Clobetasol propionate, a Nrf-2 inhibitor, sensitizes human lung cancer cells to radiation-induced killing via mitochondrial ROS-dependent ferroptosis.

Combining radiotherapy with Nrf-2 inhibitor holds promise as a potential therapeutic strategy for radioresistant lung cancer. Here, the radiosensitizing efficacy of a synthetic glucocorticoid clobetasol propionate (CP) in A549 human lung cancer cells was evaluated. CP exhibited potent radiosensitization in lung cancer cells via inhibition of Nrf-2 pathway, leading to elevation of oxidative stress. Transcriptomic studies revealed significant modulation of pathways related to ferroptosis, fatty acid and glutathione metabolism. Consistent with these findings, CP treatment followed by radiation exposure showed characteristic features of ferroptosis in terms of mitochondrial swelling, rupture and loss of cristae. Ferroptosis is a form of regulated cell death triggered by iron-dependent ROS accumulation and lipid peroxidation. In combination with radiation, CP showed enhanced iron release, mitochondrial ROS, and lipid peroxidation, indicating ferroptosis induction. Further, iron chelation, inhibition of lipid peroxidation or scavenging mitochondrial ROS prevented CP-mediated radiosensitization. Nrf-2 negatively regulates ferroptosis through upregulation of antioxidant defense and iron homeostasis. Interestingly, Nrf-2 overexpressing A549 cells were refractory to CP-mediated ferroptosis induction and radiosensitization. Thus, this study identified anti-psoriatic drug clobetasol propionate can be repurposed as a promising radiosensitizer for Keap-1 mutant lung cancers.

Withaferin-A alleviates acute graft versus host disease without compromising graft versus leukemia effect.

Acute graft versus host disease (aGvHD) contributes to a significant proportion of non-relapse mortality and morbidity in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT). Withaferin-A (WA), a phytomolecule obtained from Withania somnifera (Ashwagandha), is known to have anti-inflammatory, anti-proliferative and immunomodulatory properties. The efficacy of WA for the prevention and treatment of aGvHD was evaluated using a murine model of alloHSCT. Prophylactic administration of WA to mice mitigated the clinical symptoms of aGvHD and improved survival significantly compared to the GvHD control [HR = 0.07 (0.01-0.35); P < 0.001]. Furthermore, WA group had better overall survival compared to standard prophylactic regimen of CSA + MTX [HR = 0.19 (0.03-1.1), P < 0.05]. At the same time, WA did not compromise the beneficial GvL effect. In addition, WA administered to animals after the onset of aGvHD could reverse the clinical severity and improved survival, thus establishing its therapeutic potential. Our findings suggest that WA reduced the systemic levels of Th1, Th2 and Th17 inflammatory cytokine and increased the anti-inflammatory cytokine IL-10 levels significantly (P < 0.05). WA also inhibited lymphocytes migration to gut, liver, skin and lung and protected these organs from damage. Ex-vivo, WA inhibited proliferation of human peripheral blood mononuclear cells (hPBMCs), modulated immune cell phenotype and decreased cytokine release. In addition, WA inhibited pJAK2 and pSTAT3 protein levels in mouse splenocytes and hPBMCs. In conclusion, our study demonstrates the utility of WA for the prevention and treatment of aGvHD, which should be further evaluated in a clinical setting.

Safety, toxicity and pharmacokinetic assessment of oral Withaferin-A in mice.

Withaferin-A (WA) is the principle component of Withania somnifera (Ashwagandha). It has several biological activities including anti-cancer, anti-diabetic, neuroprotective, hepatoprotective and immune-modulatory properties. The acute and sub-acute toxicity of oral WA was investigated in mice. In the acute toxicity study, up to 2000 mg/kg of WA was well tolerated without any signs of toxicity or death. In the sub-acute toxicity study, mice were orally administered 10, 70 and 500 mg/kg of WA respectively, daily for 28 days. Upon physiological, serum chemistry, hematology and histopathogical examination, no features suggestive of drug-induced toxicity were observed at any dose levels, thereby confirming the No-Observed Adverse Effect Level (NOAEL) to be at least 500 mg/kg. Furthermore, the oral bioavailability of WA was evaluated using single intravenous and oral doses of 10 mg/kg and 70 mg/kg respectively using sparse sampling strategy. Bioanalysis was carried out using a validated LC-MS/MS method. The AUC of WA was found to be 3996.9 ± 557.6 ng/mL*h and 141.7 ± 16.8 ng/mL*h for the intravenous and oral routes of administration respectively. The oral bioavailability was determined to be 1.8%. To conclude, WA was found to be extremely safe even at high doses, with a low oral bioavailability.

