sepantronium and Glioma

Because the anti-apoptotic protein Bcl-xL is overexpressed in glioma, one might expect that inhibiting or silencing this gene would promote tumor cell killing. However, our studies have shown that this approach has limited independent activity, but may tip the balance in favor of apoptosis induction in response to other therapeutic interventions. To address this issue, we performed a pharmacological screen using a panel of signaling inhibitors and chemotherapeutic agents in Bcl-xL silenced cells. Although limited apoptosis induction was observed with a series of inhibitors for receptor tyrosine kinases, PKC inhibitors, Src family members, JAK/STAT, histone deacetylase, the PI3K/Akt/mTOR pathway, MAP kinase, CDK, heat shock proteins, proteasomal processing, and various conventional chemotherapeutic agents, we observed a dramatic potentiation of apoptosis in Bcl-xL silenced cells with the survivin inhibitor, YM155. Treatment with YM155 increased the release of cytochrome c, smac/DIABLO and apoptosis inducing-factor, and promoted loss of mitochondrial membrane potential, activation of Bax, recruitment of LC3-II to the autophagosomes and apoptosis in Bcl-xL silenced cells. We also found an additional mechanism for the augmentation of apoptosis due to abrogation of DNA double-strand break repair mediated by Rad51 repression and enhanced accumulation of γH2AX. In summary, our observations may provide a new insight into the link between Bcl-xL and survivin inhibition for the development of novel therapies for glioma. © 2016 Wiley Periodicals, Inc.

Topics: Antineoplastic Agents; Apoptosis; Autophagy; bcl-X Protein; Cell Line, Tumor; Cytochromes c; DNA Damage; Gene Silencing; Glioma; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Membrane Potential, Mitochondrial; Naphthoquinones; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Survivin; TOR Serine-Threonine Kinases

Survivin has multiple functions in the progression of cancer. The aim of the present study was to investigate the influence of YM155, a specific survivin inhibitor, on the biological behavior of U87 glioblastoma cells. The proliferative activity and growth rate of U87 cells were determined by colony formation assay and mononuclear cell direct cytotoxicity assay (MTT assay). The reconstituted basement membrane penetrating capacity was determined by cell invasion assay. The cell movement and migratory capacity were detected by wound-healing repair assay. We found that inhibition of survivin by YM155 dramatically decreased the invasive and metastatic capacities of the cells, suppressed the proliferation, decelerated the rate of growth, and reduced the number of clones on soft agar. In conclusion, YM155 has the potential to be used in the clinical treatment of GBM.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Glioma; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Naphthoquinones; Neurons; Survivin

Induction of apoptosis by the death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising antitumor therapy. However, not all tumor cells are sensitive to TRAIL, highlighting the need for strategies to overcome TRAIL resistance. Inhibitor of apoptosis family member survivin is constitutively activated in various cancers and blocks apoptotic signaling. Recently, we demonstrated that YM-155 [3-(2-methoxyethyl)-2-methyl-4,9-dioxo-1-(pyrazin-2-ylmethyl)-4,9-dihydro-3H-naphtho[2,3-d]imidazol-1-ium bromide], a small molecule inhibitor, downregulates not only survivin in gliomas but also myeloid cell leukemia sequence 1 (Mcl-1), and it upregulates proapoptotic Noxa levels. Because Mcl-1 and survivin are critical mediators of resistance to various anticancer therapies, we questioned whether YM-155 could sensitize resistant glioma cells to TRAIL. To address this hypothesis, we combined YM-155 with TRAIL and examined the effects on cell survival and apoptotic signaling. TRAIL or YM-155 individually induced minimal killing in highly resistant U373 and LNZ308 cell lines, but combining TRAIL with YM-155 triggered a synergistic proapoptotic response, mediated through mitochondrial dysfunction via activation of caspases-8, -9, -7, -3, poly-ADP-ribose polymerase, and Bid. Apoptosis induced by combination treatments was blocked by caspase-8 and pan-caspase inhibitors. In addition, knockdown of Mcl-1 by RNA interference overcame apoptotic resistance to TRAIL. Conversely, silencing Noxa by RNA interference reduced the combined effects of YM-155 and TRAIL on apoptosis. Mechanistically, these findings indicate that YM-155 plays a role in counteracting glioma cell resistance to TRAIL-induced apoptosis by downregulating Mcl-1 and survivin and amplifying mitochondrial signaling through intrinsic and extrinsic apoptotic pathways. The significantly enhanced antitumor activity of the combination of YM-155 and TRAIL may have applications for therapy of malignant glioma.

Topics: Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Cell Line, Tumor; Cell Survival; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; Gene Knockdown Techniques; Glioma; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Naphthoquinones; Protein Conformation; Proto-Oncogene Proteins c-bcl-2; Recombinant Proteins; Survivin; TNF-Related Apoptosis-Inducing Ligand

Antiapoptotic proteins are commonly overexpressed in gliomas, contributing to therapeutic resistance. We recently reported that clinically achievable concentrations of the Bcl-2/Bcl-xL inhibitor ABT-737 failed to induce apoptosis in glioma cells, with persistent expression of survivin and Mcl-1. To address the role of these mediators in glioma apoptosis resistance, we analyzed the effects of YM-155, a survivin suppressant, on survival on a panel of glioma cell lines. YM-155 inhibited cell growth and downregulated survivin and Mcl-1 in a dose- and cell line-dependent manner. While U373, LN18, LNZ428, T98G, LN229, and LNZ308 cells exhibited an IC(50) of 10 to 75 nmol/L, A172 cells were resistant (IC(50) ∼ 250 nmol/L). No correlation was found between sensitivity to YM-155 and baseline expression of survivin or cIAP-1/cIAP-2/XIAP. However, strong correlation was observed between EGF receptor (EGFR) activation levels and YM-155 response, which was confirmed using EGFR-transduced versus wild-type cells. Because we postulated that decreasing Mcl-1 expression may enhance glioma sensitivity to ABT-737, we examined whether cotreatment with YM-155 promoted ABT-737 efficacy. YM-155 synergistically enhanced ABT-737-induced cytotoxicity and caspase-dependent apoptosis. Downregulation of Mcl-1 using short hairpin RNA also enhanced ABT-737-inducing killing, confirming an important role for Mcl-1 in mediating synergism between ABT-737 and YM-155. As with YM-155 alone, sensitivity to YM-155 and ABT-737 inversely correlated with EGFR activation status. However, sensitivity could be restored in highly resistant U87-EGFRvIII cells by inhibition of EGFR or its downstream pathways, highlighting the impact of EGFR signaling on Mcl-1 expression and the relevance of combined targeted therapies to overcome the multiple resistance mechanisms of these aggressive tumors.

Topics: Apoptosis; bcl-X Protein; Biphenyl Compounds; Brain Neoplasms; Cell Line, Tumor; Down-Regulation; Drug Resistance, Neoplasm; ErbB Receptors; Gene Expression Regulation, Neoplastic; Glioma; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Microtubule-Associated Proteins; Myeloid Cell Leukemia Sequence 1 Protein; Naphthoquinones; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Sulfonamides; Survivin