| Background and objectiveLung cancer is one of the most common malignant neoplasms worldwide and its incidence has been increasing over the past few decades in some areas such as Europe, USA and Asia. Non-small cell lung cancer is the most common type in all types of lung cancer.Although surgical resection, Chemotherapy and radiofrequency ablation are common options for Non-small cell lung cancer, patients with advanced Non-small cell lung cancer are not candidates for these therapies.In addition, because of the inherently chemotherapyresistant nature of Non-small cell lung cancer, systemic cytotoxic chemotherapy agentsare minimally effective at improving patient survival.Thus, a major goal is to find new chemotherapeutic agents and more effective therapies for the treatment of Non-small cell lung cancer.Vibrio vulnificus cytolysin(VVC), a polypeptide with molecular weight 50851, is encoded by the vvhA gene. It is produced by most pathogenic strains, is water-soluble and demonstrates thermal instability.In recent years, Vibrio vulnificus cytolysin as a key factor in the pathogenesis of Vibrio vulnificus, has drawn increasing attention of researchers. VVC is extremely active and the only secreted exotoxin characteristic of vibrio vulnificus species. In addition to hemolytic activities, VVC can kill various mammalian cells. Studies showed that VVC perforated the target cell membrane and played an important role in the dissolution of cells.It was report that low concentration of VVC might induce apoptosis in various mammalian cells. However, there have been very few studies so far about the cytotoxic effects of VVC on cancer cells.In previous study, we have constructed the pET32a (+)-vvhA expression vector and also succeeded in optimizing the conditions of expression, purity and renaturation. This has also laid the foundation for further studies on biological activity of rVVC. Further studies showed that VVC is necessary to the pathogenicity of V.vulnificus. The lung is the target organ of VVC when V.vulnificus infection occurs. We also found that rVVC inhibits proliferation and induces apoptosis in A549 cell line.Based on the cytotoxic effects of VVC on various mammalian cells and our previous research results, we conducted this study to investigate the cytolytic effects and molecular mechanism of VVC in human lung adenocarcinoma cell line A549. In this study, We use of the expression vector pET32a-(+)-vvhA, obtained sufficient and high purity rVVC in the optimized conditions. We also analyzed the inhibitory effects of rVVC on A549 (human lung adenocarcinoma cells) cells in vitro, and investigated the possible mechanisms involved.Methods:1. Extraction, purification and identification of the rVVCThe pET-32a-vvhA/E.coliBL21(DE3) was recovery, culture and expression.then choose a separate plaque for PCR amplification and agarose gel electrophoresis to verify the existence of the target gene. Then the recombination proteins were expressed under the optimized inducing condition. The induced E.coli BL21(DE3) were mechanical lysised by sonication. Then the recombination proteins of V.vulnificus cytolysin(rVVC) with His-tag in the cell lysis were purified by binding to Ni-IMAC resin. The purification of recombination proteins were detected by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western-blot using antibody against His-tagged. The concentration Purify rVVC were calculated using BCA protein quantitative method. 2.The cytolytic effects of rVVC human lung adenocarcinoma cells A549 and human bronchial epithelial cells HBE2.1 The cytolytic effects of rVVC human lung adenocarcinoma cells A549(1) MTT was used to measure the inhibition ratio of A549 in response to different concentration.(2) Instant,24 and 48 hours’survival rates of A549 cells treated by rVVC were determined based on the trypan blue exclusive assay.(3) The capability of cell colony formation was detected by clone formation test.(4) The morphologic changes of A549 both directly and after HE stain was observed by inverted microscope.The ultrastructure changes of A549 were observed and photographed by using transmission electron microscope.(5) Measuring the stereological parameters of the cell area and the nuclear area (Ac,An), perimeter (Cc, Cn), Long Axis of cell and Long Axis of nuclear (Dmaj), Short Axis of cell and Short Axis of cell (Dmin), Ratio Axis of cell(Rac), Area of Cytoplasm(Acp), Ratio of Nuclear and Cytoplasm(Rnp), Form Fctor PE of cell and Form Fctor PE of nuclear (PE),Form Fctor AR of cell and Form Fctor AR of nuclear (AR), Regular Form Fotor of cell and Regular Form Fotor of nuclear(RFF), Form Irregular Idex of cell and Form Irregular Idex of nuclear (FII) in normal group and rVVC attack group (10μg/ml; 24h) to test morphological changes of the human lung adenocarcinoma cells A549 under the attack of rVVC; The ultrastructure morphological changes of the human lung adenocarcinoma cells A549 under the attack of rVVC was tested by applications of the stereological principles and methods. Measuring the stereological parameters of the mitochondrial Vv(VolumeDensity),Sv (SurfaceDensity), Nv(NumericalDensity),S(MeanSurface),V(Mean Volume).2.2 The cytolytic effects of rVVC human bronchial epithelial cells HBE(1) MTT was used to measure the inhibition ratio of HBE in response to different concentration.