H2S promotes SCC cell proliferation through the activation of Akt and Erk1/2 signaling pathways To assess the consequences of the acceleration of the cell cycle caused by NaHS, changes in the proliferation-related Akt and Erk1/2 signaling pathway molecules were detected by Western blot. The results showed that in these three SCC cell lines, compared with the total Erk1/2 and total Akt expression levels in their corresponding controls, the expressions of p-Erk1/2 and p-Akt were clearly activated by 1000 lM NaHS (Figure 3b).
Discussion More attention is paid to cancer because it is on the way to becoming the world’s leading cause of death. People often care about the etiology, treatment, and mechanisms involved. The influencing factors of the growth rate of cancer are one of the targets for cancer research. In fact, in specific regions, such as oral cavity and brain, growth rate of cancer is more important than cancer cell metastasis, because the most important organs located at this region. If cancer grows too fast, cancer cells can directly invade into the adjacent tissues/organs, affect breathing, eating, or brain function, which is enough to kill the cancer patients. For these patients with cancer, survival rates mainly depend on the precise site, and the stage of the cancer at diagnosis. Sometimes, management of cancer in no progression stage or with a very low rate of progression is very vital for the patients with cancer under such situation. Therefore, halitosis should be considered because it could be an accelerator in the development of oral SCC based on this study. Halitosis is commonly observed in dental clinics; however, knowledge regarding the harmfulness of halitosis is not sufficient currently. The psychological and social problems resulting from halitosis receive considerably more attention than its effects on health (Zalewska et al, 2012). Although H2S, one of contributors of halitosis, is considered the important endogenous gaseous regulatory molecule, researchers are mainly focused on its effects in the circulatory, nervous, digestive, and immune systems. The role of H2S in the oral cavity is often neglected and has only been indistinctly explained to be the result of bad oral hygiene, respiratory/digestive systems disorders, or oral diseases.
Oral Diseases
H2S accelerates cell cycle progression in oral SCC
Z Ma et al
4
(a)
(b)
(c)
Oral Diseases
Figure 2 NaHS accelerates cell cycle progression of the oral SCC cell line GNM. (a) GNM cells were starved for 30 h before treatment with NaHS. Cells were harvested at indicated time point and then subjected to flow cytometry analysis for DNA content and cell cycle progression. NaHS effects on cell cycle distribution at indicated time points. (b) To compare the experimental results more intuitively, the histogram was drawn to show the relative percentage of cells normalized to corresponding untreated control at the same time points in different cell cycle phases after NaHS exposure. Along with NaHS concentration increases, there was a decreasing trend in G0/G1-phase cells at the 2.5 h time point, an increasing trend in S-phase cells at the 5 h time point, and an increasing trend at the 2.5 h time point and a decreasing trend at the 7.5 h time in the G2/M phase. (c) GNM cells and WSU-HN6 cells were starved for 30 h before treatment with NaHS. Cells were harvested at indicated time points and were processed for flow cytometry analysis. Data were collected from two independent experiments. *P < 0.05, **P < 0.01 compared with their corresponding controls.
H2S accelerates cell cycle progression in oral SCC
Z Ma et al
5
(a)
Figure 3 NaHS accelerates cell cycle through regulation of RPA70, proliferating cell nuclear antigen (PCNA), and CDK4 molecules and, subsequently, promotes cell proliferation through up-regulation of Akt and Erk1/2 signaling. (a) The synchronized oral SCC cells were treated with 500 and 1000 lM NaHS for 5 h and then subjected to Western blot. The results showed that RB1 and RPA70 were clearly down-regulated by 1000 lM NaHS; PCNA was markedly up-regulated by 1000 lM NaHS in the three SCC cell lines. Meanwhile, CDK4 was clearly up-regulated by 500 lM NaHS in GNM and CAL-27 cell lines and by 1000 lM NaHS in WSU-HN6 cell line. In addition, (b) p-Akt and p-Erk1/2 were clearly up-regulated by NaHS compared with their corresponding controls
(b)
To the best of our knowledge, the long-term persistence of bacteria and fungi makes the oral cavity a challenging environment (Duarte, 2013). Many diseases, such as oral cancer, gingivalis, and periodontitis, can occur in the oral cavity, and H2S may have some effects on these diseases. In the present study, we investigated the role of the third gaseotransmitter H2S in oral SCC cell lines. Before starting the study, we explored the sources of H2S in the body. Based on published literatures, H2S is mainly produced by endogenous sulfate-reducing microorganisms in the gastrointestinal tract (Cao et al, 2010) or is released from L-cysteine by the catalysis of cystathionine-b-synthase and cystathionine-c-lyase in the tissues (Sen et al, 2012). The H2S concentration ranges in the different tissues. For example, H2S exists in human gingival fluid at maximum concentrations of 1.9 mM (Persson, 1992) and can reach as high as 3.4 mM in the gastrointestinal tract (Rose et al, 2005; Magee et al, 2000). For in vitro study, NaHS was commonly used as a donor of H2S. In liquid solution, NaHS is decompounded to Na+ and HS-, and HS- further binds with H+ to generate a H2S molecule. NaHS solution is approximately one-third H2S in its non-dissociated form (Reiffenstein et al, 1992; Shen et al, 2012). Therefore, we chose the commonly used NaHS concentrations ranging from 100 to 1000 lM for the following experiments (Cai et al, 2010), and these concentrations were physiologically relevant to the content of H2S in the human periodontal pocket (Persson, 1992). We firstly investigated the proliferative role of NaHS in oral SCC cell lines. Our CCK-8 results clearly showed that exogenous administration of NaHS at 200–1000 lM could strongly induce oral SCC cell proliferation. The optimal concentration of NaHS that induced the maximum level of proliferation was 500 lM. A high-level NaHS (1000 lM) had a lower proliferative effect on oral SCC cell proliferation compared with 500 lM NaHS administration, which indicated that H2S at a high dose might evoke an anti-proliferative or apoptotic pathway and counterbalance its function in cell proliferation. Our results are
