Bcl-XL but Not Bcl-2 Is a Potential Target in Medulloblastoma Therapy

Medulloblastoma (MB) is the most common solid tumour in children and, despite current treatment with a rather aggressive combination therapy, accounts for 10% of all deaths associated with paediatric cancer. Breaking the tumour cells’ intrinsic resistance to therapy-induced cell death should lead to less aggressive and more effective treatment options. In other tumour entities, this has been achieved by modulating the balance between the various pro- and anti-apoptotic members of the Bcl-2 family with small molecule inhibitors. To evaluate the therapeutic benefits of ABT-199 (Venetoclax), a Bcl-2 inhibitor, and ABT-263 (Navitoclax), a dual Bcl-XL/Bcl-2 inhibitor, increasingly more relevant model systems were investigated. Starting from established MB cell lines, progressing to primary patient-derived material and finally an experimental tumour system imbedded in an organic environment were chosen. Assessment of the metabolic activity (a surrogate readout for population viability), the induction of DNA fragmentation (apoptosis) and changes in cell number (the combined effect of alterations in proliferation and cell death induction) revealed that ABT-263, but not ABT-199, is a promising candidate for combination therapy, synergizing with cell death-inducing stimuli. Interestingly, in the experimental tumour setting, the sensitizing effect of ABT-263 seems to be predominantly mediated via an anti-proliferative and not a pro-apoptotic effect, opening a future line of investigation. Our data show that modulation of specific members of the Bcl-2 family might be a promising therapeutic addition for the treatment of MB.

The AAA+ ATPase RavA-ViaA complex sensitizes Escherichia coli to aminoglycosides under anaerobic low energy conservation conditions

Aminoglycosides have been used against Gram-negative bacteria for decades. Yet, uncertainties remain about various aspects of their uptake mechanism. Moreover their killing efficiency is well known to vary as a function of growth conditions and types of metabolism used by the targeted bacterium. Here we show that RavA, an AAA+ ATPase from the MoxR subfamily, associated with its VWA-containing partner, ViaA sensitize E. coli to lethal concentrations of AG, including gentamycin (Gm) and tobramycin, but not of antibiotics of other classes. We show this sensitizing effect to be due to enhanced Gm uptake in a proton motive force dependent manner. We evaluated the influence of RavA ViaA throughout a series of growth conditions, including aerobiosis and anaerobiosis. This led us to observe that the sensitizing effect of RavA ViaA varies with the respiratory chain used, i.e. RavA ViaA influence was prominent in the absence of exogenous electron acceptor or with fumarate, i.e. in poor energy conservation conditions, and dispensable in the presence of nitrate or oxygen, i.e. in high level of energy conservation. We propose RavA ViaA to be able to sense energetic state of the cell and to be used under low energy conditions for facilitating uptake of chemicals across the membrane, including Gm.

Perhexiline Therapy in Patients with Type 2 Diabetes: Dissociation Between Potentiation of Nitric Oxide Signalling and Changes in Insulin Resistance

