Photodynamic Therapy Combined with Bcl-2/Bcl-xL Inhibition Increases the Noxa/Mcl-1 Ratio Independent of Usp9X and Synergistically Enhances Apoptosis in Glioblastoma.

The purpose of this study was to assess in vitro whether the biological effects of 5-aminolevulinic acid (5-ALA)-based photodynamic therapy are enhanced by inhibition of the anti-apoptotic Bcl-2 family proteins Bcl-2 and Bcl-xL in different glioblastoma models. Pre-clinical testing of a microcontroller-based device emitting light of 405 nm wavelength in combination with exposure to 5-ALA (PDT) and the Bcl-2/Bcl-xL inhibitor ABT-263 (navitoclax) was performed in human established and primary cultured glioblastoma cells as well as glioma stem-like cells. We applied cell count analyses to assess cellular proliferation and Annexin V/PI staining to examine pro-apoptotic effects. Western blot analyses and specific knockdown experiments using siRNA were used to examine molecular mechanisms of action. Bcl-2/Bcl-xL inhibition synergistically enhanced apoptosis in combination with PDT. This effect was caspase-dependent. On the molecular level, PDT caused an increased Noxa/Mcl-1 ratio, which was even more pronounced when combined with ABT-263 in a Usp9X-independent manner. Our data showed that Bcl-2/Bcl-xL inhibition increases the response of glioblastoma cells toward photodynamic therapy. This effect can be partly attributed to cytotoxicity and is likely related to a pro-apoptotic shift because of an increased Noxa/Mcl-1 ratio. The results of this study warrant further investigation.

Current state and future perspective of drug repurposing in malignant glioma.

Malignant gliomas are still extremely difficult to treat because complete surgical resection is biologically not feasible due to the invasive nature of these diseases and the proximity of tumors to functionally sensitive areas. Moreover, adjuvant therapies are facing a strong therapeutic resistance since the central nervous system is a highly protected environment and the tumor cells display a vast intra-tumoral genetic and epigenetic variation. As a consequence, new therapeutics are urgently needed but the process of developing novel compounds that finally reach clinical application is highly time-consuming and expensive. Drug repurposing is an approach to facilitate and accelerate the discovery of new cancer treatments. In malignant glioma, like in other cancers, pre-existing physiological pathways that regulate cell growth, cell death or cell migration are dysregulated causing malignant transformation. A wide variety of drugs are clinically used to treat non-cancerous diseases interfering with these malignancy-associated pathways. Repurposed drugs have key advantages: They already have approval for clinical use by national regulatory authorities. Moreover, they are for the most part inexpensive and their side effect and safety profiles are well characterized. In this work, we provide an overview on current repurposing strategies for the treatment of malignant glioma.

Dual metabolic reprogramming by ONC201/TIC10 and 2-Deoxyglucose induces energy depletion and synergistic anti-cancer activity in glioblastoma.

BACKGROUND: Dysregulation of the metabolome is a hallmark of primary brain malignancies. In this work we examined whether metabolic reprogramming through a multi-targeting approach causes enhanced anti-cancer activity in glioblastoma. METHODS: Preclinical testing of a combined treatment with ONC201/TIC10 and 2-Deoxyglucose was performed in established and primary-cultured glioblastoma cells. Extracellular flux analysis was used to determine real-time effects on OXPHOS and glycolysis. Respiratory chain complexes were analysed by western blotting. Biological effects on tumour formation were tested on the chorioallantoic membrane (CAM). RESULTS: ONC201/TIC10 impairs mitochondrial respiration accompanied by an increase of glycolysis. When combined with 2-Deoxyglucose, ONC201/TIC10 induces a state of energy depletion as outlined by a significant decrease in ATP levels and a hypo-phosphorylative state. As a result, synergistic anti-proliferative and anti-migratory effects were observed among a broad panel of different glioblastoma cells. In addition, this combinatorial approach significantly impaired tumour formation on the CAM. CONCLUSION: Treatment with ONC201/TIC10 and 2-Deoxyglucose results in a dual metabolic reprogramming of glioblastoma cells resulting in a synergistic anti-neoplastic activity. Given, that both agents penetrate the blood-brain barrier and have been used in clinical trials with a good safety profile warrants further clinical evaluation of this therapeutic strategy.

Bcl-2/Bcl-xL inhibition predominantly synergistically enhances the anti-neoplastic activity of a low-dose CUSP9 repurposed drug regime against glioblastoma.

BACKGROUND AND PURPOSE: Drug repurposing represents a promising approach to safely accelerate the clinical application of therapeutics with anti-cancer activity. In this study, we examined whether inhibition of the anti-apoptotic Bcl-2 family proteins Bcl-2 and Bcl-xL enhances the biological effects of the repurposed CUSP9 regimen in an in vitro setting of glioblastoma. EXPERIMENTAL APPROACH: We applied 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assays to assess cellular proliferation. Annexin V/propidium iodide and tetramethylrhodamine, ethyl ester staining were used to examine apoptosis. Western blotting, RT-PCR, and specific knockdown experiments using siRNA were employed to examine molecular mechanisms of action. KEY RESULTS: Bcl-2/Bcl-xL inhibition exerted synergistic anti-proliferative effects across established, primary cultured, and stem-like glioblastoma cells when combined with CUSP9 which had been reduced to only one tenth of its proposed original concentration (CUSP9-LD). The combination treatment also led to enhanced apoptosis with loss of mitochondrial membrane potential and activation of caspases. On the molecular level, CUSP9-LD counteracted ABT263-mediated up-regulation of Mcl-1. Silencing of Mcl-1 enhanced ABT263-mediated apoptosis which indicates that down-regulation of Mcl-1 is crucial for the induction of cell death by the combination treatment. CONCLUSION AND IMPLICATIONS: These data suggest that Bcl-2/Bcl-xL inhibition enhances the susceptibility of glioblastoma cells towards CUSP9, allowing dramatic dose reduction and potentially decreased toxicity when applied clinically. A clinical trial involving the original CUSP doses (CUSP9v3) is currently ongoing in our institution (NCT02770378). The Bcl-2/Bcl-xL inhibitor ABT263 is in clinical trials and might represent a valuable adjunct to the original CUSP.

Mitochondrial matrix chaperone and c-myc inhibition causes enhanced lethality in glioblastoma.

Malignant gliomas display high levels of the transcription factor c-myc and organize a tumor specific chaperone network within mitochondria. Here, we show that c-myc along with mitochondrial chaperone inhibition displays massive tumor cell death. Inhibition of mitochondrial matrix chaperones and c-myc was established by utilizing genetic as well as pharmacological approaches. Bromodomain and extraterminal (BET) family protein inhibitors, JQ1 and OTX015, were used for c-myc inhibition. Gamitrinib was applied to interfere with mitochondrial matrix chaperones. A xenograft model was used to determine the in vivo efficacy. Combined inhibition of c-myc and mitochondrial matrix chaperones led to a synergistic reduction of cellular proliferation (CI values less than 1) in established glioblastoma, patient-derived xenograft and stem cell-like glioma cultures. The combinatorial treatment of BET inhibitors and Gamitrinib elicited massive apoptosis induction with dissipation of mitochondrial membrane potential and activation of caspases. Mechanistically, BET-inhibitors and Gamitrinib mediated a pronounced integrated stress response with a PERK-dependent up regulation of ATF4 and subsequent modulation of Bcl-2 family of proteins with down-regulation of Mcl-1 and its interacting partner, Usp9X, and an increase in pro-apoptotic Noxa. Blocking ATF4 by siRNA attenuated Gamitrinib/BET inhibitor mediated increase of Noxa. Knockdown of Noxa and Bak protected from the combinatorial treatment. Finally, the combination treatment of Gamitrinib and OTX015 led to a significantly stronger reduction of tumor growth as compared to single treatments in a xenograft model of human glioma without induction of toxicity. Thus, Gamitrinib in combination with BET-inhibitors should be considered for the development for clinical application.

Broad targeting of resistance to apoptosis in cancer.

Apoptosis or programmed cell death is natural way of removing aged cells from the body. Most of the anti-cancer therapies trigger apoptosis induction and related cell death networks to eliminate malignant cells. However, in cancer, de-regulated apoptotic signaling, particularly the activation of an anti-apoptotic systems, allows cancer cells to escape this program leading to uncontrolled proliferation resulting in tumor survival, therapeutic resistance and recurrence of cancer. This resistance is a complicated phenomenon that emanates from the interactions of various molecules and signaling pathways. In this comprehensive review we discuss the various factors contributing to apoptosis resistance in cancers. The key resistance targets that are discussed include (1) Bcl-2 and Mcl-1 proteins; (2) autophagy processes; (3) necrosis and necroptosis; (4) heat shock protein signaling; (5) the proteasome pathway; (6) epigenetic mechanisms; and (7) aberrant nuclear export signaling. The shortcomings of current therapeutic modalities are highlighted and a broad spectrum strategy using approaches including (a) gossypol; (b) epigallocatechin-3-gallate; (c) UMI-77 (d) triptolide and (e) selinexor that can be used to overcome cell death resistance is presented. This review provides a roadmap for the design of successful anti-cancer strategies that overcome resistance to apoptosis for better therapeutic outcome in patients with cancer.

Olanzapine inhibits proliferation, migration and anchorage-independent growth in human glioblastoma cell lines and enhances temozolomide's antiproliferative effect.

The poor prognosis of patients with glioblastoma fuels the search for more effective therapeutic compounds. We previously hypothesised that the neuroleptic olanzapine may enhance antineoplastic effects of temozolomide the standard chemotherapeutic agent used in this disease. This study tested this hypothesis. The anti-proliferative effect of olanzapine was examined by MTT assays and cell count analysis. Soft-agar assays were performed to examine colony-forming ability. In addition, the inhibitory effect of olanzapine on the migratory capacity of U87MG and A172 cells was analyzed by Transwell(®) assays. Moreover, staining for annexin V/propidium iodide or carboxyfluorescein succinimidyl ester was performed prior to flow cytometric analysis in order to better understand the subjacent cellular mechanism. Our initial hypothesis that olanzapine may enhance temozolomide's anti-tumor activity could be confirmed in U87MG and A172 glioblastoma cell lines. Moreover, treatment with olanzapine alone resulted in a marked anti-proliferative effect on U87MG, A172 and two glioma stem-like cells with IC50 values ranging from 25 to 79.9 µM. In U87MG cells, anchorage-independent growth was dose-dependently inhibited. In A172 cells, migration was also shown to be inhibited in a dose-dependent manner. In addition, olanzapine was shown to exert a cell line-dependent pleomorphism with respect to the induction of apoptosis, necrosis and/or cytostasis. Our data show that the neuroleptic olanzapine enhances the anti-tumor activity of temozolomide against glioblastoma cell lines. Moreover, this is the first study to show that olanzapine provides on its own anti-cancer activity in glioblastoma and thus may have potential for repurposing.

Utilization of the cellular stress response to sensitize cancer cells to TRAIL-mediated apoptosis.

INTRODUCTION: Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is a promising death ligand who has received significant attention due to its specific anti-cancer activity. Recently, a number of clinical trials involving either recombinant soluble TRAIL or agonistic death receptor (DR) antibodies have even been initiated. One major caveat in TRAIL-based anti-cancer therapies is that a considerable number of cancer cells are notorious resistant to apoptosis induction by TRAIL. Overcoming this primary or secondary evolved resistance is an utmost important goal of present cancer research. The current literature suggests that TRAIL resistance is mediated by a number of endogenous factors. AREAS COVERED: According to recent research, stress-related transcription factors have acquired a pivotal role in the sensitization of highly resistant cancer cells, for example, pancreatic cancer and glioblastoma cells, to TRAIL-mediated cell death. Out of this transcription factor family, C/EBP-homologous protein (CHOP) is linked to the control of DR-mediated apoptosis by modulation of several apoptotic and anti-apoptotic factors. Stress responses in certain organelles, such as endoplasmic reticulum (ER) and mitochondria, are potent inductors of CHOP expression. This report focuses on the influence of stress responses on endogenous or acquired resistance to extrinsic apoptosis in tumor cells and summarizes recent findings and results. The Medline and ClinicalTrials database with key words were used for this review. EXPERT OPINION: A potential novel treatment strategy for highly treatment-resistant tumors is the induction of a cellular stress response in cancer cells. The induction of an organelle-related stress response, such as nuclear, ER and mitochondrial stress, leads to a dramatic sensitization of a broad variety of cancer cells of different tumor entities to the apoptotic ligand, TRAIL. Importantly, non-neoplastic cells are not sensitized to TRAIL-mediated cell death through the unfolded protein response in most instances, suggesting that this treatment is not only of high efficacy, but even more less of unwanted toxicity in patients.

KAAD-cyclopamine augmented TRAIL-mediated apoptosis in malignant glioma cells by modulating the intrinsic and extrinsic apoptotic pathway.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic. The main obstacle in TRAIL-based therapy is that many glioma cells are resistant. In this study glioblastoma cell lines, human glioblastoma short-term cultures and human astrocytes were treated with 3-keto-N-aminoethylaminoethylcaproyldihydrocinnamoyl cyclopamine (KAAD-cyclopamine), tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) or the combination of both. Single treatment with KAAD-cyclopamine or TRAIL does not induce cytotoxicity in malignant glioma cells. However, treatment with KAAD-cyclopamine in combination with TRAIL induces rapid apoptosis in TRAIL-resistant glioma cells. Notably, normal human astrocytes were not affected by the combination treatment consisting of KAAD-cyclopamine and TRAIL. KAAD-cyclopamine led to an upregulation of death receptor 4 and 5 and down-regulation of bcl-2 and c-FLIP. Furthermore, overexpression of both bcl-2 and c-FLIP attenuated KAAD-cyclopamine facilitated TRAIL-mediated apoptosis. Taken together,we provided evidence that KAAD-cyclopamine facilitated TRAIL-mediated apoptosis at the level of the intrinsic and extrinsic apoptotic pathways in malignant glioma cells.

Genistein enhances proteasomal degradation of the short isoform of FLIP in malignant glioma cells and thereby augments TRAIL-mediated apoptosis.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer drug. One obstacle in TRAIL-based therapies is that many cancer cells, including gliomas, are resistant towards TRAIL. In this study one glioblastoma cell line, one human short-term glioblastoma culture and human astrocytes were treated with genistein, tumour necrosis factor-related apoptosis-inducing ligand or the combination of both. Single treatment with genistein or TRAIL does not induce cytotoxicity in malignant glioma cells. However, treatment with genistein in combination with TRAIL induces rapid apoptosis in TRAIL-resistant glioma cells. Notably, normal human astrocytes were not affected by the combination treatment consisting of genistein and TRAIL. Genistein enhanced proteasomal degradation of the short isoform of c-FLIP. Importantly, over-expression of only the short isoform of c-FLIP attenuated genistein TRAIL-mediated cytotoxicity. Taken together, we gave evidence that genistein facilitated TRAIL-mediated apoptosis at the level of the extrinsic apoptotic pathways in malignant glioma cells.

Stem-cell-like glioma cells are resistant to TRAIL/Apo2L and exhibit down-regulation of caspase-8 by promoter methylation.

Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L) is a promising cancer drug. However, many tumours are resistant to TRAIL-based therapies. Glioma cells with stem cell features (SCG), such as CD133 expression and neurosphere formation, have been recently identified to be more resistant to cytotoxic drugs than glioma cells lacking stem-cell-like features (NSCGs). Here we report that SCGs are completely resistant to 100-2,000 ng/ml TRAIL, whereas NSCGs revealed a moderate sensitivity to TRAIL. We found that SCGs exhibited only low levels of caspase-8 mRNA and protein, known to be indispensable for TRAIL-induced apoptosis. In addition, we detected hypermethylation of CASP8 promoter in SCGs, whereas NSCGs exhibited a non-methylated CASP8 promoter. Reexpression of caspase-8 by 5-Aza-2'-deoxycytidine was not sufficient to restore TRAIL sensitivity in SCGs cells, suggesting that additional factors cause TRAIL resistance in SCGs. Our data suggest that therapy with TRAIL, either as monotherapy or in combination with demethylating agents, is not effective in treating glioblastoma because SCGs are not targeted by such treatment.

17-AAG sensitized malignant glioma cells to death-receptor mediated apoptosis.

17-AAG is a selective HSP90-inhibitor that exhibited therapeutic activity in cancer. In this study three glioblastoma cell lines (U87, LN229 and U251) were treated with 17-AAG, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the combination of both. Treatment with subtoxic doses of 17-AAG in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces rapid apoptosis in TRAIL-resistant glioma cells, suggesting that this combined treatment may offer an attractive strategy for treating gliomas. 17-AAG treatment down-regulated survivin through proteasomal degradation. In addition, over-expression of survivin attenuated cytotoxicity induced by the combination of 17-AAG and TRAIL. In summary, survivin is a key regulator of TRAIL-17-AAG mediated cell death in malignant glioma.