Nanoemulsion potentiates the anti-cancer activity of Myricetin by effective inhibition of PI3K/AKT/mTOR pathway in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is a heterogeneous tumor with a poor prognosis and high metastatic potential, resulting in poor clinical outcomes, necessitating investigation to devise effective therapeutic strategies. Multiple studies have substantiated the anti-cancer properties of the naturally occurring flavonoid "Myricetin" in various malignancies. However, the therapeutic application of Myricetin is impeded by its poor water solubility and low oral bioavailability. To overcome this limitation, we aimed to develop nanoemulsion of Myricetin (Myr-NE) and evaluate its advantage over Myricetin alone in TNBC cells. The nanoemulsion was formulated using Capryol 90 (oil), Tween 20 (surfactant), and Transcutol HP (co-surfactant). The optimized nano-formulation underwent an evaluation to determine its size, zeta potential, morphology, stability, drug encapsulation efficiency, and in vitro release properties. The anti-cancer activity of Myr-NE was further studied to examine its distinct impact on intracellular drug uptake, cell-viability, anti-tumor signaling, oxidative stress, clonogenicity, and cell death, compared with Myricetin alone in MDA-MB-231 (TNBC) cells. The in vitro drug release and intracellular drug uptake of Myricetin was significantly increased in Myr-NE formulation as compared to Myricetin alone. Moreover, Myr-NE exhibited significant inhibition of cell proliferation, clonogenicity, and increased apoptosis with ~ 2.5-fold lower IC50 as compared to Myricetin. Mechanistic investigation revealed that nanoemulsion augmented the anti-cancer efficacy of Myricetin, most likely by inhibiting the PI3K/AKT/mTOR pathway, eventually leading to enhanced cell death in TNBC cells. The study provides substantial experimental evidence to support the notion that the Myr-NE formulation has the potential to be an effective therapeutic drug for TNBC treatment.

Dietary administration of the glycolytic inhibitor 2-deoxy-D-glucose reduces endotoxemia-induced inflammation and oxidative stress: Implications in PAMP-associated acute and chronic pathology.

Pathogen-associated molecular patterns (PAMPs) like bacterial cell wall components and viral nucleic acids are known ligands of innate inflammatory receptors that trigger multiple inflammatory pathways that may result in acute inflammation and oxidative stress-driven tissue and organ toxicity. When dysregulated, this inflammation may lead to acute toxicity and multiorgan failure. Inflammatory events are often driven by high energy demands and macromolecular biosynthesis. Therefore, we proposed that targeting the metabolism of lipopolysaccharide (LPS)-driven inflammatory events, using an energy restriction approach, can be an effective strategy to prevent the acute or chronic detrimental effects of accidental or seasonal bacterial and other pathogenic exposures. In the present study, we investigated the potential of energy restriction mimetic agent (ERMA) 2-deoxy-D-glucose (2-DG) in targeting the metabolism of inflammatory events during LPS-elicited acute inflammatory response. Mice fed with 2-DG as a dietary component in drinking water showed reduced LPS-driven inflammatory processes. Dietary 2-DG reduced LPS-induced lung endothelial damage and oxidative stress by strengthening the antioxidant defense system and limiting the activation and expression of inflammatory proteins, viz., P-Stat-3, NfκΒ, and MAP kinases. This was accompanied by decreased TNF, IL-1ß, and IL-6 levels in peripheral blood and bronchoalveolar lavage fluid (BALF). 2-DG also reduced the infiltration of PMNCs (polymorphonuclear cells) in inflamed tissues. Altered glycolysis and improved mitochondrial activity in 2-DG-treated RAW 264.7 macrophage cells suggested possible impairment of macrophage metabolism and, therefore, activation in macrophages. Taken together, the present study suggests that inclusion of glycolytic inhibitor 2-DG as a part of the diet can be helpful in preventing the severity and poor prognosis associated with inflammatory events during bacterial and other pathogenic exposures.

Biocompatible epoxysilane substituted polymer-based nano biosensing platform for label-free detection of cancer biomarker SP17 in patient serum samples.

