Vishal Trivedi

MUC1 oncoprotein activates the IκB kinase β complex and constitutive NF-κB signalling

Nuclear factor-κB (NF-κB) is constitutively activated in diverse human malignancies by mechanisms that are not understood. The MUC1 oncoprotein is aberrantly overexpressed by most human carcinomas and, similarly to NF-κB, blocks apoptosis and induces transformation. This study demonstrates that overexpression of MUC1 in human carcinoma cells is associated with constitutive activation of NF-κB p65. We show that MUC1 interacts with the high-molecular-weight IκB kinase (IKK) complex in vivo and that the MUC1 cytoplasmic domain binds directly to IKKβ and IKKγ. Interaction of MUC1 with both IKKβ and IKKγ is necessary for IKKβ activation, resulting in phosphorylation and degradation of IκBα. Studies in non-malignant epithelial cells show that MUC1 is recruited to the TNF-R1 complex and interacts with IKKβ–IKKγ in response to TNFα stimulation. TNFα-induced recruitment of MUC1 is dependent on TRADD and TRAF2, but not the death-domain kinase RIP1. In addition, MUC1-mediated activation of IKKβ is dependent on TAK1 and TAB2. These findings indicate that MUC1 is important for physiological activation of IKKβ and that overexpression of MUC1, as found in human cancers, confers sustained induction of the IKKβ–NF-κB p65 pathway.

Clotrimazole Inhibits Hemoperoxidase of Plasmodium falciparum and Induces Oxidative Stress PROPOSED ANTIMALARIAL MECHANISM OF CLOTRIMAZOLE

The mechanism of antimalarial activity of clotrimazole was studied placing emphasis on its role in inhibiting hemoperoxidase for inducing oxidative stress in Plasmodium falciparum. Clotrimazole, in the presence of H2O2, causes irreversible inactivation of the enzyme, and the inactivation follows pseudo-first order kinetics, consistent with a mechanism-based (suicide) mode. The pseudo-first order kinetic constants are ki= 2.85 μm, kinact = 0.9 min-1, and t½ = 0.77 min. The one-electron oxidation product of clotrimazole has been identified by EPR spectroscopy as the 5,5′-dimethyl-1-pyrroline N-oxide (DMPO) adduct of the nitrogen-centered radical (aN = 15 G), and as DMPO protects against inactivation, this radical is involved in the inactivation process. Binding studies indicate that the clotrimazole oxidation product interacts at the heme moiety, and the heme-clotrimazole adduct has been dissociated from the inactivated enzyme and identified (m/z 1363) by mass analysis. We found that the inhibition of hemoperoxidase increases the accumulation of H2O2 in P. falciparum and causes oxidative stress. Furthermore, the inhibition of hemoperoxidase correlates well with the inhibition of parasite growth. The results described herein indicate that the antimalarial activity of clotrimazole might be due to the inhibition of hemoperoxidase and subsequent development of oxidative stress in P. falciparum.

Immunoglobulin G signaling activates lysosome/phagosome docking

An important role of IgG antibodies in the defense against microbial infections is to promote the ingestion and killing of microbes by phagocytes. Here, we developed in vivo and in vitro approaches to ask whether opsonization of particles with IgG enhances intracellular targeting of lysosomes to phagosomes. To eliminate the effect of IgG on the ingestion process, cells were exposed to latex beads at 15–20°C, which allows engulfment of both IgG-coated and uncoated beads but prevents the fusion of lysosomes with phagosomes. Upon shifting the temperature to 37°C, phagosomes containing IgG beads matured significantly faster into phagolysosomes as judged by colocalization with lysosomal markers. The IgG effect was independent of other particle-associated antigens or serum factors. Lysosome/phagosome attachment was also quantified biochemically with a cytosol-dependent scintillation proximity assay. Interactions were enhanced significantly in reactions containing cytosol from mouse macrophages that had been exposed to IgG-coated beads, indicating that IgG signaling modulates the cytosolic-targeting machinery. Similar results were obtained with cytosol from primary human monocytes, human U-937 histiocytic lymphoma cells and from Chinese hamster ovary (CHO) cells transfected with a human IgG (Fcγ) receptor. IgG-induced activation is shown to affect the actin-dependent tethering/docking stage of the targeting process and to proceed through a pathway involving protein kinase C. These results provide a rare example of an extracellular signal controlling membrane targeting on the level of tethering and docking. We propose that this pathway contributes to the role of antibodies in the protection against microbial infections.

Interaction of heme proteins with anionic polyfluorene: insights into physiological effects, folding events, and inhibition activity.

