S.P. Lochab

Synthesis and cytotoxicity evaluation of (tetrahydro-β-carboline)-1,3,5-triazine hybrids as anticancer agents

A series of tetrahydro-beta-carbolines and 1,3,5-triazine hybrids have been synthesized and evaluated for their cytotoxicity against a panel of eight human cancer cell lines and normal human fibroblasts (NIH3T3). It led us to discovery of racemic compounds 69, 71 and 75, which are selectively cytotoxic towards KB (oral cancer) cell line with IC50 values of 105.8, 664.7 and 122.2 nM, respectively; while their enantiopure forms are less active and not selective. Enantiopure compound 42 showed 2.5 times more selectivity towards MCF7 cells over normal fibroblast NIH3T3 cells with an IC50 value of 740 nM, also arrests cell cycle in G1 phase and induces apoptosis in MCF7 and MDA MB231 cell lines.

Thermoluminescence studies of CaS : Bi nanocrystalline phosphors

The thermoluminescence (TL) studies of CaS : Bi nanocrystalline phosphors prepared by the wet chemical co-precipitation method and exposed to γ-rays have been discussed. The average grain size of the samples was estimated as 35 nm using Scherrers equation. The samples exhibit a complex TL glow curve with multiple peaks. Sample exposed to γ-rays showed that the TL intensity increases linearly as the radiation doses increased in the 1.012–40.48 mGy range. The trap parameters namely, activation energy (E), order of kinetics (b) and frequency factor (s) of the main peaks of the CaS : Bi(0.08 mole%) sample have been determined using Chens method. The effect of different dopant concentrations and different heating rates has also been discussed.

Synthesis and luminescence studies of Ce doped SrS nanostructures

Cerium doped strontium sulfide nanostructures were synthesized by the solid state diffusion method in the presence of sodium thiosulfate. XRD confirmed the single phase rocksalt structure of the synthesized samples and the average grain size using the Debye–Scherrer relation is calculated to be 55 nm. TEM micrographs reveal the agglomerated whisker-like morphology with a diametre of 55–60 nm and length of several nanometres, which is in close agreement with XRD results. The effect of dopant concentration on photoluminescence (PL) intensity has been studied. PL emission for SrS : Ce (0.5 mol%) is at 481 nm with a shoulder at 530 nm at an excitation wavelength of 430 nm, which is attributed to the transitions from the 5d state to the 4f (2f7/2, 2f5/2) states of Ce3+. Ultraviolet and visible (UV–VIS) spectroscopy shows band-to-band absorption at 273 nm (4.54 eV), which is blue shifted in comparison to the band gap of bulk SrS (4.2 eV), which may be due to quantum confinement. The effect of high energy ball milling on the grain size and PL intensity has also been investigated for the first time in the doped SrS system. The PL emission wavelength is blue shifted by 3 nm but the emission intensity decreases unexpectedly as the milling time increases, although there is a reduction in size which is evident from XRD peak broadening of the milled samples. This may be ascribed to surface defects generated by ball milling which act as killing centres, quenching the PL.

2‐D gel electrophoresis‐based proteomic analysis reveals that ormeloxifen induces G0–G1 growth arrest and ERK‐mediated apoptosis in chronic myeloid leukemia cells K562

Ormeloxifen is a nonsteroidal selective estrogen receptor modulator (SERM) and has been shown to possess anticancer activities in breast and uterine cancer. Here, we show that ormeloxifen induces apoptosis in dose‐dependent manner in a variety of leukemia cells, more strikingly in K562. 2‐DE‐gel electrophoresis of K562 cells induced with ormeloxifen showed that 57 and 30% of proteins belong to apoptosis and cell‐cycle pathways, respectively. Our data demonstrate that ormeloxifen‐induced apoptosis in K562 cells involves activation of extracellular signal‐regulated kinases (ERKs) and subsequent cytochrome c release, leading to mitochondria‐mediated caspase‐3 activation. Ormeloxifen‐induced apoptosis via ERK activation was drastically inhibited by prior treatment of K562 cells with ERK inhibitor PD98059. Ormeloxifen also inhibits proliferation of K562 cells by blocking them in G0–G1 phase by inhibiting c‐myc promoter via ormeloxifen‐induced MBP‐1 (c‐myc promoter‐binding protein) and upregulation of p21 expression. We further show that ormeloxifen‐induced apoptosis in K562 is translatable to mononuclear cells isolated from chronic myeloid leukemia (CML) patients. Thus, ormeloxifen induces apoptosis in K562 cells via phosphorylation of ERK and arrests them in G0–G1 phase by reciprocal regulation of p21 and c‐myc. Therefore, inclusion of ormeloxifen in the therapy of chronic myeloid leukemia can be of potential utility.