consistent with previous reports that showed that NaHS could alter cell cycle progression in rat intestinal epithelial cells (Deplancke and Gaskins, 2003). However, the antiproliferative effect of H2S has been reported in human gingival epithelial cells (Calenic et al, 2010), insulinsecreting beta cells (Yang et al, 2007), and human aorta smooth muscle cells (Yang et al, 2004), which suggests that the effect of H2S on cell proliferation is very complicated. It is well known that cell proliferation depends on the interactions between many cell cycle regulatory proteins, including RPA70, Rb-1, PCNA, and CDK4. RPA70 is a subunit of RPA that is a single-stranded DNA-binding protein. RPA interacts with a wide variety of protein partners, including proteins required for replication, such as PCNA and Pol a. RPA70 is involved in the initiation and elongation phases of eukaryotic DNA replication. Its expression is down-regulated in cells that enter a G1phase and exit the mitotic cycle (Zhou and Elledge, 2000; Nuss et al, 2005; Liu and Weaver, 1993). In the present study, RPA70 in oral SCC cells was clearly down-regulated by a 5-h treatment with 1000 lM NaHS, which indicated that more SCC cells entered into cell cycle process after NaHS treatment compared with the untreated controls. Proliferating cell nuclear antigen was originally identified as an antigen expressed in the nuclei of cells during the DNA synthesis phase of the cell cycle. It is under the control of E2F transcription factor-containing complexes and plays an important role for both DNA synthesis and DNA repair (Leonardi et al, 1992; Egelkrout et al, 2002; Yao et al, 2003). In the present study, PCNA was clearly up-regulated in oral SCC cells by a 5-h treatment with 1000 lM NaHS, which indicated that NaHS could induce more SCC cells into S-phase compared with untreated controls; this finding was confirmed by flow cytometry. CDK4 is a catalytic subunit of the protein kinase complex whose activity is restricted to the G1-S phase through the phosphorylation of RB1 and the subsequent release of Oral Diseases
H2S accelerates cell cycle progression in oral SCC
Z Ma et al
6
E2F transcription factors; CDK4 is important for cell cycle G1-S-phase progression (Kim et al, 2010). In the present study, CDK4 was clearly up-regulated in oral SCC cells by a 5-h treatment with either 500 lM or 1000 lM NaHS, which indicated that NaHS could push more SCC cells to pass through the G1-S transition compared with the untreated controls. This effect was caused by different doses of NaHS in the three different SCC cell lines due to their differing characteristics. Phosphorylated-retinoblastoma protein1 is a tumor suppressor protein. Hypophosphorylated RB1 is an active form of RB1 that binds and inactivates the transcriptionactivating complex E2F/DP, inhibiting cell cycle progression through the G1 phase into S phase until a cell is ready to divide (Das et al, 2005). The RB-E2F/DP complex also suppresses DNA synthesis through attracting a histone deacetylase (HDAC) protein to the chromatin. Phosphorylation of RB allows E2F/DP to dissociate from RB1 and, subsequently, activate cyclins, which push the cell through the cell cycle by activating cyclin-dependent kinases and PCNA. PCNA speeds up DNA replication and repair by helping to attach DNA polymerase to DNA (Harashima and Schnittger, 2010; de Jager et al, 2005). During the M-G1 transition, RB1 is progressively dephosphorylated by protein phosphatase 1 (PP1), returning to its growth-suppressive hypophosphorylated state (Bremner and Zacksenhaus, 2010; Vietri et al, 2006). In the present study, RB1 was clearly down-regulated in oral SCC cells by a 5-h treatment with 1000 lM NaHS, which indicated that more SCC cells entered into the M-G1 phases after NaHS treatment compared with the untreated controls. At this point, RB1 was begun to revert to its hypophosphorylated state to prepare the next cell cycle. As the consequence of accelerating cell cycle progression, the proliferative markers, including p-Erk1/2 and p-Akt, should be activated (Zimmermann and Moelling, 1999; Huber et al, 2013). Therefore, we investigated the Akt and Erk1/2 signaling pathways, which are important for cell proliferation. The Akt pathway has been shown to be activated by H2S stimulation in colon cancer cells (Cai et al, 2010). Herein, we found that NaHS also activated the Akt pathway in oral SCC cells. Our results showed that both Akt and Erk1/2 phosphorylation were required in H2S-induced cell proliferation. This result is consistent with Cai’s study, which showed that NaHS could promote colon cancer cell proliferation through the up-regulation of p-Akt and p-Erk1/2 (Cai et al, 2010). Given that H2S could promote oral SCC cells proliferation in vitro and H2S is the main contributor of halitosis, we further investigated whether halitosis patients emitted higher concentration of H2S from oral cavity and whether the size of oral SCC in halitosis patients was greater than that in oral SCC patients without halitosis. Our results showed that there is a positive correlation between the intensity of halitosis and the concentration of H2S emitted by each patient. And the mean of the tumor size s.e. of the mean was 5.71 0.60 cm3 in patients with halitosis and 7.48 0.78 cm3 in patients without halitosis. There was a trend that the oral cancer in halitosis patients was greater than that in patients without halitosis, although the difference was not significant (P = 0.056) (Supplement
Oral Diseases
Figure S1). Our explanations are (i) most people have halitosis at some points in the course of their lifetime, and it is not a specific sign of cancer. The effect of halitosis on oral cancer growth is discontinuous, and it is very hard to evaluate how much effect of halitosis on oral cancer growth, unless the tumor size is continuously monitored during a longer halitosis period. (ii) Normally, for patients with oral cancer, the first visit to a doctor is much dependent on the patient financial status and how much attention the patient pays to oral health. On measurement of oral cancer size at only one time point, it is hard to determine how often halitosis happens for the patient and how much halitosis affects oral cancer growth. (iii) Owing to the psychosocial factors, most patients with oral cancer pay more attention to their oral hygiene at the first meeting with a doctor. Under this situation, we may not get the real status of halitosis in such patients with oral cancer. (iv) Ulcer is more often found in patients with large size of oral cancer compared those with small size of oral cancer. Ulcer can cause pain in these patients, which, in turn, makes oral hygiene worse, finally leading to halitosis. That is why we find the trend that the oral cancer in halitosis patients is greater than that in no halitosis controls. Taken together, we deduce that H2S can play a proliferative role in the development of oral cancer. In summary, NaHS administration can accelerate cell cycle progression and, subsequently, promote cell proliferation in oral SCC cells. RPA70, RB1, PCNA, CDK4, p-Akt, and p-Erk1/2 are involved in this process. Our observations indicate that H2S may serve as a pro-proliferative factor for human oral SCC cells via the acceleration of SCC cell cycle progression and that H2S plays a deleterious role in oral SCC cancer development. Acknowledgements The work was supported by the research grants from National Nature Science Foundation of China (Grant No. 81271150) and Ministry of Science and Technology of China under contract International Science & Technology Cooperation Program Foundation (Grant No. 1019). No potential conflict of interests were noted.
Author contributions Z. Ma performed the cell and molecular experiments and drafted the article. Q. Bi measured H2S concentration and investigated the relationship of H2S concentration and oral SCC size. Y. Wang designed the study.
References Bose P, Brockton NT, Dort JC (2013). Head and neck cancer: from anatomy to biology. Int J Cancer 133: 2013–2023. Bracht H, Scheuerle A, Groger M et al (2012). Effects of intravenous sulfide during resuscitated porcine hemorrhagic shock. Crit Care Med 40: 2157–2167. Bremner R, Zacksenhaus E (2010). Cyclins, Cdks, E2f, Skp2, and more at the first international RB Tumor Suppressor Meeting. Cancer Res 70: 6114–6118. Cai WJ, Wang MJ, Ju LH, Wang C, Zhu YC (2010). Hydrogen sulfide induces human colon cancer cell proliferation: role of Akt, ERK and p21. Cell Biol Int 34: 565–572.