Because of the toxicity caused by the heme redox-active iron proteins, their elevated levels, localization, and accumulation in the brain, many forms of neurodegenerative diseases, such as Alzheimers disease, Parkinsons disease, and Huntingtons disease, occur as a result of which the brain becomes vulnerable to oxidative stress, ultimately resulting in neuronal death. An anionic water-soluble conjugated polyfluorene derivative poly(9,9-bis(6-sulfate hexyl) fluorene-alt-1,4-phenylene) sodium salt (P1) that binds Fe³⁺ proteins with very high selectivity and sensitivity is reported here. The photophysical properties of P1 were modified by the interaction with ferric heme-containing proteins cytochrome c (Cc), methemoglobin (MetHb), and hemin. P1 was found to be highly sensitive toward Fe³⁺ heme proteins as compared to nonmetalloproteins. We observed that the respective activities of ferric heme proteins were inhibited and proteins were unfolded, due to modification in their heme microenvironment in the presence of the polymer P1. The observations reported in this article provide the first example for the use of a water-soluble conjugated polymer in applications, such as (1) to detect small quantities of iron proteins in aqueous medium/physiological condition with the highest K(sv) values of 2.27 × 10⁸ M⁻¹ for Cc, 3.81 × 10⁷ M⁻¹ for MetHb, and 5.31 × 10⁷ M⁻¹ for hemin; (2) to study the physiological effects of heme metalloproteins; (3) to visualize the folding events in real time; and (4) the inhibition activity of metalloproteins can be selectively studied using a conjugated polymer based assay system rapidly without interference from nonmetalloproteins at biological pH. All this is achieved by generating optical events, taking advantage of the bright fluorescence of anionic polyfluorene P1 in this case, that can be observed and monitored by modification in the absorption and emission color in real time.

Methemoglobin exposure produces toxicological effects in macrophages due to multiple ROS spike induced apoptosis

Macrophages are an integral part of the immune system, required to produce a robust immune response against an infectious organism. Presence of methemoglobin in body fluids such as blood, cerebrospinal fluid and urine is associated with tissue damage. We tested cytotoxic effects of MetHb and underlying molecular events in mouse macrophage cell line J774A.1. MetHb exposure dose dependently reduced macrophage viability in an MTT assay. Light microscopy and scanning electron microscopic (SEM) observation of MetHb treated macrophage indicated death (less number of cells per field), severe damage to membrane structure and accumulation of particulate matter in the cytosol. The macrophage death during MetHb exposure was due to induction of apoptosis as indicated by annexin-V/FITC staining and DNA fragmentation analysis. MetHb treatment generated a periodic ROS spikes with time in the macrophage cytosol to develop oxidative stress. Scavenging ROS spikes with NAC, mannitol or PBN dose dependently protected macrophages against MetHb induced toxicity, apoptosis and cellular membrane damage. Our work highlighted the contributions of MetHb mediated toxicity toward macrophage and its potential role in tissue damage and immune depression during malaria and other hemolytic disorders.

In Silico Characterization of Atypical Kinase PFD0975w from Plasmodium Kinome: A Suitable Target For Drug Discovery

RIO‐2 kinase is known to regulate ribosome biogenesis and other cell cycle events. The 3D model of ATP bound and an unbound form of PFD0975w was generated using AfRIO‐2 crystal structure 1TQI, 1ZAO as template employing Modeller9v7 program. Structural characterization identified N‐terminal winged helix domain (1–84), C‐terminal kinase domain (148–275), and presence of other critical residues known for ATP binding and kinase activity. Using Q‐site and pocket finder, a number of well‐defined substrate (peptide) binding regions were identified in the catalytic core of the protein. The peptide binding regions were further validated by molecular modeling a non‐specific polyalanine peptide and a sequence‐specific peptide2 into these sites to generate a stable PFD0975w/peptide complexes. Peptide fits well into identified pocket on PFD0975w and makes extensive interaction with the protein residues. These newly identified peptide binding sites potentially give opportunity to design a specific inhibitor against PFD0975w. There are subtle but significant differences between Plasmodium falciparum and human RIO‐2 to exploit PFD0975w for drug development. In conclusion, our finding will let us to design effective chemotherapy against malaria parasite exploiting PFD0975w as a drug target.

Phagocytic uptake of oxidized heme polymer is highly cytotoxic to macrophages.

