Expression Of Ativin A/Follistatin In Glioblastoma And The Effect On U87MG In Vitro

BackgroundGliomas of astrocytic, oligodendroglial and ependymal origin account for more than 70% of all brain tumors. The most frequent (65%) and most malignant histological type is the glioblastoma (GBM, astrocytoma grade IV). Since the introduction of computerized tomography and magnetic resonance imaging, the incidence rates of brain tumors have been rather stable, with a tendency of higher rates in highly developed, industrialized countries. Despite multimodal treatment regimes, including surgical resection, irradiation, and chemotherapy, GBM have a poor prognosis as indicated by a mean postoperative survival time of less than 12 months. Pediatric high-grade gliomas represent approximately 10% of all pediatric brain tumors. Similar to adult high-grade gliomas, they behave very aggressively, and these children have a very poor prognosis despite a variety of therapies that include chemotherapy and radiotherapy.Activin A was found to be overexpressed in some kind of cancers, and was demonstrated to enhance some cancer cell lines'proliferation, also to induce the apoptosis of some cancer cell lines. This article was to study the expression of activin A and follistatin in glioma and normal brain tissues; also, to evaluated the effect of activin A/follistatin system on the proliferation or apoptosis of U87MG cell line in vitro.ObjectiveTo investigate the expression of activin A/follistatin in glioblastoma and the effect of activin A/follistatin on U87 in vitro.Methods31 cases (18 male,13 female patients, age 25 to 71, average age 41.6) of glioma samples confirmed by two pathologists were collected in Qilu hospital.8 normal brain samples (including 5male,3 female, age 41-66, average age 55.4) were collected in Qilu hospital from cerebral hemorrhage patients who needed an operative therapy. All human studies have been approved by the appropriate ethics committee of Qilu Hospital and have been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. All patients gave their informed consent prior to their inclusion in the study. The expression of Activin-A, follistatin, p-Smad2/3 was determined by immunohistochemical statining. Real-time PCR was used to detect the expression of activin A and follistatin mRNA.Cell line U87MG was kindly provided by Dr. Ronghui Li. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) with 10% heat-inactivated FCS and antibiotics (10 UI/ml penicillin and 10μg/ml streptomycin) in 75cm2 culture flasks in a humidified atmosphere with 5% CO2 in air. The growth medium was replaced 2 or 3 times a week, and cells were passaged twice a week by trypsinisation.Before the intervention with ACT A and/or follistatin, the media was replaced with DMEM with 0.5% FCS for 24h to arrest the at G0/G1 stage. Then the cells were divided into 5 groups randomly:normal control group (NG, ACT A Ong/ml, FS Ong/ml) ACT A group (AG, ACT A 3ng/ml,10ng/ml,30ng/ml),ACT A+FS group (AFG, ACT A 3ng/ml+FS100ng/ml, 30ng/ml+FS 100ng/ml).The vitality of cells was evaluated by determining the DNA synthesis of cultured cells with [3H]Thymidine incorporation assay. The apoptosis rate and the proliferation index of the cells was determined at 48 hr by flow cytometry (FCM). p-Smad2/3 expression were determined by Western blot analysis.Results1.1 Immunohistochemical statiningThe results of activin A, follistatin staining optical densities were analized by semiquantitative scoring. Compared with normal brain tissues, activin A staining was prominently overexpressed in glioblastomas (ODs 0.58±0.08 in glioblastomas vs 0.20±0.03 in normal brain tissues, p<0.05), follistatin staining was not statistically different between glioblastomas and normal brain tissues (ODs 0.31±0.04 in glioblastomas vs 0.28±0.03 in normal brain tissues, p>0.05), p-Smad2/3 was also overexpressed in glioblastomas (ODs 0.46±0.05 in glioblastoma vs 0.26±0.06 in normal brain tissues).1.2 Expression of ACT A,FSmRNA in glioblastoma tissuesACT A mRNA was sligntly expressed in normal brain tissues, but was significantly overexpessed in glioblastoma tissues(3.3 fold of normal brain tissue). Expression of FSmRNA was not significantly different between the glioblastomas and normal brain tissues.1.3 Detection of p-Smad2/3 by Western blottingThe results showed that p-smad2/3 was upgradulated in the glioblastomas compared with that of the normal brain tissues.2 Results of cell culture2.1.1 The [3H]Thymidine assayDNA synthesis was evaluated by quantifying [3H]thymidine incorporation. Ativin A (3, 10,30ng/ml) caused increase (114.27±2.4%,148.7±5.1%,224.5±5.9%,390.2±8.8% of control) of the [3H]Thymidine incorporation by U87MG cells in a dose-dependent manner. The effect of activin A was modulated by follistatin. U87MG cells were cultured with or without follistatin (100ng/mg), activin A (3,10,30 ng/ml) or both. Follistatin alone had no significant effect on DNA synthesis, but follistatin decreased activin A-induced DNA synthesis (by 41.3%,82.1% and 137.6%1, respectively).2.1.2 Flow Cytometry Analysis of Cell CycleActivin A increased the proportion of proliferating cells in a dose dependent manner (Activin A 3,30ng/ml increased U87MG proliferative index by an increase of 6.3±0.5% and 12.7±±1.2%) compared with control respectively). In contrast, follistatin alone had no significant effect on U87MG proliferative index.2.1.3 Western blot Analysis of p-Smad2/3Western blot analysis showed that the expression of p-Smad2/3 was up-regulated by activin A in a dose dependent manner.Conclusion 1. Activin A/Smad signaling was up-regulated in glioblastoma; follistatin, the natural antagonist of activin A, was not.2. Activin A can promote the proliferation of U87 in vivo, follistatin can block the effect of activin A on U87.3. The disequilibrium of activin A/follistatin system may play a role in the proliferation and apoptosis of glioma. Blocking the activin A/Smad signaling might be a therapeutic of glioblastoma.