H2S accelerates cell cycle progression in oral SCC
Z Ma et al
Calenic B, Yaegaki K, Murata T et al (2010). Oral malodorous compound triggers mitochondrial-dependent apoptosis and causes genomic DNA damage in human gingival epithelial cells. J Periodontal Res 45: 31–37. Cao Q, Zhang L, Yang G, Xu C, Wang R (2010). Butyrate-stimulated H2S production in colon cancer cells. Antioxid Redox Signal 12: 1101–1109. Das SK, Hashimoto T, Shimizu K et al (2005). Fucoxanthin induces cell cycle arrest at G0/G1 phase in human colon carcinoma cells through up-regulation of p21WAF1/Cip1. Biochim Biophys Acta 1726: 328–335. Deplancke B, Gaskins HR (2003). Hydrogen sulfide induces serum-independent cell cycle entry in nontransformed rat intestinal epithelial cells. Faseb J 17: 1310–1312. Duarte S (2013). The challenge of treating oral infections caused by biofilms Oral Hyg. Health 1: e101. Egelkrout EM, Mariconti L, Settlage SB, Cella R, Robertson D, Hanley-Bowdoin L (2002). Two E2F elements regulate the proliferating cell nuclear antigen promoter differently during leaf development. Plant Cell 14: 3225–3236. Feller L, Blignaut E (2005). Halitosis: a review. SADJ 60: 17–19. Harashima H, Schnittger A (2010). The integration of cell division, growth and differentiation. Curr Opin Plant Biol 13: 66–74. Huber RM, Rajski M, Sivasankaran B, Moncayo G, Hemmings BA, Merlo A (2013). Deltex-1 activates mitotic signaling and proliferation and increases the clonogenic and invasive potential of U373 and LN18 glioblastoma cells and correlates with patient survival. PLoS ONE 8: e57793. de Jager SM, Maughan S, Dewitte W, Scofield S, Murray JA (2005). The developmental context of cell-cycle control in plants. Semin Cell Dev Biol 16: 385–396. Kim H, Huang W, Jiang X, Pennicooke B, Park PJ, Johnson MD (2010). Integrative genome analysis reveals an oncomir/ oncogene cluster regulating glioblastoma survivorship. Proc Natl Acad Sci USA 107: 2183–2188. Kimura H, Nagai Y, Umemura K, Kimura Y (2005). Physiological roles of hydrogen sulfide: synaptic modulation, neuroprotection, and smooth muscle relaxation. Antioxid Redox Signal 7: 795–803. Lee CH, Kho HS, Chung SC, Lee SW, Kim YK (2003). The relationship between volatile sulfur compounds and major halitosis-inducing factors. J Periodontol 74: 32–37. Leonardi E, Girlando S, Serio G et al (1992). PCNA and Ki67 expression in breast carcinoma: correlations with clinical and biological variables. J Clin Pathol 45: 416–419. Liu VF, Weaver DT (1993). The ionizing radiation-induced replication protein A phosphorylation response differs between ataxia telangiectasia and normal human cells. Mol Cell Biol 13: 7222–7231. Lozano R, Naghavi M, Foreman K, Lim S, Shibuya K, Aboyans V et al (2012). Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet 380: 2095–2128. Magee EA, Richardson CJ, Hughes R, Cummings JH (2000). Contribution of dietary protein to sulfide production in the large intestine: an in vitro and a controlled feeding study in humans. Am J Clin Nutr 72: 1488–1494. Nuss JE, Patrick SM, Oakley GG et al (2005). DNA damage induced hyperphosphorylation of replication protein A. 1.
Identification of novel sites of phosphorylation in response to DNA damage. Biochemistry 44: 8428–8437. Parkin DM, Bray F, Ferlay J, Pisani P (2005). Global cancer statistics, 2002. CA Cancer J Clin 55: 74–108. Persson S (1992). Hydrogen sulfide and methyl mercaptan in periodontal pockets. Oral Microbiol Immunol 7: 378–379. Reiffenstein RJ, Hulbert WC, Roth SH (1992). Toxicology of hydrogen sulfide. Annu Rev Pharmacol Toxicol 32: 109– 134. Rose P, Moore PK, Ming SH, Nam OC, Armstrong JS, Whiteman M (2005). Hydrogen sulfide protects colon cancer cells from chemopreventative agent beta-phenylethyl isothiocyanate induced apoptosis. World J Gastroenterol 11: 3990–3997. Sen N, Paul BD, Gadalla MM et al (2012). Hydrogen sulfidelinked sulfhydration of NF-kappaB mediates its antiapoptotic actions. Mol Cell 45: 13–24. Shen Y, Guo W, Wang Z, Zhang Y, Zhong L, Zhu Y (2012). Protective effects of hydrogen sulfide in hypoxic human umbilical vein endothelial cells: a possible mitochondriadependent pathway. Int J Mol Sci 14: 13093–13108. Streeter E, Ng HH, Hart JL (2013). Hydrogen sulfide as a vasculoprotective factor. Med Gas Res 3: 9. Szabo C (2012). Roles of hydrogen sulfide in the pathogenesis of diabetes mellitus and its complications. Antioxid Redox Signal 17: 68–80. Vietri M, Bianchi M, Ludlow JW, Mittnacht S, Villa-Moruzzi E (2006). Direct interaction between the catalytic subunit of Protein Phosphatase 1 and pRb. Cancer Cell Int 6: 3. Whiteman M, Li L, Rose P, Tan CH, Parkinson DB, Moore PK (2010). The effect of hydrogen sulfide donors on lipopolysaccharide-induced formation of inflammatory mediators in macrophages. Antioxid Redox Signal 12: 1147–1154. Yang G, Sun X, Wang R (2004). Hydrogen sulfide-induced apoptosis of human aorta smooth muscle cells via the activation of mitogen-activated protein kinases and caspase-3. FASEB J 18: 1782–1784. Yang G, Yang W, Wu L, Wang R (2007). H2S, endoplasmic reticulum stress, and apoptosis of insulin-secreting beta cells. J Biol Chem 282: 16567–16576. Yao N, Coryell L, Zhang D et al (2003). Replication factor C clamp loader subunit arrangement within the circular pentamer and its attachment points to proliferating cell nuclear antigen. J Biol Chem 278: 50744–50753. Zalewska A, Zatonski M, Jablonka-Strom A, Paradowska A, Kawala B, Litwin A (2012). Halitosis–a common medical and social problem. A review on pathology, diagnosis and treatment. Acta Gastroenterol Belg 75: 300–309. Zhou BB, Elledge SJ (2000). The DNA damage response: putting checkpoints in perspective. Nature 408: 433–439. Zimmermann S, Moelling K (1999). Phosphorylation and regulation of Raf by Akt (protein kinase B). Science 286: 1741– 1744.
