The Effect Of Sterigmatocystin On Cell Cycle Distribution In Human Immortalized Bronchial Epithelial Cells In Vitro

Objective: Sterigmatocystin (ST) is a carcinogenic mycotoxin producedas a secondary metabolite by Aspergillus, Penicillium, and Bipolaris species.ST is found as a contaminant in a variety of plant food products, such asgrains,corn, beans, spices, animal feed and even in indoor environment, suchas carpet and building materials. Based on its toxicological, mutagenic andcarcinogenic effects, ST was classified as a2B carcinogen (possible humancarcinogen) by the International Agency for Research on Cancer. Animalexperiments found that ST could induce lung adenocarcinoma in mice.Besides the ability of inducing lung adenocarcinoma, previous studies havedemonstrated that ST could induce malignant transformation in human fetallung tissue in vitro, suggesting that ST was a potent lung carcinogen. Due tothe limitations of human embryos research, the relative experiments can notproceed. Therefore, further study to explore the mechanism of ST-inducedlung cancer is needed.Cell cycle regulation and apoptosis are important ways to maintain cellularhomeostasis between cell division and cell death. It is generally accepted thatthe cancer process is a result of an imbalance between cell growth and death.Cells are continually exposed to numerous forces and toxins capable ofdamaging DNA. Cells respond to DNA damage by undergoing cell cyclearrest, to facilitate DNA repair, or by developing apoptosis to eliminateexcessively damaged and potentially harmful cells. Accumulating evidencehas indicated that cell cycle arrest is the most common effects of mycotoxins(T-2toxin, Citrinin, Ochratoxin, et al). Rencently, we found that ST couldinduce G2arrest in human gastric epithelial cell line (GES-1) and humanesophageal epithelial cell line (Het-1A). Up to now, the researchs on the lungtoxicity of ST were focused on the DNA damage in human lung cancer cell line (A549cells) in vitro. However, the effect of ST on cell cycle distributionin human lung cells is still unknown.In view of these, we chose a human immortalized bronchial epithelial cellline BEAS-2B cells to evaluate the effect of ST on cell cycle distribution inlung cells, and to explore the carcinogenicity of ST on human lung cells.Methods:1Cell cultureBEAS-2B cells were cultured in DMEM/F-12, supplemented with100U/ml penicillin,100U/ml streptomycin, and10%fetal bovine serum (FBS),under5%CO2/95%air at37℃.2MTT assayThe method of MTT was employed to evaluate the level of proliferation.BEAS-2B Cells were seeded on96-well culture plates at1×104cells/well andtreated with ST ranging from0.06to240μM for24h at37℃. At the end oftreatment (24h),20μl of MTT stock solution (final concentration:0.5mg/ml)was added to each well for another4h incubation. After4h, the medium wasreplaced with150μl of dimethylsulfoxide (DMSO) to dissolve the convertedpurple dye in culture plates. The absorbance was measured on aspectrophotometer microplate reader at a wavelength of490nm. Cell viabilitywas assayed as the relative formazan formation in treated wells compared tocontrol wells [(A490treated wells/A490control wells)×100%] aftercorrection for background absorbance.3Cell group and treatmentThe cells in logarithmic growth phase were randomly divided into fourgroups: solvent control group,6μM,12μM, and24μM. The cells in solventcontrol group were treated with DMSO (all in same volume). After24h in theexperiment, the cells were harvested and assayed by Western blot and flowcytometry.4Flow cytometric (FCM) analysisAfter treated with different concentrations of ST (6,12and24μM) for24h, the Cells from different treatment groups were collected and washed twice with cold PBS, then fixed with70%ethanol to determine the distribution ofdifferent cell cycle phases.5Western blotAfter treatment with DMSO (solvent control) or the differentconcentrations of ST (6,12and24μM) for24h, the cells were harvested. Theexpression of Phospho-H2AX, CyclinA, CyclinB1CyclinD1, CyclinE1,CDK2, CDK4and CDK1/Phospho-CDK1at protein level in BEAS-2Bcellswas determined by the method of Western blot.6Statistical analysisAll experiments were performed at least three times. All data wereexpressed as the mean±standard deviation (SD). Significant differences wereanalyzed by one-way analysis of variance (ANOVA) using SPSS software.The dose-effect relationship was analyzed with correlation analysis.Differences between groups were considered to be statistically significant at P<0.05.Results:1ST inhibited growth of BEAS-2B cellsCytotoxic effect of ST on BEAS-2B cells was measured by MTT assayafter treatment. As shown in Fig.1, the cell viability in ST treated groupsdisplayed a significant decrease in a concentration-dependent manner rangingfrom12to240μM (r=-0.91P <0.05).2ST causes DNA double-strand breaks in BEAS-2B cellsThe molecular weight of γ-H2AX is14KD. After SDS-PAGEelectrophoresis and Western blot,14KD positive bands were found in lanes ofboth the solvent control and ST treatment groups. Results fromconcentration-dependent studies revealed that after treatment with differentconcentrations of ST (6,12,24μM) for24h, the expression of γ-H2AX wassignificantly increased in a concentration-dependent manner (r=0.914P<0.05).The result suggested that ST could cause DNA double-strand breaks inBEAS-2B cells in vitro. 