Preclinical investigation of ibrutinib, a Bruton's kinase tyrosine (Btk) inhibitor, in suppressing glioma tumorigenesis and stem cell phenotypes.

Standard interventions for glioma include surgery, radiation and chemotherapies but the prognosis for malignant cases such as glioblastoma multiforme remain grim. Even with targeted therapeutic agent, bevacitumab, malignant glioma often develops resistance and recurrence. Thus, developing alternative interventions (therapeutic targets, biomarkers) is urgently required. Bruton's tyrosine kinase (Btk) has been long implicated in B cell malignancies but surprisingly it has recently been shown to also play a tumorigenic role in solid tumors such as ovarian and prostate cancer. Bioinformatics data indicates that Btk is significantly higher in clinical glioma samples as compared to normal brain cells and Btk expression level is associated with stage progression. This prompts us to investigate the potential role of Btk as a therapeutic target for glioma. Here, we demonstrate Btk expression is associated with GBM tumorigenesis. Down-regulation of Btk in GBM cell lines showed a significantly reduced abilities in colony formation, migration and GBM sphere-forming potential. Mechanistically, Btk-silenced cells showed a concomitant reduction in the expression of CD133 and Akt/mTOR signaling. In parallel, Ibrutinib (a Btk inhibitor) treatment led to a similar anti-tumorigenic response. Using xenograft mouse model, tumorigenesis was significantly reduced in Btk-silenced or ibrutinib-treated mice as compared to control counterparts. Finally, our glioma tissue microarray analysis indicated a higher Btk staining in the malignant tumors than less malignant and normal brain tissues. Collectively, Btk may represent a novel therapeutic target for glioma and ibrunitib may be used as an adjuvant treatment for malignant GBM.

Fisetin suppresses ADAM9 expression and inhibits invasion of glioma cancer cells through increased phosphorylation of ERK1/2.

Fisetin (3,3',4',7-tetrahydroxyflavone) is a naturally occurring flavonoid which is widely distributed in plants. It has been reported to possess some anticancer and anti-invasive capabilities. We set out to explore the effects of fisetin on antimetastatic and its mechanism of action in GBM8401 cells. The results indicated that fisetin exhibited effective inhibition of cell migration and inhibited the invasion of GBM8401 cells under non-cytotoxic concentrations. To identify the potential targets of fisetin, human proteinase antibody array analysis was performed, and the results indicated that the fisetin treatment inhibited the expression of ADAM9 protein and mRNA, which are known to contribute to the progression of glioma cancer. Our results showed that fisetin phosphorylated ERK1/2 in a sustained way that contributed to the inhibited ADAM9 protein and mRNA expression determined by Western blot and RT-PCR. Moreover, inhibition of ERK1/2 by U0126 or transfection with the siERK plasmid significantly abolished the fisetin-inhibited migration and invasion through activation of the ERK1/2 pathway. In summary, our results suggest that fisetin might be a potential therapeutic agent against human glioma cells based on its capacity to activate ERK1/2 and to inhibit ADAM9 expression.

Nodal regulates energy metabolism in glioma cells by inducing expression of hypoxia-inducible factor 1α.

BACKGROUND: A shift in glucose metabolism from oxidative phosphorylation to anaerobic glycolysis is the biochemical hallmark of malignant cancer cells. METHODS: In the present study, we demonstrated that Nodal stimulated the expression of glycolytic enzymes and decreased reliance on mitochondrial oxidative phosphorylation in human glioma cancer cells. The shift in glucose metabolism was mediated by induction of the hypoxia-inducible factor (HIF). RESULTS: Nodal protein expression was shown to be correlated with expression levels of glucose transporter (Glut)-1, hexokinase (HK)-II, pyruvate dehydrogenase kinase (PDK)-1, the phosphorylation level of pyruvate dehydrogenase (PDH), glucose uptake, and lactate accumulation in human glioma cells. These effects were inversely correlated with mitochondrial oxygen consumption and ATP production. Knockdown of Nodal expression with specific small hairpin RNA reduced Glut-1, HK-II, and PDK-1 expressions and PDH phosphorylation. Nodal knockdown also reduced glucose uptake and lactate generation, which in turn increased mitochondrial membrane potential (Ψ), O2 utilization, and ATP synthesis. The ectopic expression of Nodal in low-expressing Nodal glioma cells resulted in the opposite results compared with those of Nodal knockdown glioma cells. Treatment of cells with recombinant Nodal increased HIF-1 expression, and this effect was regulated at the transcriptional level. Blockage of the Nodal receptor by a pharmacological inhibitor or Nodal knockdown in U87MG cells decreased HIF-1α expression. Furthermore, HIF-1α knockdown in U87MG cells decreased Glut-1, HK-II, and PDK-1 expressions and PDH phosphorylation, which were similar to results in Nodal knockdown cells. CONCLUSION: Taken together, these results suggest that Nodal affects energy metabolism through HIF-1α.

The investigation of mitogen-activated protein kinase phosphatase-1 as a potential pharmacological target in non-small cell lung carcinomas, assisted by non-invasive molecular imaging.

