Apoptosis In Glioblastoma Multiforme Cells Research Articles

Evaluation of Tyrosine Kinase Inhibitor Combinations for Glioblastoma Therapy

Glioblastoma multiforme (GBM) is the most common intracranial cancer but despite recent advances in therapy the overall survival remains about 20 months. Whole genome exon sequencing studies implicate mutations in the receptor tyrosine kinase pathways (RTK) for driving tumor growth in over 80% of GBMs. In spite of various RTKs being mutated or altered in the majority of GBMs, clinical studies have not been able to demonstrate efficacy of molecular targeted therapies using tyrosine kinase inhibitors in GBMs. Activation of multiple downstream signaling pathways has been implicated as a possible means by which inhibition of a single RTK has been ineffective in GBM. In this study, we sought a combination of approved drugs that would inhibit in vitro and in vivo growth of GBM oncospheres. A combination consisting of gefitinib and sunitinib acted synergistically in inhibiting growth of GBM oncospheres in vitro. Sunitinib was the only RTK inhibitor that could induce apoptosis in GBM cells. However, the in vivo efficacy testing of the gefitinib and sunitinib combination in an EGFR amplified/ PTEN wild type GBM xenograft model revealed that gefitinib alone could significantly improve survival in animals whereas sunitinib did not show any survival benefit. Subsequent testing of the same drug combination in a different syngeneic glioma model that lacked EGFR amplification but was more susceptible to sunitinib in vitro demonstrated no survival benefit when treated with gefitinib or sunitinib or the gefitinib and sunitinib combination. Although a modest survival benefit was obtained in one of two animal models with EGFR amplification due to gefitinib alone, the addition of sunitinib, to test our best in vitro combination therapy, did not translate to any additional in vivo benefit. Improved targeted therapies, with drug properties favorable to intracranial tumors, are likely required to form effective drug combinations for GBM.

The Inhibitor of Growth 1 (ING1) Is Involved in Trichostatin A-Induced Apoptosis and Caspase 3 Signaling in p53-Deficient Glioblastoma Cells

Prognosis for patients with glioblastoma multiforme (GBM) is poor. Inhibitors of histone deacetylases (HDACi) like trichostatin A (TSA) are promising alternatives to conventional treatment. Deficient tumor suppressor functions, such as TP53 mutations and p14(ARF)/p16(INK4a) deletions, are characteristic for GBM and can cause resistance to DNA damaging agents such as cisplatin and to HDACi like TSA. The type II tumor suppressor Inhibitor of growth 1 (ING1) is involved in DNA damage response and histone modification. We have previously shown that ING1 is downregulated in GBM and involved in glioma-induced angiogenesis and in cisplatin-induced apoptosis in malignant glioma cells. Hence, the goal of our present study was to investigate whether TSA affects ING1 protein expression and also whether modulating ING1 levels affects TSA-induced apoptosis in malignant glioma cells that contain deficient p53 function and inactive pl4(ARF)/p16(INK4a) signaling. If so, we asked, which apoptotic pathway might be the major mediator beyond this interaction. To test whether ING1 proteins function in TSA-induced apoptosis in GBM, we analyzed TSA effects in LN229 GBM cells, which harbor TP53 mutations and INK4a deletion, following ING1 knockdown by siRNA. Expression of ING1, acetylated core histones H3 and H4, and the proapoptotic proteins caspase 3 and Fas-associated death domain (FADD) was determined by Western blotting. Percentages of apoptotic cells were obtained by flow cytometry. TSA induced the major ING1 isoform p33(ING1b) and increased levels of both histone acetylation and apoptosis in LN229 cells. ING1 knockdown cells revealed marked resistance to TSA-induced apoptosis, impairment of caspase 3 activation, and suppression of FADD. The data suggest that ING1 contributes to TSA-induced apoptosis in GBM cells with deficient p53 and p14(ARF)/p16(INK4a) functions, possibly by regulating FADD/caspase 3 signaling.

Promyelocytic leukemia protein induces apoptosis due to caspase-8 activation via the repression of NFκB activation in glioblastoma

