| Glioblastoma multiforme (GBM, WHO grade IV) is the most common and lethal type of primary brain tumor with a very poor prognosis. Current therapeutic approaches for GBM are predominantly palliative, including maximal surgical resection, radiotherapy and chemotherapy. However, one of the most important barriers to successful therapy is the diffuse invasion of GBM cells, making them elusive targets for effective surgical management and giving rise to tumor recurrence. Besides, lack of significant antitumor effect of traditional therapies is frequently observed, resulting in quite low relative survival (five-year survival rates are less than5%)(Statistical report2012, The Central Brain Tumor Registry of the United States, CBTRUS). Therefore, identifying novel effective therapeutic agents for treating GBM is urgently needed.Etiology and pathophysiology of GBM are complex and currently unclear. Potential molecular targets for GBM therapies are being explored while effective ones are still largely unknown. As GBM is characterized by high proliferation rate and diffuse invasion, regulators targeting these cell behaviors may lead to immediate clinical improvement.This study is supported by Department of Systems Medicine and Bioengineering, The Methodist Hospital Research Institute (TMHRI) of the USA and Key Laboratory of the Ministry of Education for Experimental Teratology of China. In this study, novel inhibitors of glioblastoma cell growth and invasion are explored, which may serve as novel potential anti-GBM agents.Part I Identification of novel small-molecule inhibitors of glioblastoma cell growth and invasion by high-throughput screeningObjectives:To discover novel potential compounds against GBM, human glioblastoma cell line U87-luc (U87MG glioblastoma cells genetically engineered to express luciferase) were used and1,280pharmacologically active compounds from56pharmacological classes were quantitatively assessed for their effects on U87-Luc cell growth and invasion.Methods:First, the high-throughput screening plates were prepared. Each compound of the LOPAC1280TM library was mixed with U87-Luc cell conditioned media and MatrigelTM Basement Membrane Matrix (the final concentration of each compound was10μM), and the mixture was added onto the screening plates in triplicate in a cold room at4℃. Cell growth and invasion were evaluated through Bioluminescence Imaging (BLI), and Z’factor was used for quality control. Z’ factor was defined as described in the following equation:1-(3σ+3μ)/|μs-μb|, where σ represents the SD of signal (σs) or background (σb) and μ represents the mean.The signal intensity was quantified using Living Image(?) software in terms of photon counts and statistical significance was determined.Results:For the screen plates, an average Z’factor of0.513was obtained, indicating that the assay was acceptable for cell-based screening.The high-throughput screening of LOPAC1280TM resulted in an initial identification of25inhibitors of glioblastoma cell growth and invasion. It is gratifying that Carmustine, I-OMe-Tyrphostin AG538,(-)-Perillic acid,13-cis-retinoic acid, Vincristine sulfate, and Taxol, which had been studied in preclinical experiments, evaluated in clinical trials, or used in clinical as chemotherapeutic agents for tumors including GBM, were identified in our screening.Conclusions:Via a high-throughput screening, from an annotated compound library (LOPAC1280TM), we identified25novel small-molecule inhibitors of GBM cell growth and invasion, with an attempt to provide useful therapeutic leads for GBM treatment.Significance:In this study, all compounds of the LOPAC12801, which is a versatile library including the latest drug-like molecules in the fields of cell signaling and neuroscience, and reflects the most commonly screened targets in the drug discovery community, were evaluated in the screening assay. Novel inhibitors of GBM cell growth and invasion were identified in the present study, which provides a basis for the development of therapies for GBM, and may shed light on the molecular mechanisms underlying GBM cell behavior.In the high-throughput screening, we interestingly found that GW2974(LOPAC No. G0668), a dual inhibitor of the epidermal growth factor receptor (EGFR, ErbB1) and HER2(ErbB2), played a notable role in GBM cell behavior. GW2974has shown therapeutic efficacy and limited toxicity in gallbladder carcinoma and breast cancer, however, there is an apparent lack of information on the role of GW2974in GBM. This makes GW2974worth further study. Besides GW2974, other active compounds are being (or will be) investigated in our laboratory for their anti-GBM effects. The results will be reported in detail elsewhere. Part II Effects of GW2974in Glioblastoma in vitro and in vivoObjectives:GW2974, a dual EGFR and HER2kinase inhibitor, was selected from the results of the high-throught screening and evaluated for its effects in GBM in vitro and in vivo, and the possible mechanisms were explored.Methods:1. In vitro experimentsHuman