Expression Of TNFRSF6B In Glioma Tissues And Effect Of Anti-TNFRSF6B Neutralization Antibody On Cell Growth Suppression And Apoptosis Induction In Glioma Cells

Background and aimsGlioma is among the most related causes of morbidity and mortality in primary malignant brain tumors, which originates from brain glial cells and features rapid progression and high invasive ability. It has been reported that in China 3 to 6 persons are diagnosed as gliomas out of a population of 100,000 per year and the annual mortality hits a number of 30,000, making it the most frequent primary malignant brain tumor. Currently, comprehensive strategies are mainly applied in the treatment of glioma, i.e. a combination of surgical resection, radiotherapy and chemotherapy. However, the clinical curative outcomes are far from satisfactory given that glioma has a trend of infiltrative growth and has invaded the neighboring brain tissue in early stage, thus being difficult to resect completely and easy to recur. Therefore, it is urgent to discover effective prognostic biomarkers for the timely detection of high-risk factors such as recurrence and distant metastasis in order to offer comprehensive treatments and obtain longer lifespan and higher survival rate.Tumor necrosis factor receptor superfamily member 6B (TNFRSF6B), namely decoy receptor3 (DcR3) or M68 or tumor necrosis receptor factor 6 (TR6), is a member of Tumor necrosis factor receptor family. TNFRSF6B can bind to the receptor molecules of some protein ligands specifically and competitively inhibit the combination of ligands and death receptors, thus blocking several signaling pathways including apoptosis induced by these ligands. Overexperssion of TNFRSF6B has been detected in several malignant tumors and has been proved to play important roles in tumorigenesis, progression and metastasis. It has been validated by researches that there existed overexpression of TNFRSF6B mRNA and its protein among tissues and serum of various tumors including glioma. However, there are only three articles which studied the effects and mechanisms of TNFRSF6B in the tumorigenesis and progression of glioma and its proliferation and apoptosis. Consequently, in the current study, we further studied this.The present study attempted to elucidate the expression status and clinical significance of TNFRSF6B in glioma by exploring the expression levels of TNFRSF6B in glioma tissues and glioma cell lines and to determine the relationship between TNFRSF6B expression and clinical pathological parameters, further to investigate the effects and potential molecular mechanisms of TNFRSF6B in the proliferation and apoptosis of glioma so as to provide possible targets for prognosis and molecular target therapy for glioma patients.Materials and methods1. The expression of TNFRSF6B protein was detected in one hundred and twenty-five samples of glioma patients and eighteen cases of normal brain tissues by using immunohistochemistry (EnVision) and gioma tissue microarray. The relationship between the TNFRSF6B level and clinicopathological features was investigated, including the expression of glial fibrillary acidic protein (GFAP), labeling indexes (LIs) from Ki-67 and proliferating cell nuclear antigen (PCNA) survival and prognosis of patients.2. The glioma cells with high expression of TNFRSF6B protein were screened and selected for the subsequent experiments. Three glioma cell lines (U251MG, LN-308 and U87MG) were treated with anti-TNFRSF6B neutralization antibody. The survival rate of different glioma cells in diverse concentration of anti-TNFRSF6B neutralization antibody was detected by MTS, CellTiter-Blue Cell Viability Assay, colony formation in soft agar and Hoechst 33342/Propidium Iodide, (PI) double fluorescent staining. After anti-TNFRSF6B neutralization antibody treatment, the differences of glioma cell cycle were detected by Flow cytometry (FCM). The influence of anti-TNFRSF6B neutralization antibody on apoptosis in glioma was analyzed by FCM, Caspase-3/7 activity detection, Hoechst 33342/PI double fluorescent staining assay, respectively. In the meantime, to investigate the contribution of anti-TNFRSF6B neutralization antibody in the regulation of cellular signaling, we examined the signaling of MMP-2, ERK 1/2 and AKT pathways by using western blot, which are related to cell survival, apoptosis, invasion and metastasis. We explored the function of TNFRSF6B on the development and progression of glioma and the effect of anti-TNFRSF6B neutralization antibody on the biological function in glioma cells, to establish the experimental basis for the future clinical application of TNFRSF6B.3. Statistical analysis SPSS 20.0 statistical analysis software was used for the data statistics in the current project. Measurement data were presented as Mean±SEM. The difference of