| Lung cancer is one of the most common malignancies in the clinic,of which incidence rate is the highest in the big cities and still rising.Small cell lung cancer accounts for about20%of the total incidence ratewith its high malignant degree, short doubling time and early andextensive metastasis.And many cases are in the too late period to get anopportunity for an operation when diagnosed, while the side effects ofchemotherapy alone is quite big,.So new biotherapeutics will help toimprove the prevention and treatment of small cell lung cancer. At homeand abroad in recent years, studies have found various anti-tumorsubstances in various organisms. Some parasites also has significant anti-tumor activity. It is mainly reported in the literature Trichinella[1],Plasmodium[2], Acanthamoeba trophozoites[3-4], Toxoplasma gondii[5].Trichinella has been reported to be able to resist melanoma, colon cancer,liver cancer, gastric cancer, sarcoma, leukemia, cellulose, myeloma andbreast cancer[6-9]. Currently, for the small cell lung cancer surgery andsystemic chemotherapy treatment still are the major ways, and cisplatin islisted as standard chemotherapy drugs with many side effects and moreeasily acquired resistance. As a result seeking a new biologicalpreparation with fewer side effects and good effect is very important.It has been reported that Trichinella anti-tumor substances are fromits different antigens.According to the diffrent sources Trichinella isdivided into surface antigen, excretory-secretory antigen and solubleparasite antigens.And studies have shown that surface antigen andexcretory-secretory antigen protein complex are major components of theantigen[10]. Wang Xuelin et al[11]found that the Trichinella parasiteproteins inhibit a variety of tumor cells. Trichinella spiralis muscle larvae excretory-secretory protein are a biologically active substance extractedfrom cultured muscle larvae excrete secretions. In order to confirm itsinhibitory effect on small-cell lung cancer, we observed in vitro theeffects of secreted proteins from Trichinella spiralis for H446cells.Objective:Research Trichinella spiralis muscle larvae excretory-secretoryprotein in small cell lung cancer H446inhibition confirmed its anti-tumoractivity against H446small cell lung cancer cells. Further investigate theeffect of Trichinella H446small cell secretory protein on apoptosis oflung cancer, and its relationship with apoptosis-related genes andproteins.Methods:1. H446human small cell lung cancer cell lines (presented byProfessor China Medical University郑åŽå·), do subculture with10%fetal bovine serum level of priority (TBD).2. Experimental groups: control group and divided into differentconcentrations of Trichinella spiralis excretory-secretory protein group.Positive control group: free RPMI-1640medium fetal calf serum dilutedcisplatin into (12.8μg/ml,6.4μg/ml,3.2μg/ml,1.6μg/ml,0.8μg/ml,0.4μg/ml,0.2μg/ml) H446cells were cultured with different concentrations;experimental group, Trichinella spiralis excretory-secretory protein group:respectively, without FBS RPMI-1640medium extracted Trichinellaspiralis excretory-secretory protein formulated into a final concentrationof0.075mg/ml,0.15mg/ml,0.3mg/ml Trichinella excretory-secretoryprotein culture medium treated cells; also set the negative control group:10%FBS containing H446cells were cultured in complete medium.3. MTT colorimetric assay was applied to investigate the effects ofscutellarin on the proliferative activity of the H446cells in0,12,24,36,48,72hours.4. TUNEL assay apoptosis after different treatments were givenconcentration of0.15mg/ml Trichinella spiralis excretory secretory protein processing, concentration of6.4μg/ml of cisplatin treatment, andnot given any drug treatment24hours after detection apoptotic cells.5. Immunocytochemical staining: the expression of c-Myc in eachgroup were detected by Immunocytochemical staining.6. Western blot: the expression of c-Myc in each group weredetected by Western blot.7. PT-PCR: the expression of Bcl-2, Fas/Fasl in each group weredetected by PT-PCR.8. The statistical analysis was performed with SPSS17.0of statisticalsoftware. All the data was analyzed by Analysis of variance and LSD-ttest.P<0.05was considered statistically significant.Result:1. The impact of scutellarin on the value-added activity of H446cells.1.10.075mg/ml,0.15mg/ml,0.3mg/ml concentration of Trichinellaspiralis excretory-secretory proteins were treated0h,12h,24h,36h,48h,72h, the growth of H446cells was inhibited, and in a certain time and theconcentration range of time, concentration-dependent manner, throughstatistical analysis, the differences between the groups were statisticallysignificant (P <0.01).1.2Different concentrations of cisplatin and H446cells wereco-cultured0h,12h,24h,36h,48h,72h, the growth of H446cells wasinhibited, and within a certain range of time and concentration waspositively correlated with the concentration of the time, by statisticalanalysis, each and there are differences between the groups wasstatistically significant (P <0.01).2. By the TUNEL method: normal group did not see the apoptoticcells (Fig.3), is dyed brown or tan cell apoptosis, purple dye for livingcells; cisplatin group can be seen in apoptotic cells (Figure5); Trichinellaspiralis muscle larvae excretory-secretory proteins can be seen in the roleof a large group of apoptotic cells (Fig.4). The statistical software analysis, three groups were statistically significant differences compared(P <0.01); Trichinella spiralis muscle larvae excretory-secretory proteinsmore significant role in the group and was statistically significant (P<0.01).3. Under fluorescence immunochemical staining cells, the role ofexcretory-secretory protein concentration of0.3mg/ml Trichinella incontinuous culture48h, by immunocytochemistry staining, human smallcell lung visible expression c-myc, staining was mainly located in thecytoplasm, the negative control group deeply colored cytoplasmcytoplasm showing clear green. The experimental group cytoplasmcoloring is very shallow. After statistical analysis, the differences betweenthe groups were statistically significant (P <0.05).4. Western blot detection of application (Western-blot), at aconcentration of0.3mg/ml Trichinella spiralis excretory-secretory proteinunder the action of the relative expression of c-myc in human small celllung cancer cells was0.566±0.054, the positive control group at6.4μg/under ml concentrations of cisplatin, human small cell lung cancerrelative expression of c-myc was0.776±0.041, relative expression ofc-myc in the negative control group human small cell lung cancer cells1.172±0.026, expressed in the negative control group highest. Afterstatistical analysis, the differences between the groups were statisticallysignificant (P <0.05).5. RT-PCR technology to detect, at a concentration of0.3mg/mlTrichinella spiralis excretory-secretory protein under the action of therelative expression of human small cell lung cancer cells Bcl-2mRNAwas0.575±0.047, the positive control group at6.4μg/ml concentrationsof cisplatin the role of human small cell lung cancer relative expression ofBcl-2mRNA was0.776±0.082, relative expression of Bcl-2mRNAnegative control human small cell lung cancer cells0.975±0.069, inorder to express the highest amount of negative control group. Afterstatistical analysis, the differences between the groups were statistically significant (P <0.05). Human small cell lung cancer H446cells fasmRNA relative expression levels were0.769±0.061,0.817±0.121,0.975±0.115, to the highest expression level of fasl mRNA in the controlgroup. Human small cell lung cancer H446cells fasl mRNA relativeexpression levels were0.669±0.051,0.787±0.124,0.875±0.125, witha lowest expression levels of fasl mRNA in the blank control group.Analysed by ANOVA, the differences between the groups werestatistically significant (P <0.05).Conclusion:1. Trichinella spiralis excretory-secretory protein can inhibit theproliferation of human small cell lung cancer H446cells and induces itsapoptosis.2. Trichinella spiralis excretory-secretory protein induces apoptosisin H446human small cell lung cancer cells by down-regulatingexpression of Bcl-2and maybe upregulating Fas/Fasl to inhibit tumor. |
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