(2) Instant,24 and 48 hours’survival rates of HBE cells treated by rVVC were determined based on the trypan blue exclusive assay.(3) The capability of cell colony formation was detected by clone formation test.3. Study on the lethal effects and mechanism of Vibrio Vulnificus cytolysin on Human lung adenocarcinoma A549The activity of LDH in the supernatants was measured by dinitro-phenyl-hydrazine (DPNH) colorimetry. The apoptotic changes of A549cells were analyzed with Hoechst333258 fluorescence staining, and transmission electron microscope. The percentage changes of apoptosis were analyzed with flow cytometry.Apoptosis DNA Ladder was studied by Agarose Gel Electrophoresis.Westen-blot was used to detect expressions of Bcl-2 and Bax.Results1. Extraction, purification and identification of the rVVCThe fragment of vvhA gene in the positive clone E.coli BL21(DE3)were amplified by PCR, we obtain the 1356bp gene fragment on agarose gel. The rVVC protein could be highly expressed in E.coli BL21(DE3) under the optimized inducing conditions. The rVVC protein was certified purified by SDS-PAGE. A 70-kD band was observed in gels, matching the predicted molecular weight. The recombination protein rVVC expressed with His-tag was confirmed by Western-blot. The concentration of the purified protein with His-tag was 1.163mg/ml.2.The cytolytic effects of rVVC human lung adenocarcinoma cells A549 and human bronchial epithelial cells HBE2.1 The cytolytic effects of rVVC human lung adenocarcinoma cells A549(1)Through 96 plate culture method and the MTT method to detect the cell growth curve and the determination of absorbance of A549cells under the attack of different concentrations of rVVC. The cells growth inhibition rates after 24h of 5μg/ ml,10μg/ml group,15ugμml group,20μg/ml group,25μg/ml and 30μg/ml were: (30.96±2.94)%, (36.53±4.49)%, (45.97±4.21)%, (57.67±4.91)%, (61.72±5.75) %, (71.06±8.05)%; and after 48h were:(36.76±2.93)%, (40.59±5.12)%, (60.85±7.32)%, (66.32±6.46)%, (72.95±8.49)%, (79.65±8.28)%; while after 72h were:(39.14±3.89)%, (43.04±4.77)%, (61.01±5.52)%, (68.06±5.65) %, (75.68±8.16)%, (82.55±8.25)%. The inhibitory effects of rVVC on A549 cells were concentration-dependent and statistically significant compared to the control group (P<0.05).(2) Trypan blue staining method detects the cell mortality rate immediately,24h and after 48h after invasion of different concentration. The immediately lethal rate were0.00%、1.44%、2.91%、2.57%、3.35%、5.59%、5.73%(F=7.023,p=0.003), the 24h cell lethal rate were0.01%、21.74%、31.26 %、41.31%、43.44%、51.67%、62.98%(F=33.525,p=0.000);and the 48h cell lethal rate were 0.08%.31.05%、50.99%、56.90%、64.99%、55.77%、56.89% (F=25.136,p=0.000). The lethal effects of rVVC on A549 cells were concentration-dependent and statistically significant compared to the control group (p < 0.05).(3) Colony formation assay cloning efficiency is 68.40% in the control group and 44.20% in the 10μg/ml group.35.38.% decrease compared with Control group (χ2=178.526,p=0.000)(4) The A549 cells in normal group appear spindle, cytoplasm stained pink, round or oval nucleus and clear nucleolus. But the cells in rVVC attack group appear unclear structure, cell growth sparse, scarce cytoplasm stained nuclei become elongated, some condensation nuclei.After A549cells were incubated with 10μg/ml rVVC for 24 h, typical apoptotic characteristics including crescent-shaped nucleus, chromatin condensation and intracytoplasm vacuolus could be found under transmission electron microscope.(5) The comparative analysis of the morphological characteristics between A549 cells and A549cell (treated by rVVC):The cell parameters of the control group and the rVVC attack group whose concentration is 10μg/ml were as follows:control group Area of Nuclear (μm2), (157.807±45.611);Circumstances of nuclear (μm), (55.381±10.833);Area of Cell (μm2), (363.209±19.538) Circumstances of cell (μm), (104.317±21.988);Area of Cytoplasm (μm2), (204.873±108.116);Ratio of Nuclear and Cytoplasm (1.0476±0.7029); 10μg/ml rVVC group:Area of Nuclear (μm2), (102.390±31.962);Circumstances of nuclear (μm), (49.764±9.003);Area of Cell (μm2), (219.910±70.00) Circumstances of cell (μm), (89.622±22.025);Area of Cytoplasm (μm2), (117.528±50.988);Ratio of Nuclear and Cytoplasm (1.5103±4.6697).The mitochondrial