glioblastoma cell lines U87MG, U251MG and U87-Luc were maintained in a humidified tissue culture incubator at37℃of5%CO2atmosphere. Different doses of GW2974were evaluated for their effects on cell viability using CellTiter96(?) AQueous One Solution Cell Proliferation Assay (MTS assay).For the in vitro experiments, cells were divided into three groups:control group (no treatment), low-dose GW2974group (0.5μM GW2974treatment) and high-dose GW2974group (5μM GW2974treatment).U87MG and U251MG cells were treated accordingly for24h. Cell proliferation was evaluated by Bromodeoxyuridine (BrdU) incorporation assay. Cell invasive capacity was assessed using a24-well BioCoatTM MatrigelTM Invasion Chamber. Cell migration was examined by wound-healing assay.2. In vivo experimentsThe intracranial glioblastoma xenograft model was created using U87-Luc cells. The specific doses of GW2974for in vivo treatment (i.e.,30mg/kg/day and100mg/kg/day) were determined based on our preliminary studies as well as other’s previous works.For each experiment, the xenografted mice were divided into three groups: control group (vehicle treatment), low-dose GW2974group (30mg/kg/day GW2974treatment) and high-dose GW2974group (100mg/kg/day GW2974treatment). Treatment was started on the next day (day1) of tumor implantation (day0).Forty-five xenografted mice were used and two experiments were conducted in parallel. In Experiment1, thirty mice were randomly divided into the three groups (as above) of10each. Tumor growth was evaluated weekly using the Xenogen IVIS(?) Imaging System. The mice were checked twice daily and sacrificed when moribund as determined by a blinded observer, and survival was recorded.In Experiment2, three groups of mice (n=5per group) were sacrificed after two weeks treatment (on day14) and the brains were harvested, fixed, paraffin-embedded, and sectioned. The sections were stained with hematoxylin and eosin (H&E) and evaluated. To assess tumor angiogenesis, microvessel density (MVD) was determined by immunostaining of CD34. In addition, immunostaining of Fascin, a different tumor invasiveness marker, was performed to evaluate tumor invasion.3. Experiments for exploring the mechanismsDifferentially expressed proteins in cells treated with different doses of GW2974were explored by a commercial725antibody microarray platform (Panorama(?) Antibody Array-XPRESS Profiler725) and then subjected to Ingenuity(?) Pathway Analysis (IPA) Systems to identify significantly involved pathways. Results were validated using Western blot analysis. Cell invasion assay was performed with the addition of SB203580, a specific p38MAPK inhibitor with no cytotoxicity to GBM cells, to evaluated the role of p38MAPK in the regulation of cell invasion by GW2974.4. Statistical AnalysisDistributions of the data were tested for normality using D’Agostino and Pearson omnibus normality test. Statistically significant differences between groups were determined by Student’s t test or one-way ANOVA followed by Tukey’s test for normally distributed data, and data are presented as mean±SEM. The nonparametric Wilcoxon rank-sum test was used when the data were not normally distributed, and data are presented as median (range). Survival was evaluated for statistical significance by the Kaplan-Meier method and compared by using the log-rank test.All statistical analyses were performed in GraphPad Prism for Windows, and p<0.05was considered significant.Results:1. Results of the in vitro experimentsMTS assay showed that increasing doses of GW2974resulted in a gradual inhibition in cell viability24h post-treatment, with an obvious cytotoxicity appeared at10μM and above.0.5μM and5μM, which may represent the relative low and high doses of GW2974that the cells can tolerate, were therefore utilized for further analysis. These two doses of GW2974significantly reduced U87MG cell viability to89.4%and86.3%, respectively, when compared with the control (p<0.05). Similar viability changes were observed in U251MG cells, in which cell viability was reduced to92.2%and88.0%when compared to control (p<0.05).Next, the effect of GW2974on GBM cell proliferation was evaluated by BrdU incorporation. The rates of BrdU incorporation of low-dose and high-dose GW2974treated U87MG cells were significantly reduced to40.3%and39.3%, respectively, as compared with control cells (47.7%)(p<0.05). The rates of BrdU incorporation of low-dose and high-dose GW2974treated U251MG cells were reduced to35.7%and32.1%, respectively, as compared with control cells (42.7%)(p<0.05).The invasive ability of GBM cells was then determined using a MatrigelTM invasion assay. Compared with the control cells, the percentage of low-dose GW2974treated U87MG and U251MG cells that had invaded through Matrigel was significantly reduced to55.6%and48.6%, respectively (p<0.05). However, no significant difference was observed between the high-dose GW2974treated group and control.The effects of GW2974on GBM cell migration were further evaluated by wound-healing assay. In comparison with