Synthesis and characterization of bismuth doped calcium sulfide nanocrystallites

Nanosized bismuth doped calcium sulfide (CaS:Bi) particles have been synthesized by a wet chemical co-precipitation method. The average size of the nanoparticles was found to be about 30?nm. The particles were characterized by x-ray diffraction (XRD), transmission electron microscopy (TEM), UV?vis spectroscopy and fluorescence spectroscopy. The effect of particle size on the photoluminescence (PL) of CaS:Bi has been studied. The optimum dopant concentration was found to be 0.025?mol% of bismuth for maximum PL emission intensity. A comparative study between bulk CaS:Bi prepared by a reduction method and nanosized CaS:Bi made by the wet co-precipitation method has been carried out. Increase in band gap with decrease in particle size has been explained on the basis of the quantum size effect. The PL intensity of the nanoparticles was found to increase to almost twice that of the bulk particles. The effect of different dopant concentrations on emission intensity has also been studied.

E3 ubiquitin ligase Fbw7 negatively regulates granulocytic differentiation by targeting G-CSFR for degradation

Tight control between activation and attenuation of granulocyte colony stimulating factor receptor (G-CSFR) signaling is essential to regulate survival, proliferation and differentiation of myeloid progenitor cells. Previous studies demonstrated negative regulation of G-CSFR through endosomal-lysosomal routing and ubiquitin-proteasome mediated degradation. However, very few E3 ubiquitin ligases are known to target G-CSFR for ubiquitin-proteasome pathway. Here we identified F-box and WD repeat domain-containing 7 (Fbw7), a substrate recognizing component of Skp-Cullin-F box (SCF) E3 ubiquitin Ligase physically associates with G-CSFR and promotes its ubiquitin-mediated proteasomal degradation. Our data shows that Fbw7 also interacts with and degrades G-CSFR-T718 (a truncated mutant of G-CSFR found in severe congenital neutropenia/acute myeloid leukemia (SCN/AML patients)) though at a quite slower rate compared to G-CSFR. We further show that glycogen synthase kinase 3 beta (GSK3β), like Fbw7 also targets G-CSFR and G-CSFR-T718 for degradation; however, Fbw7 and GSK3β are interdependent in targeting G-CSFR/G-CSFR-T718 for degradation because they are unable to degrade G-CSFR individually when either of them is knocked down. We further show that Fbw7 mediated downregulation of G-CSFR inhibits signal transducer and activator of transcription 3 (STAT3) phosphorylation which is required for G-CSF dependent granulocytic differentiation. In addition, our data also shows that inhibition of Fbw7 restores G-CSFR signaling leading to enhanced STAT3 activity resulting in massive granulocytic differentiation. These data indicate that Fbw7 together with GSK3β negatively regulates G-CSFR expression and its downstream signaling.

Luminescence studies and effect of etching on cerium-doped CaS nanoparticles

Cerium-doped calcium sulphide nanoparticles were synthesized using the solid state diffusion method. The formed nanoparticles were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), UV-visible absorption spectroscopy and photoluminescence (PL) spectroscopy. The XRD pattern confirmed a cubic CaS phase with an average grain size of 53 nm of the formed samples. The TEM image showed non-agglomerated particles with an average size of 60 nm, which is in close agreement with the XRD result. The PL-emission spectrum showed peaks at 506 and 565 nm due to the transition from the excited state to the ground state of Ce3+. The effect of etching has been studied on the luminescent properties of CaS:Ce phosphors. With an increase in the etching time there is decrease in the size of the particles, as a result of which the PL spectrum showed a slight blue shift. The UV-visible absorption spectrum also showed a blue shift with an increase in etching time, which is in agreement with the nanosize effect.

Proteomic identification of E6AP as a molecular target of tamoxifen in MCF7 cells.

