Rho-kinase Inhibitor, Fasudil, Suppresses Glioblastoma Cell Line Progression In Vitro And In Vivo

IntroductionGlioma has the highest incidence among human intracranial tumors, and glioblastoma (GBM) is the most frequent and malignant histological type. Despite aggressive surgical resection, radiation, and chemo-therapy, the prognosis of patients remains poor. Therefore, more effective therapeutic methods are urgently required for the treatment of GBM. According to existing research, the hyperproliferation and inva-siveness of GBM cells are the major reasons for treatment failure and tumor recurrence.Although it is rarely metastasized systemically, GBM cells infiltrate contiguous and distant regions of the brain. In order to invade foreign tissue, a neoplastic cell must interrupt its adhesion with surrounding cells or tissue, cross the basal membrane, migrate through the extracellular matrix, proliferate, and generate its own capillary network. Matrix metalloproteinases (MMPs), such as collagenases, stromelysins, and gelatinases, belong to a larger family of proteases known as the metzincin superfamily. They are capable of degrading all kinds of extracellular matrix proteins. Recently, a growing number of evidence showed that MMPs play an important role in tumor invasion and migration. In particular, two gelatinases, MMP-2 and MMP-9, have attracted the most attention in terms of glioma invasion.Rho, a small GTP-binding protein, has various effects on cellular functions, including regulating the formation of contractile actin-myosin filaments, which form stress fibers and maintain focal adhesions at the rear of the cell. One important Rho target is Ser/Thr kinase, Rho-kinase (ROCK). Studies have shown that the expression of Rho/ROCK increases in GBM cell lines, T98G and U251, and correlates positively with the degree of malignancy in astrocytomas. The ROCK inhibitor, Y27632, suppresses the migration and proliferation of GBM cells. However, for the mechanism of Rho/ ROCK-dependent migration in tumor biology, previous reports mainly focused on the changes in cytoskeletal structure.Fasudil [1-(5-isoquinolinesulfonyl)-homopiperazine] is a ROCK inhibitor which has been proved to modify myosin light chain phosphorylation in smooth muscle cells. It has been used for the clinical treatment of cerebral vasospasms that occur after subarachnoid hemorrhage and associated cerebral ischemic symptoms. In addition, it is reported that fasudil may be useful for cardiovascular diseases, including angina pectoris, hypertension, pulmonary hypertension, stroke, and heart failure. However, the effect of fasudil treatment for GBM has not been investigated.To determine the effect of fasudil on GBM cells and reveal its underlying mechanisms, two human GBM cell lines, T98G and U251, were treated with fasudil at different doses. The biological properties of these tumor cells, including their morphology, proliferation, invasion, and apoptosis, were also studied. Assuming that MMPs play a critical role in tumor invasion and migration, the regulatory effect of fasudil on MMPs and their negative regulator tissue inhibitor of metalloproteinases (TIMPs) were investigated. The intra-cranial xenograft models were established. Cryosection of the tumor and survival curve analysis were used to determine the effect of fasudil on GBM in vivo.Methods and Results Part 11. Effect of fasudil on the morphological changes of GBM cellsThe T98G and U251 cells treated with various concentrations of fasudil in 24 h lost their original polygonal morphology and adopted more of a stellate morphology with an increasing number of cell processes. The cell body became thinner, and the processes presented as filopodia. These changes in GBM cells occurred in a time-and dose-dependent manner. At a higher concentration of fasudil (100μM), the cell showed complete loss of normal morphology, and the processes increased several times the length of the cell body.To determine if the effects of fasudil on GBM cell morphology were reversible, a recovery experiment was performed. It indicated that GBM cells treated with 5μM fasudil completely regained their original polygonal morphology, similar to what was observed in untreated control cells. However, cells treated at higher doses (20 and 100μM) after 24 h incubation in DMEM medium with 10% FBS cannot completely recover their normal morphology. These results indicated that the effects of fasudil are reversible at a lower dose after 24 h of fasudil treatment and suggested that high concentrations of fasudil may have a toxic effect on T98G and U251 cells.2. Effect of fasudil on migration and invasion of GBM cells2.1 Fasudil inhibits the motility of GBM cellsThe wound healing assay was used to assess the effects of fasudil on the migration capacity of GBM cells. The sites of the wound line were photographed immediately after scratching and 12 h later. The width of the wound line was measured, and the migration distances were calculated relative to the control group. According to our data, fasudil inhibited the motility of T98G and U251 in a dose-dependent manner. The inhibition effect of fasudil on GBM cell migration was dose-dependent. Although there was no difference in the low-dose group compared to the non-treated controls, a higher concentration of fasudil (20μM) inhibited T98G migration by at least 30%, while 100μM fasudil robustly inhibited migration by 50%. Similar outcomes were obtained in the migration of U251. 