cucurbitacin-i and Glioblastoma

Glioblastoma multiforme (GBM) is one of the utmost malignant tumors. Excessive angiogenesis and invasiveness are the major reasons for their uncontrolled growth and resistance toward conventional strategies resulting in poor prognosis. In this study, we found that low-dose JSI-124 reduced invasiveness and tumorigenicity of GBM cells. JSI-124 effectively inhibited VEGF expression in GBM cells. In a coculture study, JSI-124 completely prevented U87MG cell-mediated capillary formation of HUVECs and the migration of HUVECs when cultured alone or cocultured with U87MG cells. Furthermore, JSI-124 inhibited VEGF-induced cell proliferation, motility, invasion and the formation of capillary-like structures in HUVECs in a dose-dependent manner. JSI-124 suppressed VEGF-induced p-VEGFR2 activity through STAT3 signaling cascade in HUVECs. Immunohistochemistry analysis showed that the expression of CD34, Ki67, p-STAT3 and p-VEGFR2 protein in xenografts was remarkably decreased. Taken together, our findings provide the first evidence that JSI-124 effectively inhibits tumor angiogenesis and invasion, which might be a viable drug in anti-angiogenesis and anti-invasion therapies.

Topics: Animals; Apoptosis; Blotting, Western; Capillaries; Cell Line, Tumor; Cell Proliferation; Cell Survival; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Glioblastoma; Human Umbilical Vein Endothelial Cells; Humans; Mice; Neoplasm Invasiveness; Neovascularization, Pathologic; Triterpenes; Vascular Endothelial Growth Factor A

There is an urgent need for new therapeutic avenues to improve the outcome of patients with glioblastoma multiforme (GBM). Current studies have suggested that cucurbitacin I, a natural selective inhibitor of JAK2/STAT3, has a potent anticancer effect on a variety of cancer cell types. This study showed that autophagy and apoptosis were induced by cucurbitacin I. Exposure of GBM cells to cucurbitacin I resulted in pronounced apoptotic cell death through activating bcl-2 family proteins. Cells treatment with cucurbitacin I up-regulated Beclin 1 and triggered autophagosome formation and accumulation as well as conversion of LC3I to LC3II. Activation of the AMP-activated protein kinase/mammalian target of rapamycin/p70S6K pathway, but not the PI3K/AKT pathway, occurred in autophagy induced by cucurbitacin I, which was accompanied by decreased hypoxia-inducible factor 1α. Stable overexpression of hypoxia-inducible factor 1α induced by FG-4497 prevented cucurbitacin I-induced autophagy and down-regulation of bcl-2. Knockdown of beclin 1 or treatment with the autophagy inhibitor 3-methyladenine also inhibited autophagy induced by cucurbitacin I. A coimmunoprecipitation assay showed that the interaction of Bcl-2 and Beclin 1/hVps34 decreased markedly in cells treated with cucurbitacin I. Furthermore, knockdown of beclin 1 or treatment with the lysosome inhibitor chloroquine sensitized cancer cells to cucurbitacin I-induced apoptosis. Finally, a xenograft model provided additional evidence for the occurrence of cucurbitacin I-induced apoptosis and autophagy in vitro. Our findings provide new insights into the molecular mechanisms underlying cucurbitacin I-mediated GBM cell death and may provide an efficacious therapy for patients harboring GBM.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chloroquine; Drug Screening Assays, Antitumor; Female; Glioblastoma; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Signal Transduction; Transfection; Triterpenes

Malignant gliomas are highly proliferative and invasive neoplasms where total surgical resection is often impossible and effective local radiation therapy difficult. Consequently, there is a need to develop a greater understanding of the molecular events driving invasion and to identify novel treatment targets. Using microarray analysis comparing normal brain samples and mesenchymal glioblastoma multiforme (GBM), we identified over 140 significant genes involved in cell migration and invasion. The cofilin (CFL) pathway, which disassembles actin filaments, was highly up-regulated compared to normal brain. Up-regulation of LIM domain kinase 1 and 2 (LIMK1/2), that phosphorylates and inactivates cofilin, was confirmed in an additional independent data set comparing normal brain to GBM. We identified and utilized two small molecule inhibitors BMS-5 and Cucurbitacin I directed against the cofilin regulating kinases, LIMK1 and LIMK2, to target this pathway. Significant decreases in cell viability were observed in glioma cells treated with BMS-5 and Cucurbitacin I, while no cytotoxic effects were seen in normal astrocytes that lack LIMK. BMS-5 and Cucurbitacin I promoted increased adhesion in GBM cells, and decreased migration and invasion. Collectively, these data suggest that use of LIMK inhibitors may provide a novel way to target the invasive machinery in GBM.

Topics: Brain; Caspase 3; Caspase 7; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Cofilin 1; Gene Dosage; Glioblastoma; Humans; Lim Kinases; Neoplasm Invasiveness; Phosphorylation; RNA Interference; RNA, Small Interfering; Triterpenes

Glioblastoma multiforme (GBM) is characterized by an aggressive clinical course, therapeutic resistance, and striking molecular heterogeneity. GBM-derived brain tumor stem cells (BTSCs) closely model this molecular heterogeneity and likely have a key role in tumor recurrence and therapeutic resistance. Emerging evidence indicates that Janus kinase (JAK)2/signal transducer and activator of transcription (STAT)3 is an important mediator of tumor cell survival, growth, and invasion in a large group of GBM. Here, we used a large set of molecularly heterogeneous BTSCs to evaluate the translational potential of JAK2/STAT3 therapeutics.. BTSCs were cultured from GBM patients and MGMT promoter methylation, and the mutation statuses of EGFR, PTEN, and TP53 were determined. Endogenous JAK2/STAT3 activity was assessed in human GBM tissue, BTSCs, and orthotopic xenografts by immunohistochemistry and Western blotting. STAT3 short hairpin (sh)RNA, cucurbitacin-I, and WP1066 were used to inhibit JAK2/STAT3 activity in vitro and in vivo.. The JAK2/STAT3 pathway was demonstrated to be highly activated in human GBM, molecularly heterogeneous BTSCs derived from these tumors, and BTSC xenografts. STAT3 shRNA knockdown or cucurbitacin-I and WP1066 administration resulted in on-target JAK2/STAT3 inhibition and dramatically reduced BTSC survival regardless of endogenous MGMT promoter methylation or EGFR, PTEN, and TP53 mutational status. BTSC orthotopic xenografts maintained the high levels of activated JAK2/STAT3 seen in their parent human tumors. Intraperitoneal WP1066 reduced intratumoral JAK2/STAT3 activity and prolonged animal survival.. Our study demonstrates the in vitro and in vivo efficacy of on-target JAK2/STAT3 inhibition in heterogeneous BTSC lines that closely emulate the genomic and tumorigenic characteristics of human GBM.

Topics: Aged; Animals; Apoptosis; Blotting, Western; Brain Neoplasms; Cell Proliferation; Disease Progression; DNA Methylation; DNA Modification Methylases; DNA Repair Enzymes; ErbB Receptors; Female; Flow Cytometry; Glioblastoma; Humans; Immunoenzyme Techniques; Janus Kinase 2; Male; Mice; Mice, Inbred NOD; Mice, SCID; Middle Aged; Neoplastic Stem Cells; Promoter Regions, Genetic; PTEN Phosphohydrolase; Pyridines; RNA, Small Interfering; Signal Transduction; STAT3 Transcription Factor; Triterpenes; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Tyrphostins; Xenograft Model Antitumor Assays

Glioblastoma tumors are characterized by their invasiveness and resistance to therapies. The transcription factor signal transducer and activator of transcription 3 (STAT3) was recently identified as a master transcriptional regulator in the mesenchymal subtype of glioblastoma (GBM), which has generated an increased interest in targeting STAT3. We have evaluated more closely the mechanism of action of one particular STAT3 inhibitor, JSI-124 (cucurbitacin I). In this study, we confirmed that JSI-124 inhibits both constitutive and stimulus-induced Janus kinase 2 (JAK2) and STAT3 phosphorylation, and decreases cell proliferation while inducing apoptosis in cultured GBM cells. However, we discovered that before the inhibition of STAT3, JSI-124 activates the nuclear factor-κB (NF-κB) pathway, via NF-κB p65 phosphorylation and nuclear translocation. In addition, JSI-124 treatment induces the expression of IL-6, IL-8, and suppressor of cytokine signaling (SOCS3) mRNA, which leads to a corresponding increase in IL-6, IL-8, and SOCS3 protein expression. Moreover, the NF-κB-driven SOCS3 expression acts as a negative regulator of STAT3, abrogating any subsequent STAT3 activation and provides a mechanism of STAT3 inhibition after JSI-124 treatment. Chromatin immunoprecipitation analysis confirms that NF-κB p65 in addition to other activating cofactors are found at the promoters of IL-6, IL-8, and SOCS3 after JSI-124 treatment. Using pharmacological inhibition of NF-κB and inducible knockdown of NF-κB p65, we found that JSI-124-induced expression of IL-6, IL-8, and SOCS3 was significantly inhibited, showing an NF-κB-dependent mechanism. Our data indicate that although JSI-124 may show potential antitumor effects through inhibition of STAT3, other off-target proinflammatory pathways are activated, emphasizing that more careful and thorough preclinical investigations must be implemented to prevent potential harmful effects.

Topics: Cell Growth Processes; Cell Line, Tumor; Gene Knockdown Techniques; Glioblastoma; Humans; Janus Kinase 2; NF-kappa B; Phosphorylation; Signal Transduction; STAT3 Transcription Factor; Transcription Factor RelA; Triterpenes

JSI-124 (cucurbitacin I) is a selective inhibitor of Janus kinase/signal transducer and activator of transcription 3(JAK/STAT3) and has been shown to exert anti-proliferative and anti-tumor properties both in vitro and in vivo. As STAT3 activation has been implicated in the development of glioma, we investigated the therapeutic efficacy of JSI-124 on glioblastoma multiforme (GBM) by interfering with STAT3 pathway. In present study, two GBM cell lines, U251 and A172 cells, were treated with JSI-124. The results showed that the cell growth was inhibited significantly in a dose-and time-dependent manner. Further investigation illustrated that the levels of phosphorylated-STAT3 were decreased in GBM cells treated by JSI-124, concomitant with apoptosis augment and cell cycle arrest. Specially, JSI-124 induced G(2)/M accumulation via downregulation of cyclin B1 and cdc2 expression. Together these results suggested that inhibition of STAT3 by JSI-124 is a potential strategy for the development of the new glioblastoma multiforme therapeutics.

Topics: Antineoplastic Agents; Apoptosis; Brain Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; G2 Phase; Glioblastoma; Humans; Mitosis; Triterpenes