Research Of Apoptosis Mechanism Of Small Cell Lung Cancer H446 Cells Induced By Somatic Protein Of Trichinella Spiralis

Lung cancer(LC)is the leading cause of cancer-related death in the world,and the fourth cause of death in our country.Small cell lung cancer(SCLC)accounts for 15%~20% of all lung cancer and non-small cell lung cancer(NSCLC)is about 80%.SCLC is a kind of extremely malignant and lethal lung cancer with rapid growth and metastasis,and easy to develop resistance.Although the early onset of SCLC is sensitive to chemotherapeutic treatment,most patients are more likely to get worse within 6months after treatment,with a 5-year survival rate only 10%.Therefore,it is important to find a new and effective method to treat SCLC.Trichinella spiralis(T.s)is a kind of parasitic nematode worm which infects almost all mammals,including humans.It can cause the trichinellosis of Zoonosis.Researchers has already found that trichinella spiralis can inhibit the growth of tumor,and the active protein of trichinella spiralis inhibits the proliferation of tumor cells in vitro,while people recognizing the harm done to humans by trichinella spiralis,and try to control it.It provides a new direction for the research of tumor biological treatment.There is no report indicates whether the somatic protein of trinchinella spirais can inhibit the growth of small cell lung cancer.Therefore,we used small lung cancer H446 cells as research object to observe whether the somatic protein of trichinella spirais muscle larva has an effect of inducing apoptosis,and explore its mechanism.Objective:We intend to study the influence of proliferation activity and the effect on inducing apoptosis of small lung cancer H446 cells by trichinella spiralis muscle larva somatic protein,to investigate the mechanism of the small lung cancer H446 cells by trichinella spiralis muscle larva somatic protein.This study provides new ideas and experimental basis for the research and development of tumor biological preparation.Methods: 1.Detected the proliferation activity of H446 cells by MTT colorimetryThe concentration of 0.2mg/mL,0.4mg/mL,0.8mg/mL somatic protein were cultured with H446 cells for 24 h,48h,and 72 h respectively,and as the experimental group,untreated H446 cells were treated as the blank control group.The effect of different concentrations of trichinella spiralis muscle larva somatic protein on the proliferation activity of H446 cells were detected by MTT assay.2.Detected the cycle retardation of H446 cells by PI staining methodThe concentration of 0.2mg/mL,0.4mg/mL,0.8mg/mL somatic protein were cultured with H446 cells for 24 h respectively,the blank control was not treated.H446 cells were collected for PI stain,and detected the periodic distribution.There was no significant difference between the experimental group and the blank control group.3.Detected the apoptosis rate change of H446 cells by Annexin V-FITC/PI staining methodThe concentration of 0.2mg/mL,0.4mg/mL,0.8mg/mL somatic protein were cultured with H446 cells for 24 h respectively,the blank control was not treated.H446 cells were collected for Annexin V-FITC/PI stain,and detected the apoptosis rate.There was no significant difference between the experimental group and the blank control group.4.Detected the expression of Caspase-9 of H446 cells by Immunofluorescence methodThe concentration of 0.8mg/mL somatic protein were cultured with H446 cells for 24 h,the blank control was not treated.After the process of cell fixation,membrane rupture,sealing,add the antibody of Caspase-9,blocking antibody,the image was taken under the fluorescence microscope.5.Detected the apoptosis factors of mRNA transcriptional level of H446 cells by Real-time PCR methodThe concentration of 0.2mg/mL,0.4mg/mL,0.8mg/mL somatic protein were cultured with H446 cells for 24 h respectively,the blank control group were not treated,extracted nucleic acids to detect the mRNA transcriptional level of Bax,Bcl-2,Cyt C,Apaf-1,Caspase-9 and Caspase-3.6.Detected