the TNFRSF6B and GFAP expression intensity between glioma and normal brain tissues was applied with Mann-Whitney U test, while the difference of positive ratios was analyzed by using chi-square test. The difference of TNFRSF6B and GFAP expression intensity and positive ratio among various pathological grades of glioma was evaluated by using Kruskal-Wallis test. The difference of the TNFRSF6B and GFAP expression intensity in groups of distinct age and gender was tested with Mann-Whitney U test, while the difference of positive ratios was analyzed by using chi-square test as well. Spearman rank correlation was applied to inverstigate the relationship of TNFRSF6B and GFAP expression with the pathological grades of glioma. For measurement data, such as Ki-67 LI and PCNA LI, t test was performed with Gaussian distribution, including glioma tissue vs normal brain, grade Ⅰ-Ⅱ vs grade Ⅲ-Ⅳ, positive TNFRSF6B vs negative TNFRSF6B. One-Way ANOVA was used for the analysis of means in multi-samples. SNK-q test was performed with homogeneity of variance, otherwise Kruskal-Wallis H test was used, for instance, the difference of Ki-67 LI and PCNA LI in different groups of TNFRSF6B expression. Kaplan-Meier method and log-rank analysis were chosen for the survival study and linear trend for factor levels with pooled over strata was selected. In in vitro experiments, Mean±SEM was used to present the data of cell grow inhibitory ratio, number of cell clonies, rate of cell cycle and apoptosis. ANOVA and student t test were used to study the effect on different functional behavior between different groups. P value less than 0.05 was regarded as statistically significant.Results1. Positive signals of TNFRSF6B protein were located at cytoplasm and cytomembrane detected by immunohistochemistry. Among 125 glioma tissues,99 showed positivity to some extent with a positive rate of 79.2%. Only two cases were labelled as positive out of 18 normal brain tissues with a positive rate of 11.1%. The positive expression intensity of TNFRSF6B protein in glioma tissues was significantly higher than that of normal brain tissues (Z=-4.661, <0.001, Mann-Whitney U test). Meanwhile, the positive expression rate of TNFRSF6B protein in glioma tissues was also significantly higher than that of normal brain tissues (x 2=24.343, P<0.001, chi-square test). As pathological grading escalated, the expression level of TNFRSF6B in glioma became higher. Obvious positivity of (+++) was only observed in III to IV grading and there was statistical significance of TNFRSF6B expression among Ⅰ～Ⅱ grading and Ⅲ-Ⅳ grading(χ2=36.082, P< 0.001). The well differentiated group (I-II grading) featured a positive rate of 55.1% (27/49) while the poorly differentiated group (Ⅲ-Ⅳ grading) displayed a positive rate of 94.7%(72/76). The expression of TNFRSF6B was proved to be positively correlated with the pathological grading by Spearman rank correlation (rs =0.614, P< 0.01). However, no significant correlations emerged between TNFRSF6B expression and gender or age (P>0.05).2. The Ki-67 labeling index (LI) in normal brain tissue was 0.08±0.04, significantly lower than that in glioma tissue (12.47±1.16, t= 10.71, <0.001). The PCNA LI in normal tissue was 0.03±0.02, obviously lower than that in glioma tissue (10.86±1.01, t= 10.69, P<0.001). With respect to glioma tissues, the Ki-67 LI (12.47±1.16) was remarkably higher than PCNA LI (10.86±1.01,t= 5.985, <0.01). Moreover, there was a positive correlation between Ki-67 and PCNA LI (rs=0.978, P<0.01). The Ki-67 and PCNA LI increased with the growth of glioma pathology stage. Furthermore, the Ki-67 LI was associated with the pathological stage (rs=0.851, P<0.01). The PCNA LI was also positively related to glioma pathology stage (rs =0.459, P<0.01). However, both LIs of Ki-67 and PCNA were not correlated with gender or age (P>0.05). As for TNFRSF6B negative group, the expression level of Ki-67 LI was 3.29±1.04, significantly lower than that in TNFRSF6B positive group(15.09±1.34, t= 6.96, P<0.001). The PCNA LI in TNFRSF6B negative group was 2.78±0.89, obviously lower than TNFRSF6B positive group (13.17±1.17, t=7. 