BACKGROUND: Invasiveness and metastasis are the most common characteristics of non small cell lung cancer (NSCLC) and causes of tumour-related morbidity and mortality. Mitogen-activated protein kinases (MAPKs) signalling pathways have been shown to play critical roles in tumorigenesis. However, the precise pathological role(s) of mitogen-activated protein kinase phosphatase-1 (MKP-1) in different cancers has been controversial such that the up-regulation of MKP-1 in different cancers does not always correlate to a better prognosis. In this study, we showed that the induction of MKP-1 lead to a significant retardation of proliferation and metastasis in NSCLC cells. We also established that rosiglitazone (a PPARgamma agonist) elevated MKP-1 expression level in NSCLC cells and inhibited tumour metastasis. METHODS: Both wildtype and dominant negative forms of MKP-1 were constitutively expressed in NSCLC cell line H441GL. The migration and invasion abilities of these cells were examined in vitro. MKP-1 modulating agents such as rosiglitazone and triptolide were used to demonstrate MKP-1's role in tumorigenesis. Bioluminescent imaging was utilized to study tumorigenesis of MKP-1 over-expressing H441GL cells and anti-metastatic effect of rosiglitazone. RESULTS: Over-expression of MKP-1 reduced NSCLC cell proliferation rate as well as cell invasive and migratory abilities, evident by the reduced expression levels of MMP-2 and CXCR4. Mice inoculated with MKP-1 over-expressing H441 cells did not develop NSCLC while their control wildtype H441 inoculated littermates developed NSCLC and bone metastasis. Pharmacologically, rosiglitazone, a peroxisome proliferator activated receptor-gamma (PPARgamma) agonist appeared to induce MKP-1 expression while reduce MMP-2 and CXCR4 expression. H441GL-inoculated mice receiving daily oral rosiglitazone treatment demonstrated a significant inhibition of bone metastasis when compared to mice receiving sham treatment. We found that rosiglitazone treatment impeded the ability of cell migration and invasion in vitro. Cells pre-treated with triptolide (a MKP-1 inhibitor), reversed rosiglitazone-mediated cell invasion and migration. CONCLUSION: The induction of MKP-1 could significantly suppress the proliferative and metastatic abilities of NSCLC both in vitro and in vivo. Therefore, MKP-1 could be considered as a potential therapeutic target in NSCLC therapy and PPARgamma agonists could be explored for combined chemotherapy.

Osteopontin regulates human glioma cell invasiveness and tumor growth in mice.

Human malignant glioma cells are characterized by local invasion. In the present study, we investigated the role of osteopontin (OPN) in the invasiveness of human glioma cells isolated from grade IV tumors. We found that the expression levels of OPN in these cell lines paralleled matrix metalloproteinase-2 (MMP-2) expression and cell invasiveness potential. When U87MG glioma cells (with a high-OPN expression level) were stably transformed with specific small hairpin RNA to knock down OPN expression, MMP-2 secretion, cell invasiveness, and tumor growth in implanted brains were dramatically reduced. Conversely, forced expression of OPN in GBM-SKH glioma cells (which expressed OPN at a low level) increased MMP-2 secretion, enhanced cell invasiveness, and increased tumor growth in a rodent xenograft model. Expression of OPN was associated with increased expression of vimentin and decreased expression of glial fibrillary acidic protein. Treatment of glioma cells with 5-aza-2'-deoxycytidine (5-aza-dC) suppressed OPN expression in a concentration-dependent manner. Suppression of OPN expression by 5-aza-dC was associated with reductions in MMP-2 secretion, vimentin expression, cell invasion, intravasation, and tumor growth. These data suggest that OPN may play important roles in regulating cell invasion in glioma cells and that 5-aza-dC may serve as a therapeutic agent for human gliomas.

Rosiglitazone reduces cell invasiveness by inducing MKP-1 in human U87MG glioma cells.

We sought to investigate the molecular mechanisms by which rosiglitazone (RGZ) inhibits cell invasion in human glioma cells. In this study, we found that RGZ attenuated MMP-2 protein levels, MMP-2 gelatinolytic activity, and cell invasiveness through a PPAR-gamma independent pathway. RGZ increased mitogen activated protein kinase phosphatase-1 (MKP-1) expression. The addition of triptolide (a diterpenoid triepoxide, which blocked MKP-1 induction) abolished the inhibitory effects by RGZ. Furthermore, we demonstrated that the knock down of MKP-1 by MKP-1 specific small interference RNA reversed the reduction of MMP-2 secretion, and of cell invasiveness by RGZ. In contrast, the stable expression of MKP-1 in glioma cell lines decreased MMP-2 activity and cell invasiveness. These results suggest that RGZ may mediate the inhibitory effects through MKP-1 induction. Thus, MKP-1 could be a potential target in glioma therapy.

Dexamethasone reduced invasiveness of human malignant glioblastoma cells through a MAPK phosphatase-1 (MKP-1) dependent mechanism.

Dexamethasone has been shown to inhibit tumor invasiveness. In the present study, the effects of dexamethasone on matrix metalloproteinases-2 (MMP-2) secretion, cell invasiveness, and intravasation in human U87MG glioma cells were examined. Dexamethasone decreased MMP-2 secretion and cell invasiveness in human glioma cells. Incubation of cells with dexamethasone increased mitogen activated protein kinase phosphatase-1 (MKP-1) expression. Ectopic expression of MKP-1 decreased cell invasiveness in vitro and intravasation in vivo. Because expression of inducible nitric oxide synthase (iNOS) has been implicated in the progression of malignant gliomas, we next investigated the possible roles of NO(-) in MMP-2 secretion and cell invasiveness in human U87MG glioma cells. Treatment of glioma cells with nitric oxide donor, sodium nitroprusside (SNP), increased MMP-2 secretion and the capacity of cell invasion in U87MG cells. Addition of dexamethasone or ectopic expression of wild-type MKP-1 suppressed the SNP-stimulated MMP-2 activation and glioma cell invasiveness in U87MG cells. Taken together, these results suggest that dexamethasone may suppress MMP-2 secretion and cell invasion through MKP-1 induction in human glioma cells.