3ST could induce cell cycle arrest in BEAS-2B cells in vitroThe results from FCM analysis showed that, in6μM and12μM STtreatment groups, a significant increase in the proportion of cells in G2/Mphase was observed in BEAS-2B cells (P<0.05). However, the number of cellshad accumulated in the S and G2/M phase of the cell cycle in24μM STtreatment group compared to solvent control group (P<0.05).These results indicated that ST induces cell cycle arrest in BEAS-2B cellsin vitro.6μM and12μM ST treatment could induce G2/M arrest in BEAS-2Bcells, whereas24μM ST treatment could induce S and G2/M phase arrest inBEAS-2B cells.4The effect of ST on cell-cycle regulatory factor4.1ST causes modulation of G1phase regulatory proteins4.1.1ST could upregulate the expression of CyclinD1The molecular weight of CyclinD1is36KD. After SDS-PAGEelectrophoresis and Western blot,36KD positive bands were found in lanes ofboth the solvent control and ST treatment groups. Our results show that, inBEAS-2B cells, CyclinD1was increased in ST treatment groups comparedwith solvent control group in a concentration-dependent manner (r=0.992P<0.05).4.1.2No significant effect of ST on the expression of CDK4The molecular weight of CDK4is34KD. After SDS-PAGEelectrophoresis and Western blot,34KD positive bands were found in lanes ofboth the solvent control and ST treatment groups. The results showed thatthere was no significant difference in the expression of CDK4between STtreatment groups and solvent control group, especially in12μM and24μMST treatment groups (P>0.05).4.1.3ST could upregulate the expression of CyclinE1The molecular weight of CyclinE1is47KD. After SDS-PAGEelectrophoresis and Western blot,47KD positive bands were found in lanes ofboth the solvent control and ST treatment groups. The results showed thatafter treatment with different concentrations of ST (6,12,24μM) for24h, the expression of CyclinE1was significantly increased (P<0.05).4.1.4ST could upregulate the expression of CDK2The molecular weight of CDK2is33KD. After SDS-PAGEelectrophoresis and Western blot,33KD positive bands were found in lanes ofboth the solvent control and ST treatment groups. Our results show that, inBEAS-2B cells, CDK2was increased in ST treatment groups compared withsolvent control group (P<0.05).These results suggested that ST could upregulate the expressions of G1phase regulatory proteins in BEAS-2B cells.4.2ST causes modulation of S and G2/M phase regulatory proteins4.2.1ST could downregulate the expression of CyclinAThe molecular weight of CyclinA is52KD. After SDS-PAGEelectrophoresis and Western blot,52KD positive bands were found in lanes ofboth the solvent control and ST treatment groups. The results showed thatafter treatment with different concentrations of ST (12,24μM), CyclinA wasdecreased in a concentration-dependent manner (r=-0.929P<0.05). ST24μMtreatment significantly decreased the expression of CyclinA as compared withthat in ST12μM group (P<0.05).4.2.2ST could downregulate the expression of CDK1The molecular weight of CDK1is34KD. After SDS-PAGEelectrophoresis and Western blot,34KD positive bands were found in lanes ofboth the solvent control and ST treatment groups. Our results show that, inBEAS-2B cells, CDK1was decreased in ST treatment groups compared withsolvent control group in a concentration-dependent manner (r=-0.941P<0.05).4.2.3ST could upregulate the expression of p-CDK1The molecular weight of p-CDK1is34KD. After SDS-PAGEelectrophoresis and Western blot,34KD positive bands were found in lanes ofboth the solvent control and ST treatment groups. The results showed thatafter treatment with different concentrations of ST (6μM,12μM and24μM),the expression of p-CDK1was significantly increased in aconcentration-dependent manner (r=1P<0.05). 4.2.4ST could upregulate the expression of CyclinB1The molecular weight of CyclinB1is48KD. After SDS-PAGEelectrophoresis and Western blot,48KD positive bands were found in lanes ofboth the solvent control and ST treatment groups. Our results show thatCyclinB1was increased in all ST treatment groups compared with solventcontrol group (P<0.05).These results indicated that ST could cause different modulation of S andG2/M phase regulatory proteins in BEAS-2B cells.Conclusion:1ST could cause DNA double-strand breaks in BEAS-2B cells in vitro.2Low concentrations (6μM and12μM) ST treatment could induce G2/Marrest in BEAS-2B cells, High concentration (24μM) ST treatment couldinduce S and G2/M phase arrest in BEAS-2B cells.3Various concentrations of ST could upregulate the expressions of G1phaseregulatory proteins (CyclinD1, CyclinE1and CDK2), accelerate thetransmission of BEAS-2B cells from G1to S phase.412μM and24μM ST treatment could downregulate the expressions ofCyclinA, suggesting that the progression of S phase was delayed.5Various concentrations of ST could downregulate the expressions ofCDK1, but upregulate the expressions of CyclinB1and thephosphorylation levels of CDK1. These may be related with G2/M arrestinduced by ST.