parameters of the control group and the rVVC attack group whose concentration is 10μg/ml were as follows:control group:Volume Density (0.1635±0.0762);Surface Density(μm2/3), (0.1334±0.0526);Mean Volume(μm3) (2.8332±1.2543) Numerical Density(μm-3), (0.0599±0.0216); Mean Surface(μm2), (2.2755±0.6560); 10μg/ml rVVC group:Volume Density (0.1010±0.1837);Surface Density(μm2/3), (0.8941±1.4719);Mean Volume(μm3), (1.0498±2.4099);Numerical Density(μm-3), (0.3949±0.3338); Mean Surface(μm2), (6.1017±19.9348).2.1 The cytolytic effects of rWC human bronchial epithelial cells HBE(l)Through 96 plate culture method and the MTT method to detect the cell growth curve and the determination of absorbance of HBE cells under the attack of different concentrations of rVVC. The cells growth inhibition rates after 24h of 0.625μg/ml group,1.25μg/ml group,2.5μg/ml group,5μg/ml and 10μg/ml were: (14.80±2.55)%, (23.65±1.27)%, (26.38±2.12)%, (40.997±3.19)%, (48.90±7.77) %; and after 48h were:(24.66±1.46)%, (42.05±6.66)%, (45.45±8.80)%, (59.87±5.08)%, (70.84±10.72)%; while after 72h were:(34.14±3.84)%, (49.30±8.39)%, (55.18±3.99)%, (72.08±6.22)%; (77.77±7.04)%. The inhibitory effects of rVVC on A549 cells were concentration-dependent and statistically significant compared to the control group (P< 0.05).(2)Trypan blue staining method detects the cell lethal rate immediately,24h,and after 48h after invasion of different concentration. The immediately lethal rate were0.00%、1.30%、1.64%、2.16%、2.61%、4.86%(F=14.932,p=0.003);,the 24h cell lethal rate were0.00%、12.39%、19.42%、27.69%、40.85%、52.65% (F=98.395,p=0.000);and the 48h cell lethal rate were0.1%、22.03%、47.96%、55.31%、63.52%、77.48%(F=52.615,p=0.000). The lethal effects of rVVC on A549 cells were concentration-dependent and statistically significant compared to the control group (P<0.05).(3)Colony formation assay cloning efficiency is 47.40% in the control group and 29.10% in the 5μg/ml group.38.60% decrease compared with Control group (χ2=106.745, p=0.000)3. Study on the lethal effects and mechanism of Vibrio Vulnificus cytolysin on Human lung adenocarcinoma A549There were no significant change in LDH activity in all supernatants of the rVVC-treated cells (P> 0.05);the LDH activity of different groups:0 h group: 155.60±5.35 U/L; 0.5 h group151.69±3.52 U/L;2 h group156.68±7.70 U/L;4 h group 159.51±4.23 U/L; 8 h group 157.83±2.23 U/L. Fluorescence microscopic analysis showed marked morphological features of early apoptosis in A549 cells such as bright and nuclear condensation when treated with rVVC(10μg/ml) for a period of 24 h. After A549cells were incubated with 10μg/ml rVVC for24 h, typical apoptotic characteristicsincluding crescent-shaped nucleus, chromatin condensation and intracytoplasm vacuolus could be found under transmission electron microscope. The percentage of apoptotic cells (Annexin-V+/PI) in the control group was 7.01%. After treatment with 5,10 and 20μg/ml rVVC for 24 h, the percentages of apoptotic cells were 17.38%,37.30%, and 56.51%, respectively. The results of DNA integrity analysis showed that rVVC (10 and 20μg/ml) caused the digestion of genomic DNA into ladders obviously. Western blot analysis indicated that the expression of Bcl-2 protein decreased slightly, while the expression of P53 and Bax protein had a dramatically increase compared to the Control.Conclusions: 1.Vibrio vulnificus cytolysin (VVC) have significantly inhibition on proliferation of human lung adenocarcinoma cell line A549. Its inhibitory ability is connected with the concentration and action time of VVC; 24 hour IC50 for the A549 cells is 8.53965μg/ml, and used close to the IC50 of t Vibrio vulnificus cytolysin attacked A549 cells,12 hours to produce killing effect, within 48 hours, along with the prolongation of times killing effect of gradually increased,48h reach to Anti-peak, can be maintained to 120 hours. Vibrio vulnificus cytolytic toxin can affect the human lung adenocarcinoma cell line A549; Vibrio vulnificus cytolysin can damage the mitochondrial ultrastructure of A549 cells, lead to the structural basis of energy metabolism of A549cells are affected.2. The rVVC can effectively induce programmed cell death and suggests that rVVC induced apoptosis in A549 cell line is mediated by regulation of Bcl-2 and Bax protein expression.New points1. The result has demonstrated for the first time that Vibrio vulnificus cytolysin has lethal effects on human lung adenocarcinoma cell line A549;2. Through stereological studies on the ultrastructure changes of between A549 cells and A549cell (treated by rVVC).3. This experiment has conclusively demonstrated for the first time that rVVC inhibits proliferation and induces apoptosis in A549 cell line by regulating Bcl-2 and Bax protein expression;... |
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