7
Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1 Investigation of tumor size in oral SCC patients with and without halitosis.
Oral Diseases
ORIGINAL ARTICLE
Hydrogen sulfide accelerates cell cycle progression in oral squamous cell carcinoma cell lines Z Ma1, Q Bi2, Y Wang3 1
Department of Special Dentistry, Peking University School and Hospital of Stomatology, Beijing; 2Department of Oral Surgery, Hospital for Oral Disease Prevention and Treatment, Harbin; 3Central Laboratory, Peking University School and Hospital of Stomatology, Beijing, China
OBJECTIVE: To investigate the cell cycle regulator role of the third gaseous transmitter hydrogen sulfide (H2S) in three oral SCC cell lines by using NaHS, a donor of H2S. METHODS: The synchronized oral squamous cell carcinoma cell lines (Cal27, GNM, and WSU-HN6) were treated with different concentrations of NaHS and then subjected to cell proliferation, cell cycle, and Western blot analyses. RESULTS: The CCK-8 assay results showed that the exogenously administered H2S donor, NaHS, induced CAL-27, and GNM cell proliferation in a concentrationdependent manner, and the cell cycle analysis indicated that NaHS accelerated cell cycle progression of the synchronized CAL-27, GNM, and WSU-HN6 cells. Western blot analysis revealed that the cell cycle regulatory genes RPA70 and RB1 were significantly down-regulated and that proliferating cell nuclear antigen (PCNA) and CDK4 were markedly up-regulated by NaHS at specific time points in the cell cycle. In addition, our results indicated that the phosphorylation of Akt and Erk1/2 was involved in exogenous H2S-induced oral SCC cell proliferation. CONCLUSIONS: H2S is a potential pro-proliferative factor of human oral SCC cells that accelerates the progression of the SCC cell cycle; thus, H2S plays a deleterious role in oral SCC cancer development. Oral Diseases (2014) doi:10.1111/odi.12223 Keywords: hydrogen sulfide; squamous cell carcinoma; cell cycle; proliferation
Correspondence: Yixiang Wang, Central Laboratory, Peking University School and Hospital of Stomatology, 22 Zhongguancun Avenue South, Haidian District, Beijing 100081, China. Tel: +86 10 82195537, Fax: +86 10 62173402, E-mail: [email protected]. Received 7 September 2013; revised 2 January 2014; accepted 8 January 2014
Introduction Halitosis is a common symptom characterized by a noticeably unpleasant odor consisting of volatile sulfur compounds exhaled in the breath from mouth (Zalewska et al, 2012; Feller and Blignaut, 2005). Studies have indicated that halitosis causes not only psychological, but also social problems, which lead to social anxiety and depression (Zalewska et al, 2012). Although the existence of this unpleasant odor in the oral cavity is unfavorable, a more important clinical facet of halitosis is hydrogen sulfide (H2S) that contributes to that odor has been proven to serve as an important physiological regulator in multiple systems (Lee et al, 2003). At present, H2S is considered the third gaseous transmitter along with nitric oxide (NO) and carbon monoxide (CO) and plays multiple physiological and pathophysiological roles in various body bioreactions, such as synaptic potentiation (Kimura et al, 2005), vasorelaxation and cardioprotection (Streeter et al, 2013), inflammation (Whiteman et al, 2010), shock (Bracht et al, 2012), and diabetes(Szabo, 2012). Oral squamous cell carcinoma (SCC) is a subtype of head and neck squamous cell carcinoma (HNSCC) located in the oral cavity. HNSCC is the sixth most common malignancy worldwide (Parkin et al, 2005). Histologically speaking, SCC is the most common histological type of head and neck cancer, and 90% of oral cancer are SCCs (Lozano et al, 2012). Although the great progression has been made in the 20th century, the 5-year survival rate of patients with HNSCC still remains at approximately 50% and has not improved significantly over the past decades (Bose et al, 2013). The main season is that oral cavity is open to the environment, and so many factors, such as smoking, alcohol, viral/bacterial infections, and halitosis, can affect the development of oral cancer. We cannot fully understand the effects of these factors on the process of oral cancer development. To date, H2S, a main contributor to halitosis, has been proven to exert different but important functions in many systems. However, little is known regarding the role of H2S in the oral cavity, particularly with respect to oral
H2S accelerates cell cycle progression in oral SCC
Z Ma et al
2
SCC. In this study, we investigated the role of the third gaseotransmitter, namely H2S, in three oral SCC cell lines through a cell counting kit-8 (CCK-8) assay, flow cytometry, and Western blot analysis. Our data indicated that exogenous H2S could accelerate SCC cell cycle progression by promoting oral SCC cell proliferation through an increase in Akt and Erk1/2 phosphorylation.
Materials and methods Reagents Sodium hydrosulfide (NaHS) was obtained from SigmaAldrich (St Louis, MO, USA). Freshly made NaHS solution was used as the H2S donor. Antibodies against replication protein A 70 kD subunit (RPA70), phosphorylatedretinoblastoma protein1 (RB1), proliferating cell nuclear antigen (PCNA), cyclin-dependent kinase 4 (CDK4), phosphorylated-Erk1/2 (p-Erk), total-Erk1/2 (t-Erk), phosphorylated-Akt (p-Akt), and total Akt (t-Akt) were purchased from Cell Signaling Technology (Danvers, MA, USA). An antibody against b-actin was purchased from Santa Cruz Biotechnology (St Louis, MO, USA). DMEM medium and fetal bovine serum (FBS) were purchased from Invitrogen (Grand Island, NY, USA). Cell culture The human oral SCC cell lines CAL-27, GNM, and WSU-HN6 were maintained in DMEM medium supplemented with 10% FBS, 100 lg ml 1 streptomycin, and 100 IU ml 1 penicillin at 37°C 5% CO2 incubator. Cell proliferation assay Cells were cultured in 96-well tissue culture plates (1 9 104 cells per well) with 10% FBS for 24 h. Then, the cells were exposed to different concentrations of NaHS (different doses of NaHS were used in different plates) and sealed with parafilm for an additional 24 h. The cell proliferation was measured by a CCK-8 assay. Briefly, 10 ll CCK-8 solution was added to each well, and the plates were incubated for additional 1 h. The absorbance was measured using a spectrometer at a wave length of 450 nm. Cell cycle analysis by flow cytometry Cells were cultured in T25-cm2 flasks (8 9 105 cells per flask) with 5 ml of 10% FBS DMEM medium overnight. The cells were then cultured in serum-free medium for an additional 30 h. After synchronization, the cells were exposed to different concentrations of NaHS for the indicated times for cell cycle analysis. The cells were harvested by trypsinization and fixed in 70% ethanol at 4°C overnight. The fixed cells were washed twice with PBS, treated with RNase A (50 lg ml 1) for 30 min at room temperature, and then stained with propidium iodide. The stained cells were analyzed for cell cycle using a Beckman Coulter XL instrument (Beckman Coulter, Brea, CA, USA). Western blot As described above, the synchronized cells were treated with 500 and 1000 lM NaHS or with the indicated Oral Diseases
concentrations of NaHS for 5 h and were then lysed with RIPA lysis buffer supplemented with protease inhibitors. The protein concentration was determined by the bicinchoninic acid (BCA) method. Equal amounts of cell lysates were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in 5% non-fat dry milk for 1 h and probed with antibodies against RPA70, RB1, PCNA, CDK4, pErk1/2, t-Erk1/2, p-Akt, t-Akt, and b-actin separately at 4°C overnight. After incubation with the corresponding horseradish peroxidase-linked secondary antibody, the immunoreactive proteins were visualized using the ECL reagent (Applygen Technology, Beijing, China). Statistical analysis The results are expressed as means s.d. Differences between groups were analyzed by one-way ANOVA. Significance was established at the P < 0.05 level.
