Roles Of Thymosin β10 And Thymosin β15 In Lung Cancer Invasion And Metastasis

ObjectiveThymosins comprise a family of small proteins originally isolated from the bovine thymus and are divided into the following 3 main groups based on their isoelectric points:alpha(α),beta(β),and gamma(γ).Among them,the thymosinβfamily comprises highly conserved and acidic small-molecule proteins.Thymosinβ10(Tβ10) and thymosinβ4(Tβ4)are present in all mammalian species,and have G-actin binding motif "LKKTET",participate in cytoskeleton formation,and are closely associated with cell motility.Tβ4 can regulate VEGF expression and promote angiogenesis by "LKKTET" motif.Researchers have proved that the expressions of Tβ4 and Tβ10 are significantly higher in many tumors than in normal tissues.Moreover,Tβ4 and Tβ10 expressions are positively related to the degree of malignancy of these tumors.But in recent papers,Lee et al.found that Tβ10 was a tumor inhibitor.Unlike Tβ10,Thymosinβ15(Tβ15)was not detected in normal adult tissues.Tβ15 was first discovered and cloned in highly metastatic rat prostatic carcinoma Dunning cells by Bao et al.The expression of Tβ15 mRNA was correlated with the metastasis of rat prostate cancer, mouse lung cancer,human breast cancer and melanoma cell lines.Tβ15 protein expression was related to the metastasis of human prostate and breast cancer.However, roles of Tβ10 and Tβ15 related to human lung cancer remain unclear.In the present study,we detected the expressions of Tβ10 and Tβ15,analyzed the relationships among Tβ10,Tβ15 and clinicopathological factors of non-small cell lung cancer,and then analyzed the roles of Tβ10 and Tβ15 in metastasis and invasion of lung cancer cells. Materials and Methods1.The expressions of Tβ10,VEGF,VEGF-C protein and MVD,LVD were detected with immunohist0chemistry using anti-Tβ10 antibody(1:100),anti-VEGF antibody(1:100),anti-VEGF-C antibody(1:200),anti-CD34 antibody(ready to use) and anti-D2-40 antibody(ready to use)in non-small cell lung cancer samples.Three human lung cancer cell lines with different metastatic potential PG-BE1, PG-LH7 and A549 were maintained in RPMI1640 and DMEM medium,respectively, at 37℃under an atmosphere of 5%CO₂.An antisense Tβ10 recombinant plasmid was constructed using the pcDNA3.0-Tβ10 plasmid containing full-length Tβ10 cDNA and pcDNA3.0. Transfection assay was performed with PG-BE1 cells as Tβ10 overexpression was evident in this cell line using LipofectAMINE^TM2000 according to the manufacturer's instruction.The plasmid pcDNA3.0 was transfected at the same time.Positive clones were screened for using 400μg/ml G418.The cell clone which the expression level of Tβ10 mRNA was weakest was named BE1-as-Tβ10.The cells that were transfected empty plasmid(pcDNA3.0)were named BE1-EV.The mRNA expressions of Tβ10,Tβ4 andβ-actin in the cells were examined with RT-PCR.Immunohistochemical staining:Cells grown on coverslips were fixed with 4% paraformaldehyde followed by 0.2%Triton X-100,and incubated with anti-Tβ10 (1:100),anti-Tβ4(1:100)and anti-Tβ15(25μg/ml)antibodies.Immunofluorescence staining of Tβ10,Tβ4,and microfilament proteins:The cells were fixed with 4%paraformaldehyde followed by 0.2%Triton X-100.The cells were incubated with primary antibodies(anti-Tβ10,1:100 or anti-Tβ4,1:100)at 4℃overnight,followed by incubation with FITC-labeled secondary antibodies(1:100)and 0.1μg/ml YRITC-labeled phalloidin,incubation with 1μg/μl Hoechst 33342.Western Blot:Total cellular extracts were obtained by lysing the cells in a lysis buffer.The expressions of VEGF,VEGF-C,ERK1/2,pERK andβ-actin were detected with western blot using anti-VEGF(1:100),anti-VEGF-C(1:200),anti-ERK 1/2(1:200), anti-pERK(1:200)and anti-β-actin(1:200)antibodies.In vitro cell growth assays:Cells were plated in a 96-well plate and MTT was added to each well.After incubation for 4h,the medium was replaced with dimethyl sulfoxide.The OD value of each well was measured with a test wavelength of 490nm.Cell migration and invasion assays:A 24-well microchemotaxis chamber was used to assess migration and invasion.The cells were resuspended in serum-free RPMI1640 medium.The upper compartments were filled with cell suspension,and the lower compartments were filled with RPMI1640 medium supplemented with 10%FBS.The chambers were incubated at 37℃,5%CO₂ for 6h,fixing in methanol,stained with hematoxylin.For the invasion ability assay,precooled serum-free RPMI1640 medium was mixed with Matrigel(1:3 dilution).The upper compartments were filled with 100 ul of mixture.Other procedures followed the migration protocol.The chambers were incubated for 18h,and the results were observed.2.The expressions of Tβ15 protein was detected with immunohistochemistry using anti-Tβ15 antibody(25μg/ml)in lung cancer samples.Transfection assay was performed with PG-LH7 cells as Tβ15 was less evident in this cell line using LipofectAMINE^TM2000 according to the manufacturer's instruction.The mRNA expressions of Tβ15 andβ-actin in the cells were examined with RT-PCR.Cell migration assays:The chambers were incubated at 37℃,5%CO₂ for 24h, fixing in methanol,stained with hematoxylin.Other procedures followed the migration protocol above mentioned.3.Statistical analysis.The statistical package SPSS 13.0 was used for all analyses. Values ofP＞0.05 were considered statistically significant.Results1.Tβ10 was expressed in all 69 cases but with variable