Apoptosis in macrophages is responsible for immune-depression and pathological effects during malaria. Phagocytosis of PRBC causes induction of apoptosis in macrophages through release of cytosolic factors from infected cells. Heme polymer or β-hematin causes dose-dependent death of macrophages with LC50 of 132 µg/ml and 182 µg/ml respectively. The toxicity of hemin or heme polymer was amplified several folds in the presence of non-toxic concentration of methemoglobin. β-hematin uptake in macrophage through phagocytosis is crucial for enhanced toxicological effects in the presence of methemoglobin. Higher accumulation of β-hematin is observed in macrophages treated with β-hematin along with methemoglobin. Light and scanning electron microscopic observations further confirm accumulation of β-hematin with cellular toxicity. Toxicological potentiation of pro-oxidant molecules toward macrophages depends on generation of H2O2 and independent to release of free iron from pro-oxidant molecules. Methemoglobin oxidizes β-hematin to form oxidized β-hematin (βH*) through single electron transfer mechanism. Pre-treatment of reaction mixture with spin-trap Phenyl-N-t-butyl-nitrone dose-dependently reverses the β-hematin toxicity, indicates crucial role of βH* generation with the toxicological potentiation. Acridine orange/ethidium bromide staining and DNA fragmentation analysis indicate that macrophage follows an oxidative stress dependent apoptotic pathway to cause death. In summary, current work highlights mutual co-operation between methemoglobin and different pro-oxidant molecules to enhance toxicity towards macrophages. Hence, methemoglobin peroxidase activity can be probed for subduing cellular toxicity of pro-oxidant molecules and it may in-turn make up for host immune response against the malaria parasite.

Extracellular methemoglobin primes red blood cell aggregation in malaria: An in vitro mechanistic study

Toxic byproducts from infected RBC cause rheological alteration and RBC aggregation. Malaria culture supernatant has the ability to exhibit RBC aggregation. Ammonium sulfate fractionation and immunodepletion of methemoglobin from culture supernatant confirms methemoglobin as a major aggregant. In vitro treatment of RBC with methemoglobin induces irreversible high order RBC aggregates, resistant to shear stress and physical forces. Methemoglobin‐mediated ROS generation in the external micro‐environment to develop oxidative stress close to RBC membrane seems to be responsible for initiating and forming high order RBC aggregates through phosphatidyl‐serine externalization. Removal of oxidative stress through antioxidant treatment abolishes high order RBC aggregate formation. In conclusion, we discovered a novel pathway of methemoglobin‐mediated RBC aggregation and its potential role in patho‐physiological effects during malaria.

Alkyl Cinnamates Induce Protein Kinase C Translocation and Anticancer Activity against Breast Cancer Cells through Induction of the Mitochondrial Pathway of Apoptosis

Purpose The protein kinase C (PKC) family of serine-threonine kinases plays an important role in cancer cell progression. Thus, molecules that target PKC have potential as anticancer agents. The current study aims to understand the treatment of breast cancer cells with alkyl cinnamates. We have also explored the mechanistic details of their anticancer action and the underlying molecular signaling. Methods 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to measure the viability of MDAMB-231 breast cancer cells to assess the anticancer activity of these compounds. In addition, flow cytometry was performed to study the effect of alkyl cinnamates on the cell cycle and apoptosis. Immunoblotting and immunofluorescence techniques were performed to study PKC translocation, cytochrome c release, and modulation of the mitochondrial membrane potential in breast cancer cells targeted with alkyl cinnamates. Results The PKC agonist DM-2-8 translocated 16.6%±1.7% PKCα from cytosol to the plasma membrane and showed excellent anticancer activity with an half maximal inhibitory concentration (IC50) of 4.13±0.27 µg/mL against cancer cells. The treated cells had an abnormal morphology and exhibited cell cycle defects with G2/M arrest and reduced S phase. Cancer cells treated with DM-2-3, DM-2-4, or DM-2-8 underwent apoptosis as the major pathway of cell death, further confirmed by genomic DNA fragmentation. Furthermore, the mitochondrial membrane potential was perturbed, indicating involvement of the mitochondrial pathway of apoptosis. Immunolocalization studies revealed cytochrome c release from mitochondria to cytosol. Cancer cells treated with DM-2-8 and curcumin showed activation of caspase-9 and caspase-3 as downstream molecular components of the apoptotic pathway. Alkyl cinnamates also caused oxidative stress, which regulates the apoptotic machinery (DNA fragmentation), cell death, and morphological abnormalities in cancer cells. Conclusion Alkyl cinnamates specifically target cancer cells through induction of PKC translocation and the mitochondrial pathway of apoptosis, and could be promising anticancer drugs.

Skeletal hybridization and PfRIO-2 kinase modeling for synthesis of α-pyrone analogs as anti-malarial agent.

The pharmacophoric hybridization and computational design approach were applied to generate a novel series of α-pyrone analogs as plausible anti-malarial lead candidate. A putative active site in flexible loop close to wing-helix domain of PfRIO2 kinase was explored computationally to understand the molecular basis of ligand binding. All the synthesized molecules (3a-g) exhibited in vitro antimalarial activity. Oxidative stress induced by 3a-d were calculated and found to be significantly higher in case of 3b. Therefore, 3b, which shown most significant result was identified as promising lead for further SAR study to develop potent anti-malarials.