2.2 Fasudil inhibits invasion of GBM cellsThe matrigel transwell assay was conducted to determine the effect of fasudil on the invasion of GBM cells. After 24 h of invasion, the images of the cells affixed on the lower side of the membrane were taken and the cells were counted. All the groups were presented as the percentage of the control. After the cells were treated with various concentrations of fasudil, a dose-dependent effect of fasudil was observed on tumor cell invasion ability. Interestingly, unlike the migration assay, even the low-dose fasudil (5μM) repressed cell invasion. This inhibition was much more significant in high-dose groups. Thus, although low-dose fasudil had no effect on cell migration, it did inhibit cell invasion, indicating that migration inhibition may not be sufficient to explain how fasudil represses GBM invasion.3. Effect of fasudil on MMP-2 expression of GBM cells3.1 Effect of fasudil on mRNA expression of invasion-related genesTo determine the effect of fasudil on invasion-related gene expression, RT-PCR experiments were performed. After treat-ment with 20μM fasudil and with ROCK siRNA for 24 h to serve as positive controls, the total mRNA of T98G and U251 was extracted. Since MMPs and TIMPs are involved in the invasion and metastasis of tumor cells and ECM degradation is mainly mediated by the balance between them, MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-3 were detected using RT-PCR. The MMP-2 mRNA levels of T98G treated with fasudil and ROCK siRNA were apparently down-regulated by 50% compared to the untreated group. There was no significant difference in MMP-9, TIMP-1, TIMP-2, and TIMP-3 between the treated and untreated groups. U251 had similar results. Gelatin zymogram further confirmed that only MMP-2, not MMP-9, may be responsible for fasudil bioavailability in repressing GBM invasion.3.2 Fasudil inhibits MMP-2 expression of GBM cells To further confirm that fasudil inhibited MMP-2 expression, RT-PCR, western-blot analysis, and gelatin zymogram were performed. Exposure to fasudil for 24 h significantly de-creased the MMP-2 mRNA level. Detection of the corresponding proteins by western-blot and gelatin zymogram confirmed this finding. Quantitations of the active forms of MMP-2 were suppressed in a dose-dependent manner by fasudil treatment. A gelatin zymogram detecting MMP-2 in the supernatant of cultured cells and directly reflecting MMP-2 expression of T98G and U251 showed consistent results concerning the marked dose-dependent inhibition effect of fasudil. Various concentrations of fasudil down-regulated MMP-2 levels by 15.3,41.4, and 46.5% compared to the control in T98G, and by 11.7,16, and 32.4% compared to the control in U251, respectively, for 5,20,100μM. These results suggest that fasudil has a dose-dependent inhibition effect on MMP-2 expression of GBM cells.4. Effect of fasudil on cell proliferation and apoptosis of GBM cells Fasudil inhibits cell proliferation of GBM cellsTo examine the effect of fasudil on the growth of GBM cells, [3H]-thymidine incorporation was performed. According to the results, fasudil contributed to potential growth inhibition in the T98G and U251 cell lines in a dose-dependent manner. At higher concentrations (20 and 100μM), fasudil suppressed cell proliferation by 65.7 and 76.7% compared to the control in T98G and by 65.6 and 70.2% compared to the control in U251, respectively.Fasudil induces cell apoptosis of GBM cellsSince the Rho/ROCK pathway involves diverse cellular responses, including cell growth, differentiation, and survival, the ability of the fasudil to induce apoptosis was therefore examined. An annexin V/PI staining assay was used for the T98G and U251 cells. Y27632 and ROCK siRNA were used as positive controls, which remarkably induced cell apoptosis. At higher concentrations (20 and 100μM), fasudil markedly induced apoptosis of both GBM cell lines in a dose-dependent manner compared to the control group and the low-dose group. There was a 7.1-fold increased induction at 20μM and a 9.75-fold increased induction at 100μM for T98G, and a 6.5-fold induction at 20μM and 10.45-fold induction at 100μM for U251.Part 2Fasudil suppresses GBM growth and invasion in vivoTo evaluate whether fasudil inhibits GBM progression in vivo, eGFP-T98G cells were inoculated into the left caudate putamen of the nude mice. When the tumors were established, the mice were treated with fasudil (50μg/100μL PBS via tail vein) or PBS (100μL) as control. After the 14th day of administration, the animals were sacrificed and brain specimens were cryosectioned into 25μM thick sections in the coronal plane. When the eGFP-T98G glioma cells were inoculated into the caudate-putamen, GFP-expressing tumor cells were found to have infiltrated the surrounding brain tissue and most of the cerebral cortex. In contrast, the demarcation between the tumor and the surrounding tissues was clear in the fasudil treatment. The Kaplan-Meier survival curve and log rank test analysis revealed statistically significant prolongation of survival in the group receiving fasudil (median survival time,42 d) compared with the control group receiving only PBS (median survival time,35 d; log-rank test, P＜0.05). These results briefly indicate that fasudil could inhibit glioma invasion and growth in vivo.ConclusionIn the present study, we first confirmed that fasudil markedly induced the morphological changes and down-regulated the migration and invasion capability of T98G and U251. The intriguing difference caused by fasudil on migration and invasion led us to further investi- gate the underlying mechanism of how fasudil regulates GBM invasion. Then, we found that, among the common invasion-related genes (i.e., MMPs and TIMPs), MMP-2 expression was suppressed by ROCK inhibitors. In addition, the inhibitor significantly increased the rate of apoptosis and inhibited the prolife-ration of GBM cells. The cryosection of tumor and the survival time of mice further suggested that fasudil, as a well-known ROCK inhibitor, suppresses the progression of GBM cell lines in vitro and in vivo by inhibiting ROCK. In conclusion, our results demonstrated that fasudil treatment suppressed GBM progression by inhibiting invasion and inducing apoptosis in vitro and in vivo. The Rho/ROCK signaling pathway is a promising target for GBM treatment. Further research of the mechanism can lead to the discovery of the underlying pharmacological action of fasudil. The fact that fasudil is approved for human use and is tolerated without serious adverse reactions makes it an attractive anti-tumor drug candidate for the treatment of GBM.