Apoptosis and Altered Expression of P53, bax and bcl-2 induced by Formaldehyde in HELF cells

Apoptosis and Altered Expression of P53, bax and bcl-2 induced by Formaldehyde in HELF cells Sun Rongli 1,, Xiong Mengzhen 1, Zhang Juan 1,Yin Lihong 1, Pu Yuepu 1* 1 Southeast University, Nanjing, China * Corresponding email: yppu@seu.edu.cn SUMMARY Human embryonic lung fibroblast (HELF) cells were exposed to Formaldehyde(FA) at different concentrations and conducted time-dependent studies. The proliferation of HELF cells was inhibited by FA treatment in a dose- and time-dependent manner. The cell cycle analysis indicated that the proportion of cells in G0/G1 phase increased, the proportion of cells in G2/M phase decreased significantly as well as the cells in S phase when exposed to high FA concentration. Significant increase was found in the early apoptotic cells at the 25µM concentration; with exposure time increased, the early apoptosis rate decreased but still higher than the control group. The expression levels of p53 were significantly reduced after being exposed to FA, but significantly increased in high FA treated group; the bcl-2 expression showed from down to up, then down among FA treated groups; the mrna expression of bax increased and reached the highest level in lower FA treated group, then it was decreased. Moreover, the ratio of bax/bcl-2 increased significantly after being exposed to lower FA. In summary, FA is able to induce HELF cells proliferation inhibition, the G0/G1 phase arrest and cell apoptosis. The apoptosis of HELF cells induced by FA may be associated with the altered expression level of p53 mrna and an increased ratio of bax/bcl-2. KEYWORDS Cancer; Formaldehyde (FA); Toxicology 1 INTRODUCTION Formaldehyde is an important industrial chemical, widely used in the production of adhesives, treated wood, resins, and plastics (Andersen et al. 2008). Formaldehyde has long been associated with asthma, acute respiratory illness, and nasopharyngeal cancer (Speit et al. 2011). The IARC (2006) classified formaldehyde as a known human carcinogen (group 1) based on sufficient epidemiological evidence that formaldehyde causes nasopharyngeal cancer in humans (Cogliano et al. 2005). Despite formaldehyde s known respiratory toxicity, little is known about its mechanism of action related to disease. Cell apoptosis is not only a physiological process, but also a pathological defensive process. Three central and widely investigated genes involved in apoptosis are p53, bcl-2, bax. Therefore, the purpose of this article is to explore the apoptotic effect and regulation mechanism of FA on HELF cells. 2 MATERIALS/METHODS 2.1 Materials 2.1.1 Chemicals Formaldehyde(FA)( National Pharmaceutical Group Chemical Reagent company)dmem/f12 medium (HyClone, American), Fetal bovine serum(fbs)(gibco, American), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dimethyl sulphoxide (DMSO)(Biosharp, American), Ethanol(Chemical Reagent company, Nanjing ), Chloroform(Shenbo Chemical company, Shanghai), Isopropyl alcohol(national Pharmaceutical Group Chemical Reagent company), Trizol (Invitrogen, American), Annexin