Tamoxifen (Tam) is most widely used selective estrogen receptor modulator (SERM) for treatment of hormone‐responsive breast cancer. Despite being regularly used in clinical therapy for breast cancer since 1971, the mechanism of Tam action remains largely unclear. In order to gain insights into Tam‐mediated antibreast cancer actions, we applied 2DE and MS based proteomics approach to identify target proteins of Tam. We identified E6‐associated protein, i.e. E6AP (UBE3A) among others to be regulated by Tam that otherwise is upregulated in breast tumors. We confirmed our 2DE finding by immunoblotting and further show that Tam leads to inhibition of E6AP expression presumably by promoting its autoubiquitination, which is coupled with nuclear export and subsequent proteasome‐mediated degradation. Furthermore, we show that Tam‐ and siE6AP‐mediated inhibition of E6AP leads to enhanced G0–G1 growth arrest and apoptosis, which is also evident from significant upregulation of cytochrome‐c, Bax, p21, and PARP cleavage. Taken together, our data suggest that, Tam‐targeted E6AP inhibition is in fact required for Tam‐mediated antibreast cancer actions. Thus, E6AP may be a therapeutic target in breast cancer.

E3 ubiquitin ligase E6AP negatively regulates adipogenesis by downregulating proadipogenic factor C/EBPalpha.

CCAAT/Enhancer Binding Protein Alpha (C/EBPα) is a key transcription factor involved in the adipocyte differentiation. Here for the first time we demonstrate that E6AP, an E3 ubiquitin ligase inhibits adipocyte differentiation in 3T3-L1 cells as revealed by reduced lipid staining with oil red. Knock down of E6AP in mouse 3T3L1 preadipocytes is sufficient to convert them to adipocytes independent of external hormonal induction. C/EBPα protein level is drastically increased in E6AP deficient 3T3L1 preadipocytes while inverse is observed when wild type E6AP is over expressed. We show that transient transfection of wild type E6AP downregulates C/EBPα protein expression in a dose dependent manner while catalytically inactive E6AP-C843A rather stabilizes it. In addition, wild type E6AP inhibits expression of proadipogenic genes while E6AP-C843A enhances them. More importantly, overexpression of E6AP-C843A in mesenchymal progenitor cells promotes accumulation of lipid droplets while there is drastically reduced lipid droplet formation when E6AP is over expressed. Taken together, our finding suggests that E6AP may negatively control adipogenesis by inhibiting C/EBPα expression by targeting it to ubiquitin-proteasome pathway for degradation.

E6AP, an E3 ubiquitin ligase negatively regulates granulopoiesis by targeting transcription factor C/EBPα for ubiquitin-mediated proteasome degradation.

CCAAT/enhancer-binding protein alpha (C/EBPα) is an important transcription factor involved in granulocytic differentiation. Here, for the first time we demonstrate that E6-associated protein (E6AP), an E3 ubiquitin ligase targets C/EBPα for ubiquitin-mediated proteasome degradation and thereby negatively modulates its functions. Wild-type E6AP promotes ubiquitin dependent proteasome degradation of C/EBPα, while catalytically inactive E6-associated protein having cysteine replaced with alanine at amino-acid position 843 (E6AP-C843A) rather stabilizes it. Further, these two proteins physically associate both in non-myeloid (overexpressed human embryonic kidney epithelium) and myeloid cells. We show that E6AP-mediated degradation of C/EBPα protein expression curtails its transactivation potential on its target genes. Noticeably, E6AP degrades both wild-type 42 kDa CCAAT-enhancer-binding protein alpha (p42C/EBPα) and mutant isoform 30 kDa CCAAT-enhancer-binding protein alpha (p30C/EBPα), this may explain perturbed p42C/EBPα/p30C/EBPα ratio often observed in acute myeloid leukemia (AML). We show that overexpression of catalytically inactive E6AP-C843A in C/EBPα inducible K562- p42C/EBPα-estrogen receptor (ER) cells inhibits β-estradiol (E2)-induced C/EBPα degradation leading to enhanced granulocytic differentiation. This enhanced granulocytic differentiation upon E2-induced activation of C/EBPα in C/EBPα stably transfected cells (β-estradiol inducible K562 cells stably expressing p42C/EBPα-ER (K562-C/EBPα-p42-ER)) was further substantiated by siE6AP-mediated knockdown of E6AP in both K562-C/EBPα-p42-ER and 32dcl3 (32D clone 3, a cell line widely used model for in vitro study of hematopoietic cell proliferation, differentiation, and apoptosis) cells. Taken together, our data suggest that E6AP targeted C/EBPα protein degradation may provide a possible explanation for both loss of expression and/or functional inactivation of C/EBPα often experienced in myeloid leukemia.