expression levels.Tβ10 expression is only in cytoplasm.The expression rates of VEGF and VEGF-C in the 69 cases were 68.1%and 52.2%,respectively;they were expressed in the cytoplasm.Tβ10 overexpression was correlated with the expression of VEGF(P=0.004)and VEGF-C (P=0.017).The Tβ10 expression level was correlated with the stage of lung cancer(P =0.026),distant metastases(P=0.016),lymph node metastases(P=0.007),degree of differentiation of lung cancer(P=0.03)and was not correlated with age,gender,and the histological type of the cancer.The average MVD and LVD in the 69 tumor samples were 38.65±14.31 and 18.56±12.48.The MVD rate was higher in the patients with positive VEGF expression than in others with negative VEGF expression(44.96±8.57vs.25.17±14.92,P＜0.01).The LVD rate was higher in the patients with VEGF-C expression than in the patients with negative VEGF-C expression(24.67±11.54vs.11.90±9.88, P＜0.01).Higher rates of MVD and LVD were found(43.85±10.03 and 21.39±12.39, respectively)in patients with high level of Tβ10 expression,while lower rates of MVD (24.98±15.09)and LVD(11.10±9.48)were observed in patients with low level of Tβ10 expression(P＜0.01).Further analysis showed that MVD was significantly correlated with distant metastasis(P＜0.001),but not with lymph node metastases(P= 0.817),while LVD was significantly correlated with lymph node metastases(P=0.017), but not with distant metastases(P=0.079).2.Tβ10 mRNA and protein were expressed in all the LH7,BE1 and A549 cells, but Tβ10 mRNA expression in LH7 cells was significantly lower than those in BE1 and A549 cells;Tβ10 was expressed in the cytoplasm.The PG-LH7 cells stained light brown-yellow,but the PG-BE1 and A549 cells stained dark brown-yellow.We constructed Tβ10 antisense recombinant expression plasmid pcDNA3.0-as-Tβ10. pcDNA3.0-as-Tβ10 transfected BE1 ceils which the expression levels of Tβ10 mRNA and protein in it were higher than in the other two.And stable transfected clones were obtained after selection with G418.The expression of Tβ10 mRNA in BE1-as-Tβ10 cells was reduced than BE1 cells.Immunofluorescence staining of Tβ10 in it was weaker than in BE1 and BE1-EV cells.Polymeric F-actin arranged parallel could be seen in the cytoplasm of the BEI-as-Tβ10 cells.Meanwhile,the VEGF,VEGF-C and pERK protein expressions in the BE1-as-Tβ10 cells were significantly decreased.3.The results of the MTT assay showed that the proliferation of the BE1-as-Tβ10 cells was significantly lower than that of the BE1-EV and control groups(P＜0.01,P＜0.01).In the migration assay,the migration cell number of the BE1-as-Tβ10 cells(18±12.6)was significantly lower than that of the BE1-EV(48.4±9.1,P＜0.01)and control groups(41.2±14.9,P＜0.05).In the invasion assay,the invasion cell number of the antisense Tβ10 recombinant plasmid-transfected cells(7.8±3.6)was significantly lower than that of the EV-transfected(23.8±8.4,P＜0.01)and control groups(22.4±8.4,P＜0.01).4.Tβ15 protein was expressed in all 76 cases but with different expression levels. Tβ15 expression is only in cytoplasm.No Tβ15 expression was observed in the alveolar and bronchial epithelial cells in cancer-adjacent tissues.The Tβ15 expression level was correlated with advanced stage(P=0.018),lymph node metastasis(P= 0.001),and poor differentiation degree(P=0.013),respectively.5.Tβ15 mRNA was expressed in both LH7 and BE1 cells,higher Tβ15 expression was found in PG-BE1 cells known for high metastatic potential,while lower Tβ15 expression was observed in low metastatic potential PG-LH7 cells.The PG-LH7 cells stained light brown-yellow that was indicative of low expression,but the PG-BE1 cells stained dark brown-yellow that was indicative of high expression.The Tβ15 mRNA level in pEGFP-Tβ15 plasmid-transfected PG-LH7 cells(LH7-Tβ15-1 and LH7-Tβ15-2)was significantly increased.In the migration assay,the migration cell number of LH7-Tβ15-1 and LH7-Tβ15-2 cells were 96.33±6.94 and 98±10.58,and they were significantly higher than that of the EV-transfected(46±8.89)and control groups(37±8.19).Conclusions1.The Tβ10 expression level was significantly associated with advanced clinical stages,poor differentiation,lymph node and distant metastases of non-small cell lung cancer.High Tβ10 expression was associated with high expressions of VEGF and VEGF-C.Moreover,both the MVD and LVD in the group with high Tβ10 expression were significantly higher than those in the group with low Tβ10 expression.The expressions of VEGF and VEGF-C were significantly correlated with MVD and LVD.2.The levels of Tβ10 mRNA and its protein correlated positively with the metastatic potential in human tumors presently examined.The Tβ10 protein and mRNA expressions were both inhibited in human lung cancer PG-BE1 cells which was transfected with antisense Tβ10 recombinant plasmid pcDNA3.0-as-Tβ10 plasmid.The proliferation,invasion and migration abilities and other factors such as VEGF,VEGF-C, pERK of the cells which were transfected with pcDNA3.0-as-Tβ10 plasmid were decreased,and thick F-actin appeared in the ceils.These may be associated with pERK inhibition and cytoskeleton formation which were regulated by Tβ10.3.Elevated Tβ15 expression was correlated with advanced stage,lymph node metastasis,and poor differentiation degree of non-small cell lung cancer,respectively.4.Transfection of pEGFP-C1-Tβ15 plasmid into LH7 cells can significantly increase lung cancer cell migration.