Purpose: Perhexiline (Px) has previously been utilized primarily in the treatment of otherwise refractory angina pectoris and/or systolic heart failure. In recent years, Px has also shown increasing promise as a potential chemotherapeutic agent. Px inhibits carnitine palmitoyltransferases 1 and 2 (CPT1, CPT2), which control uptake of long-chain fatty acids into mitochondria and thus represent the rate-limiting steps in their metabolism. However, occasional cases of hypoglycaemia have been reported in Px-treated patients, raising the possibility that Px may also increase sensitivity to insulin. Furthermore, Px increases anti-aggregatory responses to nitric oxide (NO), an effect which may parallel insulin sensitization. No previous studies have examined either the effect of Px on insulin sensitivity, or the relationship of such putative changes with effects on NO signalling. We therefore sought to examine these relationships in patients with stable T2D and cardiovascular disease.Methods: In 30 patients with concomitant T2D and cardiovascular disease, Px was initiated, and dosage was titrated to reach therapeutic range and to prevent toxicity. Investigations were performed before and after 2 weeks, to examine changes in insulin sensitivity, platelet responsiveness to the anti-aggregatory effects of the NO donor sodium nitroprusside (SNP), as well as other markers of inflammation and modulators of NO signaling.Results: Px substantially potentiated inhibition of ADP-induced platelet aggregation by SNP (from 16.7±3.0 to 27.3±3.7%; p=0.005). Px did not change fasting blood glucose concentrations and reduced insulin sensitivity (HOMA-IR score increased from median of 4.47 to 6.08; p=0.028), via increasing fasting plasma insulin concentrations (median 16.5 to 19.0 mU/L: p=0.014). Increases in SNP responses tended (r=-0.30; p=0.11) to be reciprocally related to increases in HOMA-IR. No patient developed symptomatic hypoglycaemia, nor was there any other short-term toxicity of Px.Conclusions: In patients with stable T2D and cardiovascular disease, Px is not an insulin sensitizer, and does not normally induce hypoglycaemia. These results effectively dissociate the NO-sensitizing effect of Px from variability in insulin signaling.

Evaluation of the sensitizing effect of a disinfectant based on glutaraldehyde upon inhalation

When investigating new disinfectants, it is necessary to assess the degree of their danger by modeling the conditions of use. As is known, the main danger of disinfectants, especially volatiles, is posed by the inhalation route of entry into the body, exerting an irritating effect on the respiratory organs, as well as a general toxic and sensitizing effect. Despite reasonable restrictions on the use of disinfectants from the aldehyde class for decontamination of the surfaces of premises and equipment, state registration receives disinfectants based on glutaraldehyde (GA), intended for wide use in various areas, including everyday life, which is unacceptable. Experiments were carried out to confirm the danger of using GA-based disinfectants. Sensitizing effect of GA-based disinfectant under conditions of surface treatment by rubbing method is studied. Significant release of GA into the air after surface treatment (27.7±4.1 mg/m3) was detected. The concentration of GA in the air decreased by 4.5 times after 6 hours of ventilation. The sensitizing effect of the GA-based agent was determined on guinea pigs by a provocative ear swelling test and a leukocyte specific lysis reaction. The results of the study indicate a pronounced sensitizing effect of the agent. Keywords: glutaraldehyde, toxicity, disinfectants, disinfection, sensitization, inhalation, guinea pigs.

Resolution capability of resist patterns and throughput using alkaline treatment under ultrasonic irradiation

Alkaline treatment of the photoresist under ultrasonic irradiation has been investigated to improve the resolution capability of resist patterns with higher throughput. The selectively dissolved phenol resin for the combination of the alkaline treatment with ultrasonic irradiation was increased by 2.3 times compared to the solely alkaline treatment. The sensitizing effect of naphthoquinone diazide (sensitizer) based on phenol was increased to 0.46 against dip treatment of 0.31. As a result, resist sensitivity was increased to 26% and the resolution capability was drastically improved. Consequently, the 0.5 μm line and space resist patterns were resolved completely with fine profile by using the photoresist with a 0.7 μm resolution limit together with g-line exposure machine with a 0.6 μm resolution limit. As a consequence, a high throughput of 25 wafers/min was achieved, which was more than 25 times higher than that of electron beam (EB) lithography.