Herein, we report the results of the studies relating to developing a simple, sensitive, cost-effective, and disposable electrochemical-based label-free immunosensor for real-time detection of a new cancer biomarker, sperm protein-17 (SP17), in complex serum samples. An indium tin oxide (ITO) coated glass substrate modified with self-assembled monolayers (SAMs) of 3-glycidoxypropyltrimethoxysilane (GPTMS) was functionalized via covalent immobilization of monoclonal anti-SP17 antibodies using EDC(1-(3-(dimethylamine)-propyl)-3-ethylcarbodiimide hydrochloride) - NHS (N-hydroxy succinimide) chemistry. The developed immunosensor platform (BSA/anti-SP17/GPTMS@SAMs/ITO) was characterized via scanning electron microscopy (SEM), atomic force microscopy (AFM), contact angle (CA), Fourier transform infrared (FT-IR) spectroscopic, and electrochemical techniques such as cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) techniques. The fabricated BSA/anti-SP17/GPTMS@SAMs/ITO immunoelectrode platform was used to measure changes in the magnitude of the current of the electrodes through an electrochemical CV and DPV technique. A calibration curve between current and SP17 concentrations exhibited a broad linear detection range of (100-6000 & 50-5500 pg mL-1), with enhanced sensitivity (0.047 & 0.024 µA pg mL-1 cm-2), limit of detection (LOD) and limit of quantification (LOQ) of 47.57 & 142.9 pg mL-1 and 158.58 & 476.3 pg mL-1, by CV and DPV technique, respectively with a rapid response time of 15 min. It possessed exceptional repeatability, outstanding reproducibility, five-time reusability, and high stability. The biosensor's performance was evaluated in human serum samples, giving satisfactory findings obtained via the commercially available enzyme-linked immunosorbent assay (ELISA) technique, proving the clinical applicability for early diagnosis of cancer patients. Moreover, various in vitro studies in murine fibroblast cell line L929 have been performed to assess the cytotoxicity of GPTMS. The results demonstrated that GPTMS has excellent biocompatibility and can be used for biosensor fabrication.

A calcium and zinc composite alginate hydrogel for pre-hospital hemostasis and wound care.

We developed, characterized, and examined the hemostatic potential of sodium alginate-based Ca2+ and Zn2+ composite hydrogel (SA-CZ). SA-CZ hydrogel showed substantial in-vitro efficacy, as observed by the significant reduction in coagulation time with better blood coagulation index (BCI) and no evident hemolysis in human blood. SA-CZ significantly reduced bleeding time (≈60 %) and mean blood loss (≈65 %) in the tail bleeding and liver incision in the mice hemorrhage model (p ≤ 0.001). SA-CZ also showed enhanced cellular migration (1.58-fold) in-vitro and improved wound closure (≈70 %) as compared with betadine (≈38 %) and saline (≈34 %) at the 7th-day post-wound creation in-vivo (p < 0.005). Subcutaneous implantation and intra-venous gamma-scintigraphy of hydrogel revealed ample body clearance and non-considerable accumulation in any vital organ, proving its non-thromboembolic nature. Overall, SA-CZ showed good biocompatibility along with efficient hemostasis and wound healing qualities, making it suitable as a safe and effective aid for bleeding wounds.

Ascorbate formulation improves healing efficacy in excisional wound mice model through interplay between pro and anti-inflammatory cytokines and angiogenic markers.

BACKGROUND AND OBJECTIVE: Biomedical research in regenerative medicine prompts researchers to formulate cost-effective therapeutics for wound healing. The present study was conducted to characterize the ascorbate based formulation vis-a-vis investigating the molecular dynamics of the formulation. MATERIALS AND METHODS: To characterize the formulation, particle size, zeta potential, thermal stability, compatibility, anti-oxidant, and permeation prospective were measured using standard protocols. The in-vitro healing potential and safety formulae were evaluated using the L929 cell line. For molecular unravelling of the pharmacodynamics of formulation, an excision wound model was used, and 54 mice were randomly and equally divided into three groups, i.e., untreated, betadine-treated, and formulation-treated, to ascertain the interplay between cytokines and chemokines and their culminative role in the release of growth factors. RESULTS: The ascorbate formulae were found to be amorphous, biocompatible, safe, and long-lasting, with particle sizes and zeta potentials of 389.7 ± 0.69 nm and -38.1 ± 0.65 mV, respectively, and anti-oxidative potential. An in-vitro study revealed that the formulation has a significant (p<0.05) migration potential and is non-toxic. Expression profiling of TGF-ß, FGF-2, VEGF, and collagen III & I showed significant (p<0.05) up-regulation, whereas significant (p<0.05) down-regulation of pro-inflammatory genes like IL-1α, IL-1ß, TNF-α, IL-6, and temporal change in CCR-5 was observed in formulae-treated animals as compared to other groups. CONCLUSION: By up-regulating angiogenic and collagen-promoting growth factor gene expression while down-regulating pro-inflammatory gene expression, ascorbate formulation promotes wound healing via extracellular matrix and granulation tissue deposition with significant improvement in tensile strength.