V- FITC apoptosis detection kit, Cell cycle detection kit(keygentec, Nanjing), SYBR Green Realtime PCR Master Mix-Plus(Toyobo, Japan). 2.1.2 Apparatus SERIES 8000WJ CO 2 Incubator(Thermo scientific, Germany); fluorescence microscope(olympus Corporation, Japan); low-speed automatic balance centrifuge(jing Li Centrifuge company, Beijing,LDZ5-2); low-temperature high-speed centrifuge, PCR instrument(eppendorf company, Germany); Multifunctional microplate reader Mithras(Berthold technology, Germany, LB941); Gel Imaging System(Alpha innotech company, American, Chemilmager TM 5500); multi-purpose electrophoresis apparatus(beijing Oriental Teli Trade Center, HV3000); fluorescence quantitative PCR instrument(applied Biosystems, America, ABI 7300). 2.2 Methods 2.2.1 Cells culture The HELF cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum, 100μg/mL streptomycin and 100 units/ml penicillin. The cells were cultured at 37 C in a humidified atmosphere containing 5% CO 2. 2.2.2 Cell Treatment The cells were precultured in 5 ml of complete DMEM/F12 medium in 6-well plates (2 10 5 viable cells/well), exposed to FA at different concentrations of 0, 25, 50, 100 and 200μM for 24h; for time-dependent studies the cells were exposed to a 50μM dose of FA for desired time intervals. 2.2.3 Cell proliferation inhibition assay To assess cell viability, MTT assay was performed as follows. Briefly, 1 10 4 HELF cells per well were seeded in 96-well plates, exposed to different concentrations of FA(0-200μM) for 24h and at a concentration of 50μM FA for various time. Each concentration was tested in triplicate. After treatment, the cells were washed once with DMEM/F12 medium and incubated for 4 h in DMEM/F12 containing 10μl MTT(5mg/mL). The medium was discarded and 150μl of dimethylsulfoxide was added to each well. The absorbance was determined by 575nm absorbance using Multifunctional microplate reader Mithras LB941. Cell proliferation inhibition was obtained from the ratio of absorbance in the exposure groups to that in the control group. 2.2.4 Cell cycle analysis by flow cytometry To investigate the effect of FA on cell cycle progression, cell cycle was detected by flow cytometry. After treatment, the cells were harvested, washed in ice cold PBS once and fixed with 70% cold ethanol overnight at 20 C. The cells were centrifuged at 2000rpm for 5min, the pellet was washed twice with cold PBS and incubated with 100μl RNase A at 37 C for 30min. Finally, the suspension was stained with 400μl propidium iodine (PI) for 30 min at 4 C in dark. Cell cycle analysis was performed using a Becton Dickinson FACS Caliber flow cytometer. 2.2.5 Cell apoptosis analysis by flow cytometry After being exposed to FA, cells were stained for 15 min at room temperature in the dark with fluorescein isothiocyanate (FITC)-conjugated annexin V (1µg/mL) and PI (0.5µg/mL) in a Ca 2+ - enriched binding buffer and analyzed by a two-color flow cytometric assay. Annexin V and PI emissions were detected in the FL1 and FL2 channels of a FACS Caliber flow cytometer, using emission filters of 525 and 610 nm, respectively. Apoptosis analysis was performed using Becton Dickinson FACS Caliber. 2.2.6 Quantitative real-time PCR(Q-PCR) to determine the levels of gene expression Cultured HELF cells were plated in 6-well culture plates (2 10 5 cells per well), exposed to 50µM FA and incubated for different time(0h, 24h, 48h, 72h). Total RNA was prepared with Trizol, Q-PCR was performed using the SYBR Green PCR Master Mix and ABI7300 System. The Q-PCR primers used were: p53: forward, 5'- AGGGTTAGTTTACAATCAGC-3', reverse, 5'-GGTAGGTGCAAATGCC-3'; bcl-2:

forward, 5'-TCGATGTGATGCCTCTGCGAA GAAC-3'; reverse, 5'- ATTGCACTGCCAAACGGAGCTG-3'; bax: forward, 5'-GGTGCCTC AGGATGCG-3', reverse, 5'-GGAGTCTGTGTCCACG-3'; β-actin: forward, 5 -ATCCGCAAAG ACCTGT-3', reverse, 5'-GGGTGTAACGCAACTAAG-3'. Primers were synthesized by the Invitrogen Corporation. The reactions were performed in a 20μl volume mix containing 10μl SYBR Green Realtime PCR Master Mix-Plus, 1.2μl of forward and reverse primer, 2μl plus solution, 2μl cdna template and 3.6μl sterile, distilled-deionized water. Cycling condition was: 95 C for 3min, followed by 40 cycles at 95 C for 30s and 60 C for 1min. Each sample was run in three tubes. Relative gene expression was quantified using the comparative threshold cycle method and β-actin as an internal standard. 2.3 Statistical analysis Results were acquired using one-way Anova with spss13.0 software. Data were presented as means ± SD. Statistical significance for all tests was judged at a probability level of 0.05. 3 RESULTS 3.1 Cell proliferation inhibition assay The growth inhibitory potential of FA was determined in cultured HELF cells by MTT assay. The cell viability for 25μM FA treated group was not different compared with negative control group, significant inhibition was found after exposure to 50, 100, 200μM FA for 24h (p<0.05). For time-dependent studies, significant decreases were observed in cells treated with 50μM FA for 24h, 48h and 72h (p<0.05). Treatment of the HELF cells with FA resulted in the loss of cell viability in a doseand time-dependent manner. 3.2 Cell cycle analysis by flow cytometry FA-induced cell cycle phase distribution was analyzed by flow cytometry. No significant differences were found in the cell cycle of 25, 50µM FA treated groups compared with control group cells. However, the proportion of cells in G0/G1 phase increased significantly, the proportion of cells in G2/M phase decreased significantly as well as the cells in S phase when exposed to 100 and 200µM FA (p<0.05). For the time-dependent assay at 24h, the HELF cells exhibited a large decrease in the fraction of cells in G2/M phase and a concomitant increase in the proportion of cells in S phase. Moreover, the percentage of cells in G0/G1 phase increased significantly after being exposed to 50µM FA for 48h, 72h (p<0.05), with concomitant decreases in the percentage of both G2/M and S phases (p<0.05). 3.3 Cell apoptosis analysis by flow cytometry To investigate the mode of cytoxicity, flow cytometry was used to measure cell apoptosis. For cells exposed to various concentrations FA for 24h, significant increase was found in the early apoptotic cells at the 25µM concentration. At doses above 25µM, the early apoptosis rate were 6.06% 1.80% 4.30%, respectively (P<0.05). The late apoptotic cells have considerably increased (data not shown). The early apoptosis rate were 9.02% 8.19% 7.33% for the cells treated with 50µM FA for 24h 48h and 72h, with the exposure time increased, the apoptosis rate decreased but still higher than control group (P<0.05). 3.4 Determination of expression levels of mrna p53, bcl-2 and bax To clarify the molecular mechanism of FA-induced apoptosis, the expression levels of mrna p53, bcl-2 and bax were next detected by real-time PCR. The expression levels of p53 were significantly reduced after being exposed to 25, 50 and 100μM FA, but significantly increased in 200μM FA treated group. The bcl-2 expression decreased significantly in