Proteotoxic Stress as an Exploitable Vulnerability in Cells with Hyperactive AKT

Hyperactivity of serine-threonine kinase AKT is one of the most common molecular abnormalities in cancer, where it contributes to poor outcomes by facilitating the growth and survival of malignant cells. Despite its well-documented anti-apoptotic effects, hyperactivity of AKT is also known to be stressful to a cell. In an attempt to better elucidate this phenomenon, we observed the signs of proteotoxic stress in cells that harbor hyperactive AKT or have lost its principal negative regulator, PTEN. The activity of HSF1 was predictably elevated under these circumstances. However, such cells proved more sensitive to various regimens of heat shock, including the conditions that were well-tolerated by syngeneic cells without AKT hyperactivity. The sensitizing effect of hyperactive AKT was also seen in HSF1-deficient cells, suggesting that the phenomenon does not require the regulation of HSF1 by this kinase. Notably, the elevated activity of AKT was accompanied by increased levels of XBP1, a key component of cell defense against proteotoxic stress. Interestingly, the cells harboring hyperactive AKT were also more dependent on XBP1 for their growth. Our observations suggest that proteotoxic stress conferred by hyperactive AKT represents a targetable vulnerability, which can be exploited by either elevating the stress above the level tolerated by such cells or by eliminating the factors that enable such tolerance.

Cytotoxic activity of a unique monomeric heterogeneous two-coordinate ligand monovalent gold complex with tiopronin and a heterocyclic mercapto-tetrazole compound

Background: Importance of the role of NF-κB is recognized in situations such as malignant transformation and metastasis of cancer, and it has been suggested that inhibiting this role can be one of the cancer treatment strategies. Gold preparations such as auranofin are known to have an indirect NF-κB inhibitory effect. Objective: We synthesized a novel gold complex [tiopronin monovalent gold-5-mercapto-1-methyltetrazole, abbreviated as TPN-Au(Ⅰ)-MM4], with different physical properties and chemical structure from auranofin, and evaluated its cytotoxic activity and radiation sensitizing effect on human THP1 cells. Methods: The number of viable cells was counted by the trypan blue dye exclusion method. The cell death evaluation was performed by FITC-Annexin V+ and PI staining. In investigating the radiation sensitizing effect of TPN-Au(Ⅰ)-MM4, this compound [10 or 25 μM] was added into the culture medium 1 h before X-ray irradiation. Results: In the cells treated with 25 μM TPN-Au(Ⅰ)-MM4 for 72 h, where a decrease in the proliferation of THP1 cells were observed [The relative values of viable cells in the control group and the 25 μM treatment group were approximately 6.8 and 4.2, respectively]. In the combination of 25 μM of the compound treatment and X-ray irradiation, an increase of approximately 3.0-fold was observed in 2 Gy irradiation, and approximately 1.4-fold in 4 Gy irradiation as in comparison to the case of irradiation alone. Conclusion: These results suggest that TPN-Au(Ⅰ)-MM4 reduces the proliferation of THP1 cells through the induction of cell death, and the combined use of TPN-Au(Ⅰ)-MM4 and X-ray irradiation shows effective cytotoxicity against THP1 cells.

Identification of Novel Anthracycline Resistance Genes and Their Inhibitors

Differentially expressed genes have been previously identified by us in multidrug-resistant tumor cells mainly resistant to doxorubicin. In the present study, we exemplarily focused on some of these genes to investigate their causative relationship with drug resistance. HMOX1, NEIL2, and PRKCA were overexpressed by lentiviral-plasmid-based transfection of HEK293 cells. An in silico drug repurposing approach was applied using virtual screening and molecular docking of FDA-approved drugs to identify inhibitors of these new drug-resistant genes. Overexpression of the selected genes conferred resistance to doxorubicin and daunorubicin but not to vincristine, docetaxel, and cisplatin, indicating the involvement of these genes in resistance to anthracyclines but not to a broader MDR phenotype. Using virtual drug screening and molecular docking analyses, we identified FDA-approved compounds (conivaptan, bexarotene, and desloratadine) that were interacting with HMOX1 and PRKCA at even stronger binding affinities than 1-(adamantan-1-yl)-2-(1H-imidazol-1-yl)ethenone and ellagic acid as known inhibitors of HMOX1 and PRKCA, respectively. Conivaptan treatment increased doxorubicin sensitivity of both HMOX1- and PRKCA-transfected cell lines. Bexarotene treatment had a comparable doxorubicin-sensitizing effect in HMOX1-transfected cells and desloratadine in PRKCA-transfected cells. Novel drug resistance mechanisms independent of ABC transporters have been identified that contribute to anthracycline resistance in MDR cells.