2-deoxy-D-glucose as an adjunct to standard of care in the medical management of COVID-19: a proof-of-concept and dose-ranging randomised phase II clinical trial.

BACKGROUND: At present, no single efficacious therapeutic exists for acute COVID-19 management and a multimodal approach may be necessary. 2-deoxy-D-glucose (2-DG) is a metabolic inhibitor that has been shown to limit multiplication of SARS-CoV-2 in-vitro. We evaluated the efficacy and safety of 2-DG as adjunct to standard care in the treatment of moderate to severe COVID-19 patients. METHODS: We conducted a randomized, open-label, phase II, clinical study to evaluate the efficacy, safety, and tolerability of 2-DG administered as adjunct to standard of care (SOC). A total of 110 patients between the ages of 18 and 65 years with moderate to severe COVID-19 were included. Patients were randomized to receive 63, 90, or 126 mg/kg/day 2-DG in addition to SOC or SOC only. Times to maintaining SpO2 ≥ 94% on room air, discharge, clinical recovery, vital signs normalisation, improvement by 1 and 2 points on WHO clinical progression scale, negative conversion on RT-PCR, requirement for intensive care, and mortality were analyzed to assess the efficacy. RESULTS: Patients treated with 90 mg/kg/day 2-DG plus SOC showed better outcomes. Time to maintaining SpO2 ≥ 94% was significantly shorter in the 2-DG 90 mg compared to SOC (median 2.5 days vs. 5 days, Hazard ratio [95% confidence interval] = 2.3 [1.14, 4.64], p = 0.0201). Times to discharge from isolation ward, to clinical recovery, and to vital signs normalization were significantly shorter for the 2-DG 90 mg group. All three doses of 2-DG were well tolerated. Thirty-three (30.3%) patients reported 65 adverse events and were mostly (86%) mild. CONCLUSIONS: 2-DG 90 mg/kg/day as adjunct to SOC showed clinical benefit over SOC alone in the treatment of moderate to severe COVID-19. The promising trends observed in current phase II study is encouraging for confirmatory evaluation of the efficacy and safety of 2-DG in a larger phase III trial. TRIAL REGISTRATION: CTRI, CTRI/2020/06/025664. Registered 5th June 2020, http://ctri.nic.in/Clinicaltrials/pdf_generate.php?trialid=44369&EncHid=&modid=&compid=%27,%2744369det%27 .

Protective Effect of Organ Preservation Fluid Supplemented With Nicorandil and Rutin Trihydrate: A Comparative Study in a Rat Model of Renal Ischemia.

OBJECTIVES: The objective of organ preservation is sustained viability of detached/removed/isolated organs and subsequent successful posttransplant outcomes. Nicorandil (an ATP-sensitive potassium channel opener) is an efficacious agent to preserve lungs and heart. Rutin trihydrate (an antioxidant) inhibits free radical-mediated cytotoxicity and lipid peroxidation. We aimed to evaluate the efficacy of nicorandil and rutin trihydrate to enhance kidney preservation. MATERIALS AND METHODS: We prepared 2 versions of organ preservation fluid, supplemented with either nicorandil or rutin trihydrate, and used 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assays to evaluate the efficacy of these solutions in vitro (HEK293 human embryonic kidney cells), according to various cellular parameters such as ATP levels, reactive oxygen species, and cell viability. We also investigated the in vivo preservation efficacy in a rat model of renal ischemia and evaluated the immunohistological expression of apoptotic markers (caspase 3) in preserved rat kidney. RESULTS: We observed significant improvement of intracellular ATP levels (32 999 ± 1454 pmol/cell, n = 3; P < .05) in cells preserved in the nicorandil- supplemented solution compared with Custodiol solution (23 216 ± 1315 pmol/cell). Reactive oxygen species declined 1.25-fold (P < .05) in the presence of rutin trihydrate. Cell viability assays revealed a 4.8-fold increase in viability of renal cells preserved in the solutions supplemented with nicorandil or rutin trihydrate after 24-hour incubation compared with controls. In vivo, there were significant effects on serum creatinine (0.5480 ± 0.052, 0.956 ± 0.043 mg/dL) and blood urea nitrogen (85.36 ± 4.64, 92.85 ± 3.15 mg/dL) with the nicorandil and rutin trihydrate solutions, respectively. We observed suppressed expression of the apoptotic marker caspase 3 in groups treated with the 2 supplemented preservation fluids. CONCLUSIONS: Our results showed that solutions of organ preservation fluid supplemented with either nicorandil or rutin trihydrate can ameliorate cellular problems/dysfunction and facilitate sustained impro - vement of tissue survival and subsequent organ viability.