Promyelocytic leukemia (PML) protein plays an essential role in the induction of apoptosis; its expression is reduced in various cancers. As the functional roles of PML in glioblastoma multiforme (GBM) have not been clarified, we assessed the expression of PML protein in GBM tissues and explored the mechanisms of PML-regulated cell death in GBM cells. We examined the PML mRNA level and the expression of PML protein in surgical GBM specimens. PML-regulated apoptotic mechanisms in GBM cells transfected with plasmids expressing the PML gene were examined. The protein expression of PML was significantly lower in GBM than in non-neoplastic tissues; approximately 10% of GBM tissues were PML-null. The PML mRNA levels were similar in both tissue types. The overexpression of PML activated caspase-8 and induced apoptosis in GBM cells. In these cells, PML decreased the expression of transactivated forms of NFkappaB/p65, and c-FLIP gene expression was suppressed. Therefore, PML-induced apoptosis resulted from the suppression of the transcriptional activity of NFkappaB/p65. PML overexpression decreased phosphorylated IkappaBalpha and nuclear NFkappaB/p65 and increased the expression of the suppressor of cytokine signaling (SOCS-1). A proteasome inhibitor blocked the reduction of activated p65 by PML. The reduction of PML is associated with the pathogenesis of GBM. PML induces caspase-8-dependent apoptosis via the repression of NFkappaB activation by which PML facilitates the proteasomal degradation of activated p65 and the sequestration of p65 with IkappaBalpha in the cytoplasm. This novel mechanism of PML-regulated apoptosis may represent a therapeutic target for GBM.

Resistance of human glioblastoma multiforme cells to growth factor inhibitors is overcome by blockade of inhibitor of apoptosis proteins

Multiple receptor tyrosine kinases (RTKs), including PDGFR, have been validated as therapeutic targets in glioblastoma multiforme (GBM), yet inhibitors of RTKs have had limited clinical success. As various antiapoptotic mechanisms render GBM cells resistant to chemo- and radiotherapy, we hypothesized that these antiapoptotic mechanisms also confer resistance to RTK inhibition. We found that in vitro inhibition of PDGFR in human GBM cells initiated the intrinsic pathway of apoptosis, as evidenced by mitochondrial outer membrane permeabilization, but downstream caspase activation was blocked by inhibitor of apoptosis proteins (IAPs). Consistent with this, inhibition of PDGFR combined with small molecule inactivation of IAPs induced apoptosis in human GBM cells in vitro and had synergistic antitumor effects in orthotopic mouse models of GBM and in primary human GBM neurospheres. These results demonstrate that concomitant inhibition of IAPs can overcome resistance to RTK inhibitors in human malignant GBM cells, and suggest that blockade of IAPs has the potential to improve treatment outcomes in patients with GBM.

The Effect of Triptolide on Apoptosis of Glioblastoma Multiforme (GBM) Cells

Triptolide, derived from the traditional Chinese herb, Tripterygium wilfordii, sensitizes cancer cells to apoptosis. Glioblastoma multiforme (GBM), which accounts for most cases of central nervous malignancy, has a very poor prognosis and lacks effective therapeutic inventions. We, therefore, investigated the effects of different concentrations of, and different periods of exposure to, triptolide on cell proliferation and apoptosis in the glioma cell lines, U251MG and U87MG, and in normal human fetal astrocytes. Cell proliferation was investigated by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay and growth curve analysis, and apoptosis was assessed from genomic DNA fragmentation. Triptolide showed dose-dependent inhibition of cell proliferation and induction of apoptosis in glioma cells. It also increased the ratio of the pro-apoptotic protein, Bax, to the anti-apoptotic protein, Bcl-2. Since U87MG has the wild-type p53 gene whereas U251MG harbours a mutated p53 gene, our results indicate that triptolide induces apoptosis in GBM cells via a p53-independent pathway. The dose-dependent inhibition of cell proliferation and induction of apoptosis by triptolide may involve upregulation of Bax and downregulation of Bcl-2.

Inhibitory Effect of Triptolide on Glioblastoma Multiforme In Vitro

This study investigated the effect of triptolide, derived from the traditional Chinese herb Tripterygium wilfordii, on the growth of glioblastoma multiforme (GBM) cells. Glioma cell lines U251MG and U87MG and normal human fetal astrocytes were exposed to various concentrations of triptolide, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium and colony formation assays were used to measure cell growth and survival. Cell apoptosis was determined using annexin V. Levels of the oncogenic transformation-related proteins Ras-guanosine triphosphate (Ras-GTP), extracellular signal-regulated kinase (ERK) and Akt were determined by Western blotting. Triptolide caused a dose-dependent decrease in proliferation and increase in apoptosis in the glioma cell lines. Since U87MG has a wildtype p53 gene while U251MG harbours a mutated p53 gene, these results indicate that triptolide induces apoptosis in GBM cells via a p53-independent pathway. Treatment of GBM cells with triptolide attenuated both the Ras/ERK and the Ras/Akt signalling pathways. This could provide a theoretical basis for triptolide treatment in GBM, but further animal studies and clinical research are necessary.

An accelerated senescence response to radiation in wild-type p53 glioblastoma multiforme cells

Radiotherapy is one of the few treatment options available for glioblastoma multiforme (GBM); however, the basis for its overall ineffectiveness in GBM is not fully understood. The present study was designed to explore the nature of the response to ionizing radiation in GBM cells to gain insight into the basis for the general failure of radiotherapy in the treatment of this disease. The response to fractionated radiotherapy was examined in GBM cell lines with differing p53 status. A viable cell number was determined during an 8-day period; accelerated senescence was based on beta-galactosidase staining and cell morphology; apoptosis was evaluated by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay and fluorescence-activated cell-sorter analysis, whereas the expression of cell-cycle regulatory proteins was monitored by Western blot analysis. Based on clonogenic survival, the wild-type p53 U87 cells and mutant p53 T98 cells demonstrated essentially identical sensitivity to fractionated radiotherapy; however, neither cell line underwent apoptosis, and the primary response to irradiation was growth arrest. The wild-type p53 GBM cells showed clear evidence of accelerated senescence in response to irradiation. In contrast, senescence was not evident in mutant p53 GBM cells or GBM cells in which p53 function was abrogated by the viral E6 protein. The T98 (mutant p53) cells demonstrated a relatively robust proliferative recovery whereas both the rate and extent of recovery were attenuated in the wild-type p53 U87 cells. Both accelerated senescence and conventional growth arrest are likely to represent alternative responses to apoptosis in irradiated GBM cells.

Glucose Withdrawal Induces Oxidative Stress followed by Apoptosis in Glioblastoma Cells but not in Normal Human Astrocytes

Tumor cells rely preferentially on anaerobic glycolysis rather than on respiration for ATP generation, a phenomenon known as the Warburg effect. We explored the effects of glucose withdrawal on glioblastoma multiforme-derived cell lines and their nontransformed counterparts, normal human astrocytes. We found that glucose withdrawal induces extensive apoptosis in glioblastoma multiforme cells but not in normal astrocytes. In all cells examined, ATP levels are sustained on glucose withdrawal due to elevation of fatty acid oxidation and ensuing respiration; however, we show that oxidative stress generated in the mitochondrial respiratory chain is the direct cause of cell death in glioblastoma multiforme cells. Oxidative stress that only occurs in glioblastoma multiforme cells underlies the selective susceptibility to glucose withdrawal-induced apoptosis documented in the malignant cells. This study implicates glycolysis as a potentially efficient and selective target for glioblastoma multiforme treatment.

Proteasome inhibitor PS-341 causes cell growth arrest and apoptosis in human glioblastoma multiforme (GBM)

The proteasome plays a pivotal role in controlling cell proliferation, apoptosis, and differentiation in a variety of normal and tumor cells. PS-341, a novel boronic acid dipeptide that inhibits 26S proteasome activity, has prominent effects in vitro and in vivo against several solid tumors. We examined its antiproliferation, proapoptotic effects using three human glioblastoma multiforme (GBM) cell lines and five primary GBM explants. PS-341 markedly inhibited proliferation of GBM cell lines and explants in liquid and soft agar culture. These cells developed a G2/M cell cycle arrest with a concomitant decreased percentage of cells in S phase ( approximately 2-fold), associated with an increased expression of p21(WAF1), p27(KIP1), as well as cyclin B1 and decreased levels of CDK2, CDK4, and E2F4. About 35-40% of the cells became apoptotic when exposed to PS-341 (10(-7) M, 24-48 h) as shown by Annexin V analysis; in concert with these findings, immunobloting showed a C-terminal 85 kDa apoptotic fragment of poly ADP-ribose polymerase (PARP), and a decreased level of Bcl2 and Bcl-xl. PS-341 downregulated the expression of Bcl-2 and Bcl-xl in protein levels at an early time of treatment. These changes occurred irrespective of the p53 mutational status of the cells. PS-341 activated JNK/c-Jun signaling in GBM cells, and the JNK inhibitor SP600125 blocked the JNK signaling to reverse partially the PS-341 growth inhibition. PS-341 (10(-7) M, 24 h) decreased nuclear NF-kappaB levels as shown by Western blot, and reduced transcriptional activity of NF-kappaB as measured by reporter assays in these transformed cells. Also, PS-341 enhanced TRAIL (TNF-related apoptosis-inducing ligand) and TNFalpha (tumor necrosis factor alpha) induced cell death and apoptosis (two- to five-fold) in GBM cells. In summary, PS-341 has profound effects on growth and apoptosis of GBM cells, suggesting that PS-341 may be an effective therapy for patients with gliomas.

Inhibition of constitutively active Stat3 suppresses proliferation and induces apoptosis in glioblastoma multiforme cells.

Glioblastoma multiforme (GBM), the most common and malignant central nervous system tumor in humans, is highly proliferative and resistant to apoptosis. Stat3, a latent transcription factor being activated by aberrant cytokine or growth factor signaling, acts as a suppressor of apoptosis in a number of cancer cells. Here we report that GBM tumors and cell lines contain high levels of constitutively activated Stat3 when compared with normal human astrocytes, white matter, and normal tissue adjacent to tumor. The persistent activation of Stat3 is in part, attributable to an autocrine action of interleukin-6 in the GBM cell line U251. Janus kinase inhibitor AG490 inhibits Stat3 activation with a concomitant reduction in steady-state levels of Bcl-X(L), Bcl-2 and Mcl-1 proteins and induces apoptosis in U251 cells as revealed by Poly (ADP-ribose) polymerase cleavage and Annexin-V staining. Expression of a dominant negative mutant Stat3 protein or treatment with AG490 markedly reduces the proliferation of U251 cells by inhibiting the constitutive activation of Stat3. These results provide evidence that constitutive activation of Stat3 contributes to the pathogenesis of glioblastoma by promoting both proliferation and survival of GBM cells. Therefore, targeting Stat3 signaling may provide a potential therapeutic intervention for GBM.

Expression and localization of cyclin-dependent kinase 5 in apoptotic human glioma cells

Cyclin-dependent kinase 5 (Cdk5), a member of the cyclin-dependent kinase family, is expressed predominately in mature neurons and is implicated in neurite extension, neuronal migration, and neuronal differentiation. Cdk5 protein expression also has been associated with apoptosis in a number of nonneuronal model systems. In normal brain, substrates for Cdk5 include neurofilament and tau proteins. Because human tumors of glial origin can express neuronal proteins, we examined whether Cdk5 and its activator protein, P35, are present in early passage human glioblastoma multiforme (GBM) cells lines and primary tumor specimens. Here we report the expression of Cdk5 and an "active" proteolytic form of P35 in human GBM cells and demonstrate kinase activity of the holoenzyme. We also show that Cdk5 kinase activity and expression of its activator protein, P35, is increased in the human GBM cell line M059J after exposure to ionizing radiation and that P35 is localized within M059J cells undergoing apoptosis. These results suggest a possible role for Cdk5 in mediating apoptosis in human GBM cells.

Induction of p53-dependent, insulin-like growth factor-binding protein-3-mediated apoptosis in glioblastoma multiforme cells by a protein kinase Calpha antisense oligonucleotide.

Protein kinase Calpha (PKCalpha) expression is related to tumor progression in glioblastoma multiforme (GBM), the most common malignant brain tumor in adults. To determine whether PKCalpha regulates an anti-apoptotic survival pathway in GBM, A172 GBM cells were treated with a PKCalpha-selective antisense oligonucleotide. PKCalpha antisense oligonucleotide treatment was accompanied by reduction in PKCalpha levels and the induction of wild-type p53 and insulin-like growth factor-binding protein-3 (IGFBP3) 24-72 h after treatment, a period that coincided with the appearance of apoptotic cell death as detected by DNA fragmentation. There were no significant changes in the levels of Bcl-XL, Bax, and p21(WAF1). Induction of p53 after PKCalpha down-regulation was not associated with increased mRNA expression, but increased IGFBP3 levels were accompanied by increased mRNA levels. Recombinant human IGFBP3 induced an apoptotic effect that was similar to the PKCalpha antisense oligonucleotide, and its effect was blocked by IGF-I. These results suggest that one mechanism by which PKCalpha produces its antiapoptotic activity in GBM cells is by suppressing the p53-mediated activation of IGFBP3.

Protein kinase C as a target for cancer therapy.

Antisense and Nucleic Acid Drug DevelopmentVol. 7, No. 3 Protein Kinase C as a Target for Cancer TherapyROBERT I. GLAZERROBERT I. GLAZERSearch for more papers by this authorPublished Online:30 Jan 2009https://doi.org/10.1089/oli.1.1997.7.235AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited byHow and Why to Apply the Latest Technology*Induction of p53-Dependent, Insulin-Like Growth Factor-Binding Protein-3-Mediated Apoptosis in Glioblastoma Multiforme Cells by a Protein Kinase Cα Antisense Oligonucleotide1 February 1999 | Molecular Pharmacology, Vol. 55, No. 2Antisense Oligonucleotides Specific for the HER2/neu Oncogene Inhibit the Growth of Human Breast Carcinoma Cells That Overexpress HER2/neuJournal of Surgical Research, Vol. 77, No. 1Post-translational modification of proteins and the discovery of new medicineCurrent Opinion in Biotechnology, Vol. 8, No. 6 Volume 7Issue 3Jun 1997 To cite this article:ROBERT I. GLAZER.Protein Kinase C as a Target for Cancer Therapy.Antisense and Nucleic Acid Drug Development.Jun 1997.235-238.http://doi.org/10.1089/oli.1.1997.7.235Published in Volume: 7 Issue 3: January 30, 2009PDF download