Uttam Pati

Design, synthesis and characterization of some bioactive conjugates of curcumin with glycine, glutamic acid, valine and demethylenated piperic acid and study of their antimicrobial and antiproliferative properties

The monoesters of curcumin, a symmetric diphenol with valine and glycine have been prepared by a novel solid phase synthesis and its diesters with valine, glutamic acid and demethylenated piperic acid have been prepared by solution phase method. The assessment of their antimicrobial and anticancer (antiproliferative) activities suggested that diesters of curcumin are relatively more active than curcumin itself due to their increased solubility, slow metabolism and better cellular uptake. Furthermore, significant observation was that monoesters of curcumin have even better antimicrobial activity than their corresponding diesters, emphasizing the role of free phenolic group. The conjugate of curcumin with demethylenated piperic acid in which methylenedioxy ring was open also shows enhanced activity than the corresponding piperic acid conjugate, emphasizing the role of free phenolics in the transport or in the binding processes.

Paraoxonase gene polymorphism and coronary artery disease in indian subjects

The human serum HDL-linked paraoxonase enzyme limits the LDL peroxidation by preventing transformation of LDL into biologically active atherogenic particles. Paraoxonase serum activity varies among individuals due to an Gln/Arg polymorphism with low (A phenotype) and high activity (B phenotype). The present study correlates the paraoxonase enzyme activity and the paraoxonase gene polymorphism among 200 Indians with or without coronary artery disease (CAD). We analyzed the PON enzyme activity and have identified A and B phenotypes by Alwl restriction mapping. In 120 CAD patients, the genotypes A and B constituted 75 and 25%, where as in 80 control subjects, the genotypes A and B constituted 25 and 17%, respectively. The frequency of AB genotype is higher in CAD subjects with or without diabetes, than in controls. Arg allele frequency was higher (0.45) in CAD subjects than in controls (0.17). The conventional risk factors and the family history of CAD did not affect the genotype frequency distribution among Indians. In conclusion, paraoxonase polymorphism may have been involved in the predisposition to CAD through a mechanism other than lipid oxidation.

CHIP Chaperones Wild Type p53 Tumor Suppressor Protein

Wild type p53 exists in a constant state of equilibrium between wild type and mutant conformation and undergoes conformational changes at elevated temperature. We have demonstrated that the co-chaperone CHIP (carboxyl terminus of Hsp70-interacting protein), which suppressed aggregation of several misfolded substrates and induced the proteasomal degradation of both wild type and mutant p53, physically interacts with the amino terminus of WT53 and prevented it from irreversible thermal inactivation. CHIP preferentially binds to the p53 mutant phenotype and restored the DNA binding activity of heat-denatured p53 in an ATP-independent manner. In cells under elevated temperatures that contained a higher level of p53 mutant phenotype, CHIP restored the native-like conformation of p53 in the presence of geldanamycin, whereas CHIP-small interfering RNA considerably increased the mutant form. Further, under elevated temperatures, the levels of CHIP and p53 were higher in nucleus, and chromatin immunoprecipitation shows the presence of p53 and CHIP together upon the DNA binding site in the p21 and p53 promoters. We propose that CHIP might be a direct chaperone of wild type p53 that helps p53 in maintaining wild type conformation under physiological condition as well as help resurrect p53 mutant phenotype into a folded native state under stress condition.

Gallium compound GaQ3-induced Ca2+ signalling triggers p53-dependent and -independent apoptosis in cancer cells

BACKGROUND AND PURPOSE A novel anti‐neoplastic gallium complex GaQ3 (KP46), earlier developed by us, is currently in phase I clinical trial. GaQ3 induced S‐phase arrest and apoptosis via caspase/PARP cleavage in a variety of cancers. However, the underlying mechanism of apoptosis is unknown. Here, we have explored the mechanism(s) of GaQ3‐induced apoptosis in cancer cells, focusing on p53 and intracellular Ca2+ signalling.

Chaperoning of Mutant p53 Protein by Wild-type p53 Protein Causes Hypoxic Tumor Regression * □

Background: Hypoxia-induced p53 is transcriptionally inactive, and its molecular conformation and functional status in hypoxic tumors are unknown. Results: WT p53 exists in mutant conformation in hypoxic tumors, and its conformation is oxygen-dependent. WT p53 functions as a molecular chaperone. Conclusion: WT p53 chaperones and rescues mutant p53 in hypoxic tumors. Significance: p53 chaperone therapy causes regression of hypoxic tumor xenografts through WT p53 chaperone activity. Mutant (Mt) p53 abrogates tumor suppression functions of wild-type (WT) p53 through mutant-specific, gain-of-function effects, and patients bearing Mt p53 are chemoresistant. The dominant negative effect of p53 mutants results from their aggregation propensity which causes co-aggregation of WT p53. We explored the mechanism of p53 inactivation in hypoxia and hypothesized whether WT p53 could rescue Mt p53 in hypoxic tumors. WT p53 exists in mutant conformation in hypoxic core of MCF-7 solid tumors, and its conformation is oxygen-dependent. Under simulated hypoxia in cells, WT p53 undergoes conformational change in acquiring mutant conformation. An in vivo chaperone assay shows that WT p53 functions as a molecular chaperone in rescuing conformational and structural p53 mutants in cancer cells both at the transcription and proteome levels. WT p53 chaperone therapy is further shown to cause significant regression of tumor xenografts through reconversion of the mutant phenotype to wild-type p53. The chaperone function of WT p53 is directly linked to the induction of apoptosis in both cancer cells and tumor xenografts. As oncogenic p53 mutants are linked to chemoresistance in hypoxic tumors, p53 chaperone therapy will introduce new dimensions to existing cancer therapeutics. We propose that in cancer cells, WT p53 chaperoning may either exist as a cellular event to potentially reