Bortezomib administered prior to temozolomide depletes MGMT, chemosensitizes glioblastoma with unmethylated MGMT promoter and prolongs animal survival.

BACKGROUND: Resistance to temozolomide (TMZ) is due in part to enhanced DNA repair mediated by high expression of O6-methyl guanine DNA methyltransferase (MGMT) that is often characterised by unmethylated promoter. Here, we investigated pre-treatment of glioblastoma (GBM) cells with the 26S-proteasome inhibitor bortezomib (BTZ) as a strategy to interfere with MGMT expression and thus sensitise them to TMZ. METHODS: Cell lines and patient GBM-derived cells were examined in vitro, and the latter also implanted orthotopically into NOD-SCID C.B.-Igh-1b/lcrTac-Prkdc mice to assess efficacy and tolerability of BTZ and TMZ combination therapy. MGMT promoter methylation was determined using pyrosequencing and PCR, protein signalling utilised western blotting while drug biodistribution was examined by LC-MS/MS. Statistical analysis utilised Analysis of variance and the Kaplan-Meier method. RESULTS: Pre-treatment with BTZ prior to temozolomide killed chemoresistant GBM cells with unmethylated MGMT promoter through MGMT mRNA and protein depletion in vitro without affecting methylation. Chymotryptic activity was abolished, processing of NFkB/p65 to activated forms was reduced and corresponded with low MGMT levels. BTZ crossed the blood-brain barrier, diminished proteasome activity and significantly prolonged animal survival. CONCLUSION: BTZ chemosensitized resistant GBM cells, and the schedule may be amenable for temozolomide refractory patients with unmethylated MGMT promoter.

The Styryl Benzoic Acid Derivative DC10 Potentiates Radiotherapy by Targeting the xCT-Glutathione Axis.

Metastatic tumors with moderate radiosensitivity account for most cancer-related deaths, highlighting the limitations of current radiotherapy regimens. The xCT-inhibitor sulfasalazine (SAS) sensitizes cancer cells to radiotherapy by blocking cystine uptake via the xCT membrane antiporter, and thereby glutathione (GSH) synthesis protecting against radiation-induced oxidative stress. The expression of xCT in multiple tumor types implies it as a target generic to cancer rather than confined to few subtypes. However, SAS has limited clinical potential as a radiosensitizer due to side effects and low bioavailability. Using SAS as a starting point, we previously developed synthetic xCT-inhibitors through scaffold hopping and structure optimization aided by structure-activity relationship analysis (SAR). Notably, the compound DC10 exhibited inhibition of GSH synthesis. In this study, we validated DC10 as a radiosensitizer in the xCT-expressing cancer cell lines A172, A375 and MCF7, and mice harboring melanoma xenografts. After DC10 treatment, we measured 14C-cystine uptake in the cancer cells using liquid scintillation counting, and intracellular GSH levels and reactive oxygen species (ROS) using luminescence assays. We performed immunoblotting of H2AX and ATM to assess DNA damage after treatment with DC10 and radiotherapy. We then assessed the effect of adding DC10 to radiation upon cancer cell colony formation. Blood samples from mice treated with DC10 underwent biochemical analysis to assess toxicity. Finally, mice with A375 melanomas in the flank, received DC10 and radiotherapy in combination, as monotherapies or no treatment. Notably, DC10 reduced cystine uptake and GSH synthesis and increased ROS levels in a dose-dependent manner. Furthermore, DC10 interacted synergistically with radiation to increase DNA damage and reduce tumor cell colony formation. Mice receiving DC10 were clinically unaffected, whereas blood samples analysis to assess bone marrow suppression, liver or kidney toxicity revealed no significant differences between treated mice and untreated controls. Importantly, DC10 potentiated the anti-tumor efficacy of radiation in mice with melanoma xenografts. We conclude that DC10 is well tolerated and acts as a radiosensitizer by inhibiting cystine uptake, leading to GSH depletion and increased oxidative stress. Our findings demonstrate the feasibility of using synthetic xCT-inhibitors to overcome radioresistance.

Bortezomib abrogates temozolomide-induced autophagic flux through an ATG5 dependent pathway.

Introduction: Glioblastoma (GBM) is invariably resistant to temozolomide (TMZ) chemotherapy. Inhibiting the proteasomal pathway is an emerging strategy to accumulate damaged proteins and inhibit their lysosomal degradation. We hypothesized that pre-treatment of glioblastoma with bortezomib (BTZ) might sensitize glioblastoma to temozolomide by abolishing autophagy survival signals to augment DNA damage and apoptosis. Methods: P3 patient-derived glioblastoma cells, as well as the tumour cell lines U87, HF66, A172, and T98G were investigated for clonogenic survival after single or combined treatment with temozolomide and bortezomib in vitro. We investigated the requirement of functional autophagy machinery by utilizing pharmacological inhibitors or CRISPR-Cas9 knockout (KO) of autophagy-related genes -5 and -7 (ATG5 and ATG7) in glioblastoma cells and monitored changes in autophagic flux after temozolomide and/or bortezomib treatments. P3 wild-type and P3 ATG5-/- (ATG5 KO) cells were implanted orthotopically into NOD-SCID mice to assess the efficacy of bortezomib and temozolomide combination therapy with and without functional autophagy machinery. Results: The chemo-resistant glioblastoma cells increased autophagic flux during temozolomide treatment as indicated by increased degradation of long-lived proteins, diminished expression of autophagy markers LC3A/B-II and p62 (SQSTM1), increased co-localisation of LC3A/B-II with STX17, augmented and no induction of apoptosis. In contrast, bortezomib treatment abrogated autophagic flux indicated by the accumulation of LC3A/B-II and p62 (SQSTM1) positive autophagosomes that did not fuse with lysosomes and thus reduced the degradation of long-lived proteins. Bortezomib synergistically enhanced temozolomide efficacy by attenuating cell proliferation, increased DNA double-strand breaks, and apoptosis in an autophagy-dependent manner. Abolishing autophagy in ATG5 KOs reversed the bortezomib-induced toxicity, rescued glioblastoma cell death and reduced animal survival. Discussion: We conclude that bortezomib abrogates temozolomide induced autophagy flux through an ATG5 dependent pathway.

Structure-Activity-Relationship-Aided Design and Synthesis of xCT Antiporter Inhibitors.

The xCT antiporter is a cell membrane protein involved in active counter-transportation of glutamate (outflux) with cystine (influx) over the human cell membrane. This feature makes the xCT antiporter a crucial element of the biosynthesis of the vital free radical scavenger glutathione. The prodrug sulfasalazine, a medication for the treatment of ulcerative colitis, was previously proven to inhibit the xCT antiporter. Starting from sulfasalazine, a molecular scaffold jumping followed by SAR-assisted design and synthesis provided a series of styryl hydroxy-benzoic acid analogues that were biologically tested in vitro for their ability to decrease intracellular glutathione levels using four different cancer cell lines: A172 (glioma), A375 (melanoma), U87 (glioma) and MCF7 (breast carcinoma). Depletion of glutathione levels varied among the compounds as well as among the cell lines. Flow cytometry using propidium iodide and the annexin V marker demonstrated minimal toxicity in normal human astrocytes for a promising candidate molecule (E)-5-(2-([1,1'-biphenyl]-4-yl)vinyl)-2-hydroxybenzoic acid.

Meclofenamate causes loss of cellular tethering and decoupling of functional networks in glioblastoma.

BACKGROUND: Glioblastoma cells assemble to a syncytial communicating network based on tumor microtubes (TMs) as ultra-long membrane protrusions. The relationship between network architecture and transcriptional profile remains poorly investigated. Drugs that interfere with this syncytial connectivity such as meclofenamate (MFA) may be highly attractive for glioblastoma therapy. METHODS: In a human neocortical slice model using glioblastoma cell populations of different transcriptional signatures, three-dimensional tumor networks were reconstructed, and TM-based intercellular connectivity was mapped on the basis of two-photon imaging data. MFA was used to modulate morphological and functional connectivity; downstream effects of MFA treatment were investigated by RNA sequencing and fluorescence-activated cell sorting (FACS) analysis. RESULTS: TM-based network morphology strongly differed between the transcriptional cellular subtypes of glioblastoma and was dependent on axon guidance molecule expression. MFA revealed both a functional and morphological demolishment of glioblastoma network architectures which was reflected by a reduction of TM-mediated intercellular cytosolic traffic as well as a breakdown of TM length. RNA sequencing confirmed a downregulation of NCAM and axon guidance molecule signaling upon MFA treatment. Loss of glioblastoma communicating networks was accompanied by a failure in the upregulation of genes that are required for DNA repair in response to temozolomide (TMZ) treatment and culminated in profound treatment response to TMZ-mediated toxicity. CONCLUSION: The capacity of TM formation reflects transcriptional cellular heterogeneity. MFA effectively demolishes functional and morphological TM-based syncytial network architectures. These findings might pave the way to a clinical implementation of MFA as a TM-targeted therapeutic approach.

COVID-19 and the environment: A critical review and research agenda.

The current Coronavirus infection (COVID-19) outbreak has had a substantial impact on many aspects of general life. Although a number of studies have been published on the topic already, there has not been a critical review of studies on the impacts of COVID-19 by and on environmental factors. The current study fills this gap by presenting a critical analysis of 57 studies on the nexus between COVID-19 and the environment, published in nine journals up to May 2020. Majority of the studies in our sample are published in Science of the Total Environment (74%), and studies used mostly descriptive statistics and regression as research methods. We identified four underlying research clusters based on a systematic content analysis of the studies. The clusters are: (1) COVID-19 and environmental degradation, (2) COVID-19 and air pollution, (3) COVID-19 and climate/metrological factors and (4) COVID-19 and temperature. Besides a critical analysis of the studies in each cluster, we propose research questions to guide future research on the relationship between COVID-19 and the environment.