Results Exogenous H2S promotes oral SCC cell proliferation To investigate the effect of H2S on the oral SCC cell lines GNM and CAL-27, the cell viability was determined using CCK-8 after the cells had been treated with increasing concentrations (0–1000 lM) of NaHS for 24 h. The results showed that H2S enhanced cell proliferation in a dose-dependent manner. Compared with the untreated control, the cells treated with NaHS at 200, 500 and 1000 lM showed significantly increased cell proliferation by 22.1 3.8%, 27.0 4.2%, and 16.1 4.5% in GNM cells (P < 0.01) and 12.4 1.3%, 17.3 1.1%, and 18.5 3.4% in CAL-27 cells (P < 0.05), respectively (Figure 1). Exogenous H2S accelerates cell cycle progression in oral SCC cell lines To verify the effect of H2S on the proliferation of GNM, CAL-27, and WSU-HN6 cells, cell cycle entry was
Figure 1 Effect of NaHS on cell proliferation. Cell proliferation was induced by NaHS treatment for 24 h using CCK-8 kit in oral SCC CAL-27 and GNM cells. NaHS-induced cell proliferation was in a dose-dependent manner. Data represent the means s.d. of two independent experiments analyzed using one-way ANOVA. *P < 0.05, **P < 0.01 compared with corresponding untreated controls
H2S accelerates cell cycle progression in oral SCC
Z Ma et al
analyzed using flow cytometry. The results showed that the cell cycle process was markedly changed when the cells were treated with NaHS. For GNM cells, compared with the corresponding untreated controls, 500 and 1000 lM NaHS exposure for 2.5 h significantly decreased the G0/G1-phase population [from 49.3 1.7% to 40.9 0.2% (P < 0.05) in 500 lM NaHS-treated cells and to 22.1 0.6% (P < 0.01) in 1000 lM NaHS-treated cells] and increased the G2/Mphase population [from 9.7 0.8% to 24.7 0.4% (P < 0.01) in 500 lM NaHS-treated GNM cells and to 40.5 3.5% (P < 0.01) in 1000 lM NaHS-treated cells]. Meanwhile, the S population was markedly decreased from 41.0 2.5% to 34.4 0.2% (P < 0.05) in 500 lM and to 36.4 2.7% (P < 0.05) in 1000 lM NaHS-treated cells. When the cells were treated with 500 and 1000 lM NaHS for 5 h, the cells in the G0/G1-phase changed [from 32.5 1.1% to 51.6 3.3% (P < 0.01) in 500 lM and to 26.9 0.6% (P < 0.01) in 1000 lM NaHS-treated cells] and in the G2/M-phase decreased [from 36.4 3.1% to 11.5 0.6% (P < 0.01) in 500 lM and to 2.4 1.9% (P < 0.01) in 1000 lM NaHS-treated cells], and the cells in the S-phase significantly increased [from 31.4 0.4% to 36.8 2.1% (P < 0.05) in 500 lM and to 70.6 0.7% (P < 0.01) in 1000 lM NaHS-treated cells]. When the cells were treated with NaHS for 7.5 h, the G0/G1-phase population changed [from 45.8 1.1% to 47.8 3.3% in 500 lM and to 15.1 0.6% (P < 0.01) in 1000 lM NaHS-treated cells], while the S-phase population significantly decreased [from 34.4 0.4% to 38.4 2.1% in 500 lM and to 40.8 0.7% (P < 0.01) in 1000 lM NaHS-treated cells]. In the cells treated with 500 lM NaHS for 7.5 h, the G2/M-phase population decreased slightly (from 19.8 0.6% to.13.8 1.1%, P < 0.05), while after treatment with 1000 lM NaHS for 7.5 h, the G2/M-phase population clearly increased (from 19.8 0.6% to 44.1 0.2%, P < 0.01) in comparison with the untreated control cells (Figure 2a). To compare the experimental results more intuitively, we drew the following histogram. The relative percentage of cells in the different cell cycle phases after NaHS exposure was normalized to the corresponding untreated controls at the same time point, and the results clearly showed the following dose-dependent trends: along with NaHS concentration increases, there was a decreasing trend in G0/G1-phase cells at the 2.5 h time point, an increasing trend in S-phase cells at the 5 h time point, and an increasing trend at the 2.5 h time point, and a decreasing trend at the 7.5 h time in the G2/ M phase (Figure 2b). Similar results were found in NaHS-treated CAL-27 and WSU-HN6 cells. For the CAL-27 cells, with the 5-h treatment with 1000 lM NaHS, the percentage of cells in S-phase was significantly increased (19.6 0.2%) compared with the untreated control (13.5 0.6%, P < 0.05). For WSU-HN6 cells, the percentage of G2/M-phase cells markedly increased from zero to 15.8 1.1% (P < 0.01), while the percentage of S-phase cells significantly decreased from 39.8 0.6% to 15.0 1.3% (P < 0.05) (Figure 2c).
H2S accelerates cell cycle progression via the downregulation of RB1 and RPA70 and the up-regulation of PCNA and CDK4 To understand the underlying mechanism of the H2Sinduced SCC cell proliferation, cell cycle-related genes, including RB1, RPA70, CDK4, and PCNA, were investigated by Western blot after oral SCC cells were treated with NaHS for 5 h. The results showed that in these three SCC cell lines, compared with their corresponding controls, the expression of RB1 and RPA70 was clearly down-regulated by 1000 lM NaHS. Meanwhile, PCNA was markedly up-regulated by 1000 lM NaHS. CDK4 was clearly up-regulated by 500 lM NaHS in the GNM and CAL-27 cells and by 1000 lM NaHS in the WSUHN6 cells (Figure 3a).