25μM FA treated group, increased and peaked in 100μM FA treated group, after then it was reduced in 200μM FA treated group. The mrna expression of bax increased and reached the highest level in 25μM FA treated group, it was then decreased. Moreover, the ratio of bax/bcl-2 increased significantly after being exposed to 25μM FA (P<0.05). 4 DISCUSSION Formaldehyde is present in the environment as a result of natural processes or from man-made sources such as motor vehicle exhaust, wood burning stoves, and cigarette smoke. It is also a natural metabolite of living organisms (Yu PH et al. 2001, 2003), and can be found in all cells, tissues and body fluids. The IARC classified formaldehyde as a known human carcinogen (group 1) to cause nasopharyngeal cancer, recently, the known effect of formaldehyde on the human cancer burden would increase markedly because the IARC revised its categorization of formaldehyde to be a known human leukemogen (Baan et al. 2009; Goldstein, 2011; Thompson and Grafstrom, 2011; Zhang et al. 2010). Studies have demonstrated an association between cell cycle regulation and cancer, inhibition of the cell cycle has been appreciated as a target for the management of cancer (Mcdonald et al. 2011; Owa et al. 2001). Studies have shown that cellular proliferation is controlled by the cell cycle and a dysregulated cell cycle is a hallmark of cancer (Sherr, 1996; Sandhu and Slingerland, 2000). In this study, the results demonstrated that the FA treatment inhibited the proliferation of HELF cells in a dose- and time-dependent manner. Meanwhile, FA treatment caused a dose-dependent and time-dependent increase in arrest of HELF cells in G 0 /G 1 phase of the cell cycle, this increase in the G 0 /G 1 phase cell population was accompanied by a concomitant decrease in the S phase and G 2 /M phase cell populations. This FA induced cell proliferation inhibition may be due to the G 0 /G 1 phase arrest. Cell apoptosis is not only a physiological process, but also a pathological defensive process. Firstly, apoptosis is induced by injurious stimuli of lesser amplitude than those causing necrosis in the same cells, and secondly, that it occurs more readily in cell populations prepared for apoptosis (Mark and Gerald, 1997). In current study, though the early apoptosis rate of HELF cells treated with 25µM FA increased significantly compared with control, the early apoptosis rate for higher concentrations treated groups didn t increase, this might be due to the dose and time we used. With the exposure time increased, the early apoptosis rate decreased but still higher than control group (P<0.05). Under normal conditions, p53 levels are maintained at a low state by virtue of extremely short-half life of the polypeptide. Following DNA damage, levels of p53 rise dramatically within minutes. This then leads to the activation of a number of genes and triggers apoptosis. Bcl-2 family members are of particular importance as they are potent regulators of apoptosis, the ratio of bax/bcl-2 plays an important role in determining whether cells will undergo apoptosis (Xu et al. 2008; Mukherjee et al. 2011; Tenuzz et al. 2009; Weaver and Liu, 2008). In this study, the expression level of p53 decreased significantly after being exposed to 25, 50, 100μM FA and increased significantly in 200μM FA treated group compared with control group; the bcl-2 expression decreased significantly in 25μM FA treated group, increased in 50μM FA group and peaked in 100μM FA treated group, then decreased significantly in 200μM FA treated group; the bax expression increased and reached the highest level in 25μM FA treated group, it was then decreased. Moreover, the bax/bcl- 2 increased significantly after being exposed to 25μM FA. Therefore, it suggests that the changed ratio of bax/bcl-2 may play important roles in FA-induced apoptosis in HELF cells.

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