the control cells, low-dose GW2974 remarkably decreased the relative migration distances (percentage) of U87MG and U251MG cells to42.5%and51.6%, respectively (p<0.05), whereas5μM GW2974abrogated this effect and resulted in a relative migration distances comparable to that of control.2. Results of the in vivo experimentsFor Experiment1, the tumors rapidly grew in the control mice, resulting in the death of all the mice by day22. In comparison with the control, the mice receiving low-dose GW2974showed a remarkable delay in tumor growth (p<0.05) with a100%survival rate by day27. The high-dose GW2974also decelerated tumor growth significantly (p<0.05), however, the mice in this group began to die on day20, and all had died by day23. Compared with the control group, the life spans of mice in the low-dose GW2974group were significantly prolonged (p<0.01), whereas no change was observed in the high-dose GW2974group.For Experiment2, brain slices of mice were obtained on day14and subjected to histological examination to assess tumor invasion and angiogenesis. Compared with the control group, the tumors in low-dose GW2974group exhibited a regular contour, indicating that the invasion to peri-tumor areas was inhibited. However, the tumors of mice in the high-dose GW2974group exhibited irregular tumor borders, and tumor satellite nodules could be found in the adjacent brain tissues. This was further confirmed by Fascin immunostaining. Compared with the control group, the tumors in the low-dose GW2974group showed an overall decrease in Fascin staining intensity (p<0.05), whereas this decrease was totally abrogated in the high-dose GW2974group. MVD was quantitated to evaluate how GW2974interfered with tumor angiogenesis. In both low-dose and high-dose GW2974groups, MVD was significantly inhibited as compared with the vehicle treated control group (p<0.05).3. Results of the experiments for exploring the mechanismsThe results of antibody microarray and IPA system revealed that responses of GBM cells to different doses of GW2974were mediated by distinct signaling pathways. To validate the array-determined changes, we performed conventional Western blot for individual key proteins within these pathways. Data showed that phospho-EGFR (p-EGFR) and phospho-ERK (p-ERK) were decreased in the cells treated with both low-dose GW2974group and high-dose GW2974group when compared with the control group (p<0.05). Phospho-p38(p-p38), on the other hand, was only increased in the high-dose GW2974group as compared with the control group (p<0.05). No difference in p-p38levels was observed between the low-dose GW2974group and control group. P38mitogen-activated protein kinase (MAPK) pathway was therefore conjectured to play a role in GBM cell invasion induced by the high dose GW2974.To test our hypotheses, cell invasion assay was performed with the addition of SB203580, which is a specific p38MAPK inhibitor that is extremely useful in delineating the function of p38and has shown no apparent cytotoxicity. Remarkably, combination of SB203580with5μM GW2974resulted in a cell invasion comparable to that of0.5μM GW2974treated cells. No significant difference was observed between control group and the group treated with SB203580alone. This result indicated that p38-MAPK pathway might be a connecting link between EGFR/HER2inhibition and GBM cell invasion.Conclusions:1. Low-dose and high-dose GW2974inhibit GBM cell proliferation, whereas only low-dose GW2974inhibited GBM cell invasion. Similar results were observed in an intracranial GBM xenograft model, where though both doses of GW2974slowed tumor growth, no improvement in survival was observed in mice treated with high-dose GW2974presumably due to the augmentation of tumor invasion.2. GW2974exerts different effects on GBM invasion depending on drug dosage.3. P38MAPK pathway might contribute to the differential regulation of cell invasion by GW2974. Significance:Mutations and amplifications of EGFR and its family members are implicated in a number of cancers, including GBM. In this study, we found for the first time that EGFR/HER2inhibitor GW2974played a dose-related role on GBM invasion. Our results indicated that in clinical trials administration and dosing of EGFR/HER2inhibitors such as GW2974ought to be re-evaluated, if possible, taking into consideration the possibility of opposing effects exerted by different doses. This might help in understanding why conventional cytotoxic chemotherapy or radiotherapy for GBM that has focused on growth inhibition often results in unsatisfactory outcomes. Based on the results presented here, it is suggested that tumor invasion should be seriously considered when chemotherapy is applied. In comparison to tumor growth, the invasive potential of GBM may be more correlated with the prognosis and therapeutic results. Inhibiting GBM invasion and elucidating the underlying mechanisms are therefore critical in promoting therapeutic efficacy and avoiding side effects. |
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