the apoptosis factors protein expression of H446 cells by Western Blot methodThe concentration of 0.2mg/mL,0.4mg/mL,0.8mg/mL somatic protein were cultured with H446 cells for 24 h respectively,the blank control group were not treated,extracted total cell protein to detect the protein expression level of Bax,Bcl-2,Cyt C,Apaf-1,Caspase-9 and Caspase-3.7.Statistical analysisAll data of experiment were analyzed using SPSS19.0 statistical software package.The single factor variance analysis test was used for data processing.P< 0.05 showed statistically significant difference.Results: 1.MTT colorimetry detect the proliferation activity of H446 cellsDifferent concentration(0.2mg/mL,0.4mg/mL,0.8mg/mL)somatic protein were cultured with H446 cells for 24 h,48h,and 72 h,compared to the blank control group,the proliferation activity of H446 cells was obvious suppression(P?0.05),and showed dose-time dependence.2.PI staining method detect the cycle arrest of H446 cellsPI staining results suggested that somatic protein arrested H446 cells at G0/G1 phase.0.2mg/mL,0.4mg/mL,0.8mg/mL somatic protein were cultured with H446 cells for 24 h,the proportion of G0/G1 phase was(46.66±0.38)%,(55.64±0.93)%,(58.63±0.47)%respectively,and the blank control group was(44.23±1.7)%;the proportion of S phase was(46.55±1.17)%,(43.75±1.49)%,(33.72±2.24)%respectively,and the blank control group was(47.34±1.11)%.The cell proportion of G0/G1 phase of 0.2mg/mL,0.4mg/mL,0.8mg/mL somatic protein were higher than the blank control group(P?0.05),the cell proportion of S phase of 0.2mg/mL,0.4mg/mL,0.8mg/mL somatic protein were lower than the blank control group(P?0.05),and showed dose-time dependence.3.Annexin V-FITC/PI staining method detect the apoptosis rate of H446 cellsAnnexin V-FITC/PI staining results showed that different concentration(0.2mg/mL,0.4mg/mL,0.8mg/mL)somatic protein were cultured with H446 cells for 24 h,induced apoptosis of H446 cells and in late apoptosis mainly.0.2mg/mL,0.4mg/mL,0.8mg/mL somatic protein group of late apoptosis rate were(8.13±0.14)%,(10.7±1.60)%,(22.63±1.52)% respectively,the blank control group was(3.89±0.47)%.The H446 cells of late apoptosis rate in the experimental group was higher than the blank control group(P?0.05),and showed dose-time dependence.4.Immunofluorescence detect the expression of Caspase-9 of H446 cellsImmunofluorescence results showed that 0.8mg/mL somatic protein were cultured with H446 cells for 24 h,the Caspase-9 showed green fluorescence,while the fluorescence intensity of the blank control group was not obvious;DAPI staining was bright blue,and the intensity was higher than the blank control group.Fluorescence localization suggested that the Caspase-9 was expressed in both cytoplasm and nucleus.5.Real-time PCR detect the mRNA transcriptional level of mitochondria-related apoptosis factors of H446 cellsReal-time PCR results showed that 0.2mg/mL,0.4mg/mL,0.8mg/mL somatic protein group of the mRNA transcriptional level of Bax,Cyt-C,Apaf-1,Caspase-9 and Caspase-3 were up-regulated,while the mRNA transcriptional levels of Bcl-2 were down-regulated(P< 0.05),compared with the blank control group,the difference was statistically significant.6.Western Blot detect the protein expression of mitochondria-related apoptosis factors of H446 cellsWestern Blot results showed that 0.2mg/mL,0.4mg/mL,0.8mg/mL somatic protein group of the protein expression level of Bax,Cyt-C,Apaf-1,Caspase-9 and Caspase-3 were up-regulated,while the protein expression level of Bcl-2 were down-regulated(P< 0.05),compared with the blank control group,the difference was statistically significant.Conclusion: 1.Trichinella spiralis muscle larva somatic protein has inhibitory effect on the proliferation activity of small cell lung cancer H446 cells,and can induce the apoptosis of H446 cells.2.Trichinella spiralis muscle larva somatic protein may be the possible to induce the apoptosis of H446 cells by Caspase-dependent manner through endogenous pathways of mitochondrial apoptosis.