06, P<0.001). As expected, Ki-67 LI was positive correlated with TNFRSF6B expression(rs= 0.459, P<0.01). Simultaneously, PCNA LI was also positive correlated with TNFRSF6B expression (rs= 0.477, P< 0.01).3. There was a negative correlation between the expression of GFAP protein and pathological grades of glioma (rs=0.632, P<0.01). There was a statistically significant difference of GFAP protein expression between normal brain and glioma tissues (P<0.01). And the expression ratio of GFAP protein was 100.0% in normal brain tissue, much higher as compared with the rate of 81.6% in low grades (Ⅰ and Ⅱ), and 39.7% in high grades (III and IV). Moreover, significant difference was found between grade I, II and grade III, IV. Expression of GFAP protein was lowest with the rate of 25%in grade IV among all the four grades. In addition, TNFRSF6B was negatively correlated with GFAP protein (rs=-0.397, P<0.01). GFAP protein expression was negatively related to LIs of Ki-67 (rs=-0.465, P<0.01) and PCNA (rs =-0.483, P<0.01).4. In this current study,76 patients had complete survival data. The LIs of Ki-67 and PCNA were both related to the survival, which demonstrated the reliability of the current survival data. In order to clarify the correlation between expression of TNFRSF6B and patient survival, TNFRSF6B expression has been detected in 76 samples. The result showed that the survival of patients with negative expression of TNFRSF6B was 50±1.79 months, slightly longer than 47.45±3.11 months of the positive expression group, however, there was no statistical significance.5. The cell growth inhibitory effect of anti-TNFRSF6B neutralization antibody was evaluated by using MTS tetrazolium assay and the result revealed that cell proliferation was not influenced in the hIgGl controls in all 3 cell lines tested. However, after treatment with anti-TNFRSF6B neutralization antibody, a large reduction in proliferation of glioma cells was examined at 48,72 and 96 h in all the three cell lines tested and the most potent effect occurred at 96 h. In the 3 cell lines, anti-TNFRSF6B neutralization antibody offered the most powerful impact on U251MG cells, with the proliferation rate of 70.667±5.033% at 96 h post treatment. To confirm these results, the effect on cell viability was verified by using a fluorimetric resorufin viability assay and Hoechst 33342/PI double fluorescent chromatin staining, which largely mirrored the results from MTS assay and the cell growth inhibitory effect of anti-TNFRSF6B neutralization antibody showed a time and dosage dependent manner on glioma cells. Furthermore, soft agar colony formation assay showed that in the group treated by anti-TNFRSF6B neutralization, colony rate was 12.333±4.933%, significantly lower than that in the blank (31.333±3.055%) and negative hlgGl groups (30.667±4.726%, P<0.001) in glioma U251MG cells. Growth inhibition by anti-TNFRSF6B neutralization antibody in the glioma cells was accompanied by cell cycle G1/S arrest, which was indicated by a decrease in DNA synthesis and retardation of cell growth. Anti-TNFRSF6B neutralization antibody inhibited DNA synthesis and increased the percentage of U251MG cells in GO-G1 phase.After 96 h treatment, the percentage of GO-G1 phase cells was 59.333±1.528% in anti-TNFRSF6B neutralization antibody treated group compared with 49.667±2.082% in blank and 50.667±1.528% in hIgGl controls. This increase was coupled with the reduced percentage of cells in S phase. The percentage of S phase cells was 20.667±0.577% in anti-TNFRSF6B neutralization antibody treated group compared with 34.667±1.155% in blank and 35.667±3.055% in hIgG1 control. Similar, but slightly weaker effect of anti-TNFRSF6B neutralization antibody on the cell cycle was also observed in other two cell lines LN-308 and U87MG.6.After treatment with anti-TNFRSF6B neutralization antibody, the caspase-3/7 activities of all the three glioma cell lines tested were significantly increased with a time and dosage dependent manner. Consistent with the result of cell proliferation assay, the most powerful effect of anti-TNFRSF6B neutralization antibody on caspase-3/7 activity occurred in U251MG cells, with an optimum dose of 0.8mg/L and a time point 96 hours post treatment up to highest activity. Meanwhile, by using Hoechst 33342/PI double fluorescent staining, we found a significant increase in apoptosis in the group treated with anti-TNFRSF6B neutralization antibody, which was comparable to the result of caspase-3/7 activity detection.7.The signaling of MMP-2, ERK and AKT pathways were examined by using western blot, which were related to cell survival, apoptosis, invasion and metastasis. These pathways were influenced little with hIgGl control in glioma cells. However, the expression level of MMP-2, phospho-ERKl/2 and phospho-AKT were down-regulated by anti-TNFRSF6B neutralization antibody at 96 h post treatment.Conclusions1. TNFRSF6B is closely associated with the pathological grading, GFAP and cell proliferation in glioma, suggesting the significant role of abnormal expression of TNFRSF6B in the oncogenesis and development of glioma.2. It should be highly noted that the overexpression of TNFRSF6B in glioma might be of significance in cell proliferation and apoptosis of glioma cells.3. The apoptosis of glioma cells can be induced by inhibiting the expression of TNFRSF6B, thus lowering the cell proliferation rate.4. It can be expected that neutralizing antibody of TNFRSF6B might be a promising option for molecular target therapy for glioma patients.