Acute and sub-acute oral toxicity assessment of 5-hydroxy-1,4-naphthoquinone in mice.

5-hydroxy-1,4-naphthoquinone (5NQ) or juglone is a bioactive molecule found in walnuts and has shown therapeutic effects in various disease models. Limited information is available regarding the toxicity of 5NQ, thereby limiting the clinical development of this drug. In the present study, oral acute (50, 300 and 2000 mg/kg) and sub-acute toxicity (5, 15 and 50 mg/kg) was assessed in mice to evaluate the safety of 5NQ. The acute toxicity study identified 118 mg/kg as the point-of-departure dose (POD) for single oral administration of 5NQ using benchmark dose modeling (BMD). Repeated administration of 5NQ at doses of 15 and 50 mg/kg/day caused reduction in food consumption and body weight of mice along with alterations in liver and renal function. Histopathological assessment revealed significant damage to hepatic and renal tissues at all doses in the acute toxicity study, and at higher doses of 15 and 50 mg/kg in the sub-acute toxicity study. We observed dose dependent mortality in sub-acute toxicity study and the no observed adverse effect level (NOAEL) was established as < 5 mg/kg/day. Modeling the survival response in sub-acute toxicity study identified 1.74 mg/kg/day as the POD for repeated administration of 5NQ. Serum levels of aspartate aminotransferase (AST) were most sensitive to 5NQ administration with a lower limit of BMD interval (BMDL) of 1.1 × 10-3 mg/kg/day. The benchmark doses reported in the study can be further used to determine a reference dose of 5NQ for human risk assessment.

Comparison of different autoanalyzers for the determination of lymphocyte and neutrophil counts in mouse blood.

OBJECTIVES: Autoanalyzers are used in clinical haematology for analysis of blood samples in clinical as well as in nonclinical studies. The results from these analyzers vary from machine to machine. In this study, we compared the lymphocyte and neutrophil count of mouse blood between ADVIA 2120i, Horiba Yumizen H2500 and CellaVision analyzers against manual counting as gold standard. METHODS: Blood samples from 28 female BALB/c mice were collected and analyzed. Agreement between different autoanalyzers and manual counting were determined by Bland-Altman method. RESULTS: A high level of agreement was found between CellaVision and manual technique for lymphocyte (bias=4.75, 95% limits of agreement -14 to 24) and neutrophil count (bias=0.68 [-17 to 19]). Agreement in lymphocyte count was also observed between ADVIA and manual counting, but to a lesser extent compared to CellaVision (bias=13.9 [-10.45 to 38.27]). However, no agreement was observed for ADVIA (Neutrophils), Horiba (lymphocytes and neutrophils) with manual counting. CONCLUSIONS: Our data suggests that CellaVision could be used for the differential counting of neutrophil and lymphocytes in mouse blood sample.

Leishmanial aspartyl-tRNA synthetase: Biochemical, biophysical and structural insights.

Aminoacyl tRNA synthetases (aaRSs) are integral components of protein biosynthesis along with several non-canonical cellular processes. Inhibition studies of aaRSs presented these enzymes as promising drug targets in many pathogens, however aspartyl tRNA synthetase has not been studied in trypanosomatids despite its essentiality. Hence, full-length ORF of Leishmania donovani aspartyl tRNA synthetase (LdaspRS) was cloned and purified to homogeneity followed by molecular mass determination. The aminoacylation assay established that the purified protein performs its function optimally at physiological pH and temperature. The kinetic parameters of LdaspRS revealed the affinity of l-aspartate towards the enzyme to be very much lower than the cofactor. Our study also highlights the moonlighting function of LdaspRS to stimulate the pro-inflammatory cytokines and nitric oxide generation by host macrophage. Furthermore, CD and intrinsic tryptophan fluorescence measurements showed the changes in structural conformation at varying pH, denaturants and ligands. The modelled LdaspRS structure presented all the specific characteristics of class II aaRSs, while in silico study suggested binding of pyrimidine-derived inhibitors in its cofactor binding site with high affinity followed by validation using MD simulation. Altogether, this study could provide a platform for exploring LdaspRS to develop potential therapeutics against leishmaniasis.