Gamma-Linolenic Acid (GLA) Protects against Ionizing Radiation-Induced Damage: An In Vitro and In Vivo Study.

Radiation is pro-inflammatory in nature in view of its ability to induce the generation of reactive oxygen species (ROS), cytokines, chemokines, and growth factors with associated inflammatory cells. Cells are efficient in repairing radiation-induced DNA damage; however, exactly how this happens is not clear. In the present study, GLA reduced DNA damage (as evidenced by micronuclei formation) and enhanced metabolic viability, which led to an increase in the number of surviving RAW 264.7 cells in vitro by reducing ROS generation, and restoring the activities of desaturases, COX-1, COX-2, and 5-LOX enzymes, TNF-α/TGF-ß, NF-kB/IkB, and Bcl-2/Bax ratios, and iNOS, AIM-2, and caspases 1 and 3, to near normal. These in vitro beneficial actions were confirmed by in vivo studies, which revealed that the survival of female C57BL/6J mice exposed to lethal radiation (survival~20%) is significantly enhanced (to ~80%) by GLA treatment by restoring altered levels of duodenal HMGB1, IL-6, TNF-α, and IL-10 concentrations, as well as the expression of NF-kB, IkB, Bcl-2, Bax, delta-6-desaturase, COX-2, and 5-LOX genes, and pro- and anti-oxidant enzymes (SOD, catalase, glutathione), to near normal. These in vitro and in vivo studies suggest that GLA protects cells/tissues from lethal doses of radiation by producing appropriate changes in inflammation and its resolution in a timely fashion.

Therapeutic natural compounds Enzastaurin and Palbociclib inhibit MASTL kinase activity preventing breast cancer cell proliferation.

Microtubule-associated serine/threonine kinase-like (MASTL) regulates mitotic progression and is an attractive target for the development of new anticancer drugs. In this study, novel inhibitory molecules were screened against MASTL kinase, a protein involved in cell proliferation in breast cancer. Natural source-derived drugs Enzastaurin and Palbociclib were selected to identify their role as MASTL kinase inhibitors. Cytotoxic activity, kinase activity, and other cell-based assays of Enzastaurin and Palbociclib were evaluated on human breast cancer (MCF-7) cells. The potential natural compounds caused cytotoxicity in MCF-7 cells in a dose- and time-dependent manner. Further analysis by Annexin V and PI staining indicated that both drugs are potent inducers of apoptosis. Enzastaurin induced G2/M phase arrest, while Palbociclib caused G1 arrest. MASTL kinase activity was significantly abrogated with both the compounds showing EC50 values of 17.13 µM and 10.51 µM, respectively. Taken together, these data strongly suggest that Enzastaurin and Palbociclib possess the ability to inhibit MASTL kinase activity and induce cell death in breast cancer cells, thus exhibiting significant therapeutic potential.

Amifostine analog, DRDE-30, alleviates radiation induced lung damage by attenuating inflammation and fibrosis.

BACKGROUND: Radiotherapy of thoracic neoplasms and accidental radiation exposure often results in pneumonitis and fibrosis of lungs. Here, we investigated the potential of amifostine analogs: DRDE-07, DRDE-30, and DRDE-35, in alleviating radiation-induced lung damage. METHODS: C57BL/6 mice were exposed to 13.5 Gy thoracic irradiation, 30 min after intraperitoneal administration of the analogs, and assessed for modulation of the pathological response at 12 and 24 weeks. KEY FINDINGS: DRDE-07, DRDE-30 and DRDE-35 increased the survival of irradiated mice from 20% to 30%, 80% and 70% respectively. Reduced parenchymal opacity (X-ray CT) in the lungs of DRDE-30 pre-treated mice corroborated well with the significant decrease in Ashcroft score (p < 0.01). Two-fold increase in SOD and catalase activities (p < 0.05), coupled with a 50% increase in GSH content and a 60% decrease in MDA content (p < 0.05) suggested restoration of the antioxidant defence system. A 20% to 40% decrease in radiation-induced apoptotic and mitotic death in the lung tissue (micronuclei: p < 0.01), resulted in attenuated lung and vascular permeability (FITC-Dextran leakage) by 50% (p < 0.01), and a commensurate reduction (~50%) in leukocyte infiltration in the injured tissue (p < 0.05). DRDE-30 abrogated the activation of pro-inflammatory NF-κB and p38/MAPK signaling cascades, suppressing the release of pro-inflammatory cytokines (IL-1ß: p < 0.05; TNF-α: p < 0.05; IL-6: p < 0.05) and up-regulation of CAMs on the endothelial cell surface. Reduction in hydroxyproline content (p < 0.01) and collagen suggested inhibition of lung fibrosis which was associated with attenuation of TGF-ß/Smad pathway-mediated-EMT. CONCLUSION: DRDE-30 could be a potential prophylactic agent against radiation-induced lung injury.

Glycolytic inhibitor 2-deoxy-d-glucose attenuates SARS-CoV-2 multiplication in host cells and weakens the infective potential of progeny virions.

AIMS: Virus-infected host cells switch their metabolism to a more glycolytic phenotype, required for new virion synthesis and packaging. Therefore, we investigated the effect and mechanistic action of glycolytic inhibitor 2-Deoxy-d-glucose (2-DG) on virus multiplication in host cells following SARS-CoV-2 infection. MAIN METHODS: SARS-CoV-2 induced change in glycolysis was examined in Vero E6 cells. Effect of 2-DG on virus multiplication was evaluated by RT-PCR (N and RdRp genes) analysis, protein expression analysis of Nucleocapsid (N) and Spike (S) proteins and visual indication of cytopathy effect (CPE), The mass spectrometry analysis was performed to examine the 2-DG induced change in glycosylation status of receptor binding domain (RBD) in SARS-CoV-2 spike protein. KEY FINDINGS: We observed SARS-COV-2 infection induced increased glucose influx and glycolysis, resulting in selectively high accumulation of the fluorescent glucose analog, 2-NBDG in Vero E6 cells. 2-DG inhibited glycolysis, reduced virus multiplication and alleviated cells from virus-induced cytopathic effect (CPE) in SARS-CoV-2 infected cells. The progeny virions produced from 2-DG treated cells were found unglycosylated at crucial N-glycosites (N331 and N343) of the receptor-binding domain (RBD) in the spike protein, resulting in production of defective progeny virions with compromised infective potential. SIGNIFICANCE: The mechanistic study revealed that the inhibition of SARS-COV-2 multiplication is attributed to 2-DG induced glycolysis inhibition and possibly un-glycosylation of the spike protein, also. Therefore, based on its previous human trials in different types of Cancer and Herpes patients, it could be a potential molecule to study in COVID-19 patients.

Mitochondrial uncoupler DNP induces coexistence of dual-state hyper-energy metabolism leading to tumor growth advantage in human glioma xenografts.

Introduction: Cancer bioenergetics is an essential hallmark of neoplastic transformation. Warburg postulated that mitochondrial OXPHOS is impaired in cancer cells, leading to aerobic glycolysis as the primary metabolic pathway. However, mitochondrial function is altered but not entirely compromised in most malignancies, and that mitochondrial uncoupling is known to increase the carcinogenic potential and modifies treatment response by altering metabolic reprogramming. Our earlier study showed that transient DNP exposure increases glycolysis in human glioma cells (BMG-1). The current study investigated the persistent effect of DNP on the energy metabolism of BMG-1 cells and its influence on tumor progression in glioma xenografts. Methods: BMG-1 cells were treated with 2,4-dinitrophenol (DNP) in-vitro, to establish the OXPHOS-modified (OPM-BMG) cells. Further cellular metabolic characterization was carried out in both in-vitro cellular model and in-vivo tumor xenografts to dissect the role of metabolic adaptation in these cells and compared them with their parental phenotype. Results and Discussion: Chronic exposure to DNP in BMG-1 cells resulted in dual-state hyper-energy metabolism with elevated glycolysis++ and OXPHOS++ compared to parental BMG-1 cells with low glycolysis+ and OXPHOS+. Tumor xenograft of OPM-BMG cells showed relatively increased tumor-forming potential and accelerated tumor growth in nude mice. Moreover, compared to BMG-1, OPM-BMG tumor-derived cells also showed enhanced migration and invasion potential. Although mitochondrial uncouplers are proposed as a valuable anti-cancer strategy; however, our findings reveal that prolonged exposure to uncouplers provides tumor growth advantage over the existing glioma phenotype that may lead to poor clinical outcomes.

Sphingosine kinase inhibitor, SKI-II confers protection against the ionizing radiation by maintaining redox homeostasis most likely through Nrf2 signaling.

Exposure to ionizing radiation (IR) set a series of deleterious events causing acute radiation syndrome and mortality, posing the need for a potent and safe radio-protective drug. IR induces cell death predominantly by causing oxidative stress and macromolecular damage. The pre-existing antioxidant defence machinery of the cellular system plays a crucial role in protecting the cells against oxidative stress by activation of Nrf2. The current study was undertaken to investigate the radio-protective potential of sphingosine kinase inhibitor (SKI-II), which was demonstrated to activate Nrf2 signaling. The safety and efficacy of SKI-II were evaluated with cell cytotoxicity, proliferation index, and clonogenic survival assays in different cell lines, namely Raw 264.7, INT-407, IEC-6 and NIH/3T3 cell lines. A safe dose of SKI-II was found radio-protective in all the cell lines linked with the activated antioxidant defence system, thereby resulting in the amelioration of IR induced oxidative stress. SKI-II pretreatment also significantly reduced DNA damage, micronuclei expression, and accelerated DNA repair kinetics as compared to IR exposed cells. Reduced oxidative stress and enhanced DNA repair significantly reduced apoptosis and suppressed the pro-death signaling associated with IR exposure. Furthermore, the in-vitro observation was verified in the in-vivo model (C57 BL/6). The Intra-peritoneal (IP) administration of SKI-II, 2 h before a lethal dose of IR exposure (7.5 Gy) resulted in 75% survival. These results imply that SKI-II ameliorates IR-induced oxidative stress and cell death by inducing anti-oxidant defence system and DNA repair pathways, thus strengthening its potential to be used as radiation countermeasure.

PTEN inhibitor bpV(HOpic) confers protection against ionizing radiation.

Exposure to Ionizing radiation (IR) poses a severe threat to human health. Therefore, there is an urgent need to develop potent and safe radioprotective agents for radio-nuclear emergencies. Phosphatidylinositol-3-kinase (PI3K) mediates its cytoprotective signaling against IR by phosphorylating membrane phospholipids to phosphatidylinositol 3,4,5 triphosphate, PIP3, that serve as a docking site for AKT. Phosphatase and Tensin Homolog on chromosome 10 (PTEN) antagonizes PI3K activity by dephosphorylating PIP3, thus suppressing PI3K/AKT signaling that could prevent IR induced cytotoxicity. The current study was undertaken to investigate the radioprotective potential of PTEN inhibitor (PTENi), bpV(HOpic). The cell cytotoxicity, proliferation index, and clonogenic survival assays were performed for assessing the radioprotective potential of bpV(HOpic). A safe dose of bpV(HOpic) was shown to be radioprotective in three radiosensitive tissue origin cells. Further, bpV(HOpic) significantly reduced the IR-induced apoptosis and associated pro-death signaling. A faster and better DNA repair kinetics was also observed in bpV(HOpic) pretreated cells exposed to IR. Additionally, bpV(HOpic) decreased the IR-induced oxidative stress and significantly enhanced the antioxidant defense mechanism in cells. The radioprotective effect of bpV(HOpic) was found to be AKT dependant and primarily regulated by the enhanced glycolysis and associated signaling. Furthermore, this in-vitro observation was verified in-vivo, where administration of bpV(HOpic) in C57BL/6 mice resulted in AKT activation and conferred survival advantage against IR-induced mortality. These results imply that bpV(HOpic) ameliorates IR-induced oxidative stress and cell death by inducing AKT signaling mediated antioxidant defense system and DNA repair pathways, thus strengthening its potential to be used as a radiation countermeasure.