3
H2S promotes SCC cell proliferation through the activation of Akt and Erk1/2 signaling pathways To assess the consequences of the acceleration of the cell cycle caused by NaHS, changes in the proliferation-related Akt and Erk1/2 signaling pathway molecules were detected by Western blot. The results showed that in these three SCC cell lines, compared with the total Erk1/2 and total Akt expression levels in their corresponding controls, the expressions of p-Erk1/2 and p-Akt were clearly activated by 1000 lM NaHS (Figure 3b).
Discussion More attention is paid to cancer because it is on the way to becoming the world’s leading cause of death. People often care about the etiology, treatment, and mechanisms involved. The influencing factors of the growth rate of cancer are one of the targets for cancer research. In fact, in specific regions, such as oral cavity and brain, growth rate of cancer is more important than cancer cell metastasis, because the most important organs located at this region. If cancer grows too fast, cancer cells can directly invade into the adjacent tissues/organs, affect breathing, eating, or brain function, which is enough to kill the cancer patients. For these patients with cancer, survival rates mainly depend on the precise site, and the stage of the cancer at diagnosis. Sometimes, management of cancer in no progression stage or with a very low rate of progression is very vital for the patients with cancer under such situation. Therefore, halitosis should be considered because it could be an accelerator in the development of oral SCC based on this study. Halitosis is commonly observed in dental clinics; however, knowledge regarding the harmfulness of halitosis is not sufficient currently. The psychological and social problems resulting from halitosis receive considerably more attention than its effects on health (Zalewska et al, 2012). Although H2S, one of contributors of halitosis, is considered the important endogenous gaseous regulatory molecule, researchers are mainly focused on its effects in the circulatory, nervous, digestive, and immune systems. The role of H2S in the oral cavity is often neglected and has only been indistinctly explained to be the result of bad oral hygiene, respiratory/digestive systems disorders, or oral diseases.
Oral Diseases
H2S accelerates cell cycle progression in oral SCC
Z Ma et al
4
(a)
(b)
(c)
Oral Diseases
Figure 2 NaHS accelerates cell cycle progression of the oral SCC cell line GNM. (a) GNM cells were starved for 30 h before treatment with NaHS. Cells were harvested at indicated time point and then subjected to flow cytometry analysis for DNA content and cell cycle progression. NaHS effects on cell cycle distribution at indicated time points. (b) To compare the experimental results more intuitively, the histogram was drawn to show the relative percentage of cells normalized to corresponding untreated control at the same time points in different cell cycle phases after NaHS exposure. Along with NaHS concentration increases, there was a decreasing trend in G0/G1-phase cells at the 2.5 h time point, an increasing trend in S-phase cells at the 5 h time point, and an increasing trend at the 2.5 h time point and a decreasing trend at the 7.5 h time in the G2/M phase. (c) GNM cells and WSU-HN6 cells were starved for 30 h before treatment with NaHS. Cells were harvested at indicated time points and were processed for flow cytometry analysis. Data were collected from two independent experiments. *P < 0.05, **P < 0.01 compared with their corresponding controls.
H2S accelerates cell cycle progression in oral SCC
Z Ma et al
5
(a)
Figure 3 NaHS accelerates cell cycle through regulation of RPA70, proliferating cell nuclear antigen (PCNA), and CDK4 molecules and, subsequently, promotes cell proliferation through up-regulation of Akt and Erk1/2 signaling. (a) The synchronized oral SCC cells were treated with 500 and 1000 lM NaHS for 5 h and then subjected to Western blot. The results showed that RB1 and RPA70 were clearly down-regulated by 1000 lM NaHS; PCNA was markedly up-regulated by 1000 lM NaHS in the three SCC cell lines. Meanwhile, CDK4 was clearly up-regulated by 500 lM NaHS in GNM and CAL-27 cell lines and by 1000 lM NaHS in WSU-HN6 cell line. In addition, (b) p-Akt and p-Erk1/2 were clearly up-regulated by NaHS compared with their corresponding controls
(b)
To the best of our knowledge, the long-term persistence of bacteria and fungi makes the oral cavity a challenging environment (Duarte, 2013). Many diseases, such as oral cancer, gingivalis, and periodontitis, can occur in the oral cavity, and H2S may have some effects on these diseases. In the present study, we investigated the role of the third gaseotransmitter H2S in oral SCC cell lines. Before starting the study, we explored the sources of H2S in the body. Based on published literatures, H2S is mainly produced by endogenous sulfate-reducing microorganisms in the gastrointestinal tract (Cao et al, 2010) or is released from L-cysteine by the catalysis of cystathionine-b-synthase and cystathionine-c-lyase in the tissues (Sen et al, 2012). The H2S concentration ranges in the different tissues. For example, H2S exists in human gingival fluid at maximum concentrations of 1.9 mM (Persson, 1992) and can reach as high as 3.4 mM in the gastrointestinal tract (Rose et al, 2005; Magee et al, 2000). For in vitro study, NaHS was commonly used as a donor of H2S. In liquid solution, NaHS is decompounded to Na+ and HS-, and HS- further binds with H+ to generate a H2S molecule. NaHS solution is approximately one-third H2S in its non-dissociated form (Reiffenstein et al, 1992; Shen et al, 2012). Therefore, we chose the commonly used NaHS concentrations ranging from 100 to 1000 lM for the following experiments (Cai et al, 2010), and these concentrations were physiologically relevant to the content of H2S in the human periodontal pocket (Persson, 1992). We firstly investigated the proliferative role of NaHS in oral SCC cell lines. Our CCK-8 results clearly showed that exogenous administration of NaHS at 200–1000 lM could strongly induce oral SCC cell proliferation. The optimal concentration of NaHS that induced the maximum level of proliferation was 500 lM. A high-level NaHS (1000 lM) had a lower proliferative effect on oral SCC cell proliferation compared with 500 lM NaHS administration, which indicated that H2S at a high dose might evoke an anti-proliferative or apoptotic pathway and counterbalance its function in cell proliferation. Our results are
consistent with previous reports that showed that NaHS could alter cell cycle progression in rat intestinal epithelial cells (Deplancke and Gaskins, 2003). However, the antiproliferative effect of H2S has been reported in human gingival epithelial cells (Calenic et al, 2010), insulinsecreting beta cells (Yang et al, 2007), and human aorta smooth muscle cells (Yang et al, 2004), which suggests that the effect of H2S on cell proliferation is very complicated. It is well known that cell proliferation depends on the interactions between many cell cycle regulatory proteins, including RPA70, Rb-1, PCNA, and CDK4. RPA70 is a subunit of RPA that is a single-stranded DNA-binding protein. RPA interacts with a wide variety of protein partners, including proteins required for replication, such as PCNA and Pol a. RPA70 is involved in the initiation and elongation phases of eukaryotic DNA replication. Its expression is down-regulated in cells that enter a G1phase and exit the mitotic cycle (Zhou and Elledge, 2000; Nuss et al, 2005; Liu and Weaver, 1993). In the present study, RPA70 in oral SCC cells was clearly down-regulated by a 5-h treatment with 1000 lM NaHS, which indicated that more SCC cells entered into cell cycle process after NaHS treatment compared with the untreated controls. Proliferating cell nuclear antigen was originally identified as an antigen expressed in the nuclei of cells during the DNA synthesis phase of the cell cycle. It is under the control of E2F transcription factor-containing complexes and plays an important role for both DNA synthesis and DNA repair (Leonardi et al, 1992; Egelkrout et al, 2002; Yao et al, 2003). In the present study, PCNA was clearly up-regulated in oral SCC cells by a 5-h treatment with 1000 lM NaHS, which indicated that NaHS could induce more SCC cells into S-phase compared with untreated controls; this finding was confirmed by flow cytometry. CDK4 is a catalytic subunit of the protein kinase complex whose activity is restricted to the G1-S phase through the phosphorylation of RB1 and the subsequent release of Oral Diseases
H2S accelerates cell cycle progression in oral SCC
Z Ma et al
6
E2F transcription factors; CDK4 is important for cell cycle G1-S-phase progression (Kim et al, 2010). In the present study, CDK4 was clearly up-regulated in oral SCC cells by a 5-h treatment with either 500 lM or 1000 lM NaHS, which indicated that NaHS could push more SCC cells to pass through the G1-S transition compared with the untreated controls. This effect was caused by different doses of NaHS in the three different SCC cell lines due to their differing characteristics. Phosphorylated-retinoblastoma protein1 is a tumor suppressor protein. Hypophosphorylated RB1 is an active form of RB1 that binds and inactivates the transcriptionactivating complex E2F/DP, inhibiting cell cycle progression through the G1 phase into S phase until a cell is ready to divide (Das et al, 2005). The RB-E2F/DP complex also suppresses DNA synthesis through attracting a histone deacetylase (HDAC) protein to the chromatin. Phosphorylation of RB allows E2F/DP to dissociate from RB1 and, subsequently, activate cyclins, which push the cell through the cell cycle by activating cyclin-dependent kinases and PCNA. PCNA speeds up DNA replication and repair by helping to attach DNA polymerase to DNA (Harashima and Schnittger, 2010; de Jager et al, 2005). During the M-G1 transition, RB1 is progressively dephosphorylated by protein phosphatase 1 (PP1), returning to its growth-suppressive hypophosphorylated state (Bremner and Zacksenhaus, 2010; Vietri et al, 2006). In the present study, RB1 was clearly down-regulated in oral SCC cells by a 5-h treatment with 1000 lM NaHS, which indicated that more SCC cells entered into the M-G1 phases after NaHS treatment compared with the untreated controls. At this point, RB1 was begun to revert to its hypophosphorylated state to prepare the next cell cycle. As the consequence of accelerating cell cycle progression, the proliferative markers, including p-Erk1/2 and p-Akt, should be activated (Zimmermann and Moelling, 1999; Huber et al, 2013). Therefore, we investigated the Akt and Erk1/2 signaling pathways, which are important for cell proliferation. The Akt pathway has been shown to be activated by H2S stimulation in colon cancer cells (Cai et al, 2010). Herein, we found that NaHS also activated the Akt pathway in oral SCC cells. Our results showed that both Akt and Erk1/2 phosphorylation were required in H2S-induced cell proliferation. This result is consistent with Cai’s study, which showed that NaHS could promote colon cancer cell proliferation through the up-regulation of p-Akt and p-Erk1/2 (Cai et al, 2010). Given that H2S could promote oral SCC cells proliferation in vitro and H2S is the main contributor of halitosis, we further investigated whether halitosis patients emitted higher concentration of H2S from oral cavity and whether the size of oral SCC in halitosis patients was greater than that in oral SCC patients without halitosis. Our results showed that there is a positive correlation between the intensity of halitosis and the concentration of H2S emitted by each patient. And the mean of the tumor size s.e. of the mean was 5.71 0.60 cm3 in patients with halitosis and 7.48 0.78 cm3 in patients without halitosis. There was a trend that the oral cancer in halitosis patients was greater than that in patients without halitosis, although the difference was not significant (P = 0.056) (Supplement
Oral Diseases
Figure S1). Our explanations are (i) most people have halitosis at some points in the course of their lifetime, and it is not a specific sign of cancer. The effect of halitosis on oral cancer growth is discontinuous, and it is very hard to evaluate how much effect of halitosis on oral cancer growth, unless the tumor size is continuously monitored during a longer halitosis period. (ii) Normally, for patients with oral cancer, the first visit to a doctor is much dependent on the patient financial status and how much attention the patient pays to oral health. On measurement of oral cancer size at only one time point, it is hard to determine how often halitosis happens for the patient and how much halitosis affects oral cancer growth. (iii) Owing to the psychosocial factors, most patients with oral cancer pay more attention to their oral hygiene at the first meeting with a doctor. Under this situation, we may not get the real status of halitosis in such patients with oral cancer. (iv) Ulcer is more often found in patients with large size of oral cancer compared those with small size of oral cancer. Ulcer can cause pain in these patients, which, in turn, makes oral hygiene worse, finally leading to halitosis. That is why we find the trend that the oral cancer in halitosis patients is greater than that in no halitosis controls. Taken together, we deduce that H2S can play a proliferative role in the development of oral cancer. In summary, NaHS administration can accelerate cell cycle progression and, subsequently, promote cell proliferation in oral SCC cells. RPA70, RB1, PCNA, CDK4, p-Akt, and p-Erk1/2 are involved in this process. Our observations indicate that H2S may serve as a pro-proliferative factor for human oral SCC cells via the acceleration of SCC cell cycle progression and that H2S plays a deleterious role in oral SCC cancer development. Acknowledgements The work was supported by the research grants from National Nature Science Foundation of China (Grant No. 81271150) and Ministry of Science and Technology of China under contract International Science & Technology Cooperation Program Foundation (Grant No. 1019). No potential conflict of interests were noted.
Author contributions Z. Ma performed the cell and molecular experiments and drafted the article. Q. Bi measured H2S concentration and investigated the relationship of H2S concentration and oral SCC size. Y. Wang designed the study.
References Bose P, Brockton NT, Dort JC (2013). Head and neck cancer: from anatomy to biology. Int J Cancer 133: 2013–2023. Bracht H, Scheuerle A, Groger M et al (2012). Effects of intravenous sulfide during resuscitated porcine hemorrhagic shock. Crit Care Med 40: 2157–2167. Bremner R, Zacksenhaus E (2010). Cyclins, Cdks, E2f, Skp2, and more at the first international RB Tumor Suppressor Meeting. Cancer Res 70: 6114–6118. Cai WJ, Wang MJ, Ju LH, Wang C, Zhu YC (2010). Hydrogen sulfide induces human colon cancer cell proliferation: role of Akt, ERK and p21. Cell Biol Int 34: 565–572.
H2S accelerates cell cycle progression in oral SCC
Z Ma et al
Calenic B, Yaegaki K, Murata T et al (2010). Oral malodorous compound triggers mitochondrial-dependent apoptosis and causes genomic DNA damage in human gingival epithelial cells. J Periodontal Res 45: 31–37. Cao Q, Zhang L, Yang G, Xu C, Wang R (2010). Butyrate-stimulated H2S production in colon cancer cells. Antioxid Redox Signal 12: 1101–1109. Das SK, Hashimoto T, Shimizu K et al (2005). Fucoxanthin induces cell cycle arrest at G0/G1 phase in human colon carcinoma cells through up-regulation of p21WAF1/Cip1. Biochim Biophys Acta 1726: 328–335. Deplancke B, Gaskins HR (2003). Hydrogen sulfide induces serum-independent cell cycle entry in nontransformed rat intestinal epithelial cells. Faseb J 17: 1310–1312. Duarte S (2013). The challenge of treating oral infections caused by biofilms Oral Hyg. Health 1: e101. Egelkrout EM, Mariconti L, Settlage SB, Cella R, Robertson D, Hanley-Bowdoin L (2002). Two E2F elements regulate the proliferating cell nuclear antigen promoter differently during leaf development. Plant Cell 14: 3225–3236. Feller L, Blignaut E (2005). Halitosis: a review. SADJ 60: 17–19. Harashima H, Schnittger A (2010). The integration of cell division, growth and differentiation. Curr Opin Plant Biol 13: 66–74. Huber RM, Rajski M, Sivasankaran B, Moncayo G, Hemmings BA, Merlo A (2013). Deltex-1 activates mitotic signaling and proliferation and increases the clonogenic and invasive potential of U373 and LN18 glioblastoma cells and correlates with patient survival. PLoS ONE 8: e57793. de Jager SM, Maughan S, Dewitte W, Scofield S, Murray JA (2005). The developmental context of cell-cycle control in plants. Semin Cell Dev Biol 16: 385–396. Kim H, Huang W, Jiang X, Pennicooke B, Park PJ, Johnson MD (2010). Integrative genome analysis reveals an oncomir/ oncogene cluster regulating glioblastoma survivorship. Proc Natl Acad Sci USA 107: 2183–2188. Kimura H, Nagai Y, Umemura K, Kimura Y (2005). Physiological roles of hydrogen sulfide: synaptic modulation, neuroprotection, and smooth muscle relaxation. Antioxid Redox Signal 7: 795–803. Lee CH, Kho HS, Chung SC, Lee SW, Kim YK (2003). The relationship between volatile sulfur compounds and major halitosis-inducing factors. J Periodontol 74: 32–37. Leonardi E, Girlando S, Serio G et al (1992). PCNA and Ki67 expression in breast carcinoma: correlations with clinical and biological variables. J Clin Pathol 45: 416–419. Liu VF, Weaver DT (1993). The ionizing radiation-induced replication protein A phosphorylation response differs between ataxia telangiectasia and normal human cells. Mol Cell Biol 13: 7222–7231. Lozano R, Naghavi M, Foreman K, Lim S, Shibuya K, Aboyans V et al (2012). Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet 380: 2095–2128. Magee EA, Richardson CJ, Hughes R, Cummings JH (2000). Contribution of dietary protein to sulfide production in the large intestine: an in vitro and a controlled feeding study in humans. Am J Clin Nutr 72: 1488–1494. Nuss JE, Patrick SM, Oakley GG et al (2005). DNA damage induced hyperphosphorylation of replication protein A. 1.
Identification of novel sites of phosphorylation in response to DNA damage. Biochemistry 44: 8428–8437. Parkin DM, Bray F, Ferlay J, Pisani P (2005). Global cancer statistics, 2002. CA Cancer J Clin 55: 74–108. Persson S (1992). Hydrogen sulfide and methyl mercaptan in periodontal pockets. Oral Microbiol Immunol 7: 378–379. Reiffenstein RJ, Hulbert WC, Roth SH (1992). Toxicology of hydrogen sulfide. Annu Rev Pharmacol Toxicol 32: 109– 134. Rose P, Moore PK, Ming SH, Nam OC, Armstrong JS, Whiteman M (2005). Hydrogen sulfide protects colon cancer cells from chemopreventative agent beta-phenylethyl isothiocyanate induced apoptosis. World J Gastroenterol 11: 3990–3997. Sen N, Paul BD, Gadalla MM et al (2012). Hydrogen sulfidelinked sulfhydration of NF-kappaB mediates its antiapoptotic actions. Mol Cell 45: 13–24. Shen Y, Guo W, Wang Z, Zhang Y, Zhong L, Zhu Y (2012). Protective effects of hydrogen sulfide in hypoxic human umbilical vein endothelial cells: a possible mitochondriadependent pathway. Int J Mol Sci 14: 13093–13108. Streeter E, Ng HH, Hart JL (2013). Hydrogen sulfide as a vasculoprotective factor. Med Gas Res 3: 9. Szabo C (2012). Roles of hydrogen sulfide in the pathogenesis of diabetes mellitus and its complications. Antioxid Redox Signal 17: 68–80. Vietri M, Bianchi M, Ludlow JW, Mittnacht S, Villa-Moruzzi E (2006). Direct interaction between the catalytic subunit of Protein Phosphatase 1 and pRb. Cancer Cell Int 6: 3. Whiteman M, Li L, Rose P, Tan CH, Parkinson DB, Moore PK (2010). The effect of hydrogen sulfide donors on lipopolysaccharide-induced formation of inflammatory mediators in macrophages. Antioxid Redox Signal 12: 1147–1154. Yang G, Sun X, Wang R (2004). Hydrogen sulfide-induced apoptosis of human aorta smooth muscle cells via the activation of mitogen-activated protein kinases and caspase-3. FASEB J 18: 1782–1784. Yang G, Yang W, Wu L, Wang R (2007). H2S, endoplasmic reticulum stress, and apoptosis of insulin-secreting beta cells. J Biol Chem 282: 16567–16576. Yao N, Coryell L, Zhang D et al (2003). Replication factor C clamp loader subunit arrangement within the circular pentamer and its attachment points to proliferating cell nuclear antigen. J Biol Chem 278: 50744–50753. Zalewska A, Zatonski M, Jablonka-Strom A, Paradowska A, Kawala B, Litwin A (2012). Halitosis–a common medical and social problem. A review on pathology, diagnosis and treatment. Acta Gastroenterol Belg 75: 300–309. Zhou BB, Elledge SJ (2000). The DNA damage response: putting checkpoints in perspective. Nature 408: 433–439. Zimmermann S, Moelling K (1999). Phosphorylation and regulation of Raf by Akt (protein kinase B). Science 286: 1741– 1744.
7
Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1 Investigation of tumor size in oral SCC patients with and without halitosis.
Oral Diseases