Mild mitochondrial uncoupling protects from ionizing radiation induced cell death by attenuating oxidative stress and mitochondrial damage.

Ionizing radiation (IR) induced mitochondrial dysfunction is associated with enhanced radiation stimulated metabolic oxidative stress that interacts randomly with intracellular bio-macromolecules causing lethal cellular injury and cell death. Since mild mitochondrial uncoupling emerged as a valuable therapeutic approach by regulating oxidative stress in most prevalent human diseases including ageing, ischemic reperfusion injury, and neurodegeneration with comparable features of IR inflicted mitochondrial damage. Therefore, we explored whether mitochondrial uncoupling could also protect from IR induced cytotoxic insult. Our results showed that DNP, BHT, FCCP, and BAM15 are safe to cells at different concentrations range depending on their respective mitochondrial uncoupling potential. Pre-incubation of murine fibroblast (NIH/3T3) cells with the safe concentration of these uncouplers followed by gamma (γ)-radiation showed significant cell growth recovery, reduced ROS generation, and apoptosis, compared to IR treatment alone. We observed that DNP pre-treatment increased the surviving fraction of IR exposed HEK-293, Raw 264.7 and NIH/3T3 cells. Additionally, DNP pre-treatment followed by IR leads to reduced total and mitochondrial oxidative stress (mos), regulated calcium (Ca2+) homeostasis, and mitochondrial bioenergetics in NIH/3T3 cells. It also significantly reduced macromolecular oxidation, correlated with the regulated ROS generation and antioxidant defence system. Moreover, DNP facilitated DNA repair kinetics evidenced by reducing the number of γ-H2AX foci formation and fragmented nuclei with time. DNP pre-incubation restrained the radiation induced pro-apoptotic factors and inhibits apoptosis. Our findings raise the possibility that mild mitochondrial uncoupling with DNP could be a potential therapeutic approach for radiation induced cytotoxic insult associated with an altered mitochondrial function.

Non-Enzymatic Protein Acetylation by 7-Acetoxy-4-Methylcoumarin: Implications in Protein Biochemistry.

BACKGROUND: The semi-synthetic acetoxycoumarins are known to acetylate proteins using novel enzymatic Calreticulin Transacetylase (CRTAase) system in cells. However, the nonenzymatic protein acetylation by polyphenolic acetates is not known. OBJECTIVE: To investigate the ability of 7-acetoxy-4-methyl coumarin (7-AMC) to acetylate proteins non-enzymatically in the test tube. METHODS: We incubated 7-AMC with BSA and analyzed the protein acetylation using Western blot technique. Further, BSA induced biophysical changes in the spectroscopic properties of 7-AMC was analyzed using Fluorescence spectroscopy. RESULTS: Using pan anti-acetyl lysine antibody, herein we demonstrate that 7-AMC acetylates Bovine Serum Albumin (BSA) in time and concentration dependent manner in the absence of any enzyme. 7-AMC is a relatively less fluorescent molecule compared to the parental compound, 7- hydroxy-4-methylcoumarin (7-HMC), however the fluorescence of 7-AMC increased by two fold on incubation with BSA, depending on the time of incubation and concentration of BSA. Analysis of the reaction mixture of 7-AMC and BSA after filtration revealed that the increased fluorescence is associated with the compound of lower molecular weight in the filtrate and not residual BSA, suggesting that the less fluorescent 7-AMC undergoes self-hydrolysis in the presence of protein to give highly fluorescent parental molecule 7-HMC and acetate ion in polar solvent (phosphate buffered saline, PBS). The protein augmented conversion of 7-AMC to 7-HMC was found to be linearly related to the protein concentration. CONCLUSION: Thus protein acetylation induced by 7-AMC could also be non-enzymatic in nature and this molecule can be exploited for quantification of proteins.

Interleukin-6 confers radio-resistance by inducing Akt-mediated glycolysis and reducing mitochondrial damage in cells.

Interleukin-6 (IL-6)-induced glycolysis and therapeutic resistance is reported in some cell systems; however, the mechanism of IL-6-induced glycolysis in radio-resistance is unexplored. Therefore, to investigate, we treated Raw264.7 cells with IL-6 (1 h prior to irradiation) and examined the glycolytic flux. Increased expression of mRNA and protein levels of key glycolytic enzymes was observed after IL-6 treatment, which conferred glycolysis dependent resistance from radiation-induced cell death. We further established that IL-6-induced glycolysis is activated by Akt signalling and knocking down Akt or inhibition of pan Akt phosphorylation significantly abrogated the IL-6-induced radio-resistance. Moreover, reduction of IL-6-induced pAkt level suppressed the expression of Hexokinase-2 and its translocation to the mitochondria, thereby inhibiting the glycolysis-induced resistance to radiation. IL-6-induced glycolysis also minimized the radiation-induced mitochondrial damage. These results suggest that IL-6-induced glycolysis observed in cells may be responsible for IL-6-mediated therapeutic radio-resistance in cancer cells, partly by activation of Akt signalling.

Dietary 2-deoxy-D-glucose impairs tumour growth and metastasis by inhibiting angiogenesis.

Accumulating evidence suggests the antiangiogenic potential of the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) among the anticancerous properties of this drug. In the present studies, we investigated the antiangiogenic effects of dietary 2-DG on tumour (Lewis lung carcinoma [LLC]) as well as ionising radiation-induced angiogenesis in mouse models. Dietary 2-DG reduced the serum vascular endothelial growth factor levels (∼40%) in LLC-bearing mice along with a significant inhibition of tumour growth and metastases. In vivo Matrigel plug assays showed significant decrease in vascularisation, Fluorescein isothiocyanate (FITC)-dextran fluorescence and factor VIII-positive cells in the plugs from 2-DG-fed mice, supporting the notion that dietary 2-DG significantly suppresses the tumour-associated and radiation-induced angiogenesis. 2-DG inhibited the glucose usage and lactate production as well as ATP levels of human umbilical vein endothelial cells (HUVECs) in a concentration-dependent manner, accompanied by growth inhibition and loss of viability in vitro. Furthermore, 2-DG inhibited the capillary-like tube formation in Matrigel as well as migration and transwell invasion by HUVECs, which are functional indicators of the process of angiogenesis. These results suggest that dietary 2-DG inhibits processes related to angiogenesis, which can impair the growth and metastasis of tumours.

Hexokinase II inhibition by 3-bromopyruvate sensitizes myeloid leukemic cells K-562 to anti-leukemic drug, daunorubicin.

An increased metabolic flux towards Warburg phenotype promotes survival, proliferation and causes therapeutic resistance, in leukemic cells. Hexokinase-II (HK-II) is expressed predominantly in cancer cells, which promotes Warburg metabolic phenotype and protects the cancer cells from drug-induced apoptosis. The HK-II inhibitor 3- Bromopyruvate (3-BP) dissociates HK-II from mitochondrial complex, which leads to enhanced sensitization of leukemic cells to anti-leukemic drugs. In the present study, we analyzed the Warburg characteristics viz. HK-II expression, glucose uptake, endogenous reactive oxygen species (ROS) level of leukemic cell lines K-562 and THP-1 and then investigated if 3-BP can sensitize the leukemic cells K-562 to anti-leukemic drug Daunorubicin (DNR). We found that both K-562 and THP-1 cells have multi-fold high levels of HK-II, glucose uptake and endogenous ROS with respect to normal PBMCs. The combined treatment (CT) of 3-BP and DNR showed synergistic effect on the growth inhibition (GI) of K-562 and THP-1 cells. This growth inhibitory effect was attributed to 3-BP induced S-phase block and DNR induced G2/M block, resulted in reduced proliferation due to CT. Further, CT resulted in low HK-II level in mitochondrial fraction, high intracellular calcium and elevated apoptosis as compared with individual treatment of DNR and 3-BP. Moreover, CT caused enhanced DNA damage and hyperpolarized mitochondria, leading to cell death. Taken together, these results suggest that 3-BP synergises the anticancer effects of DNR in the chronic myeloid leukemic cell K-562, and may act as an effective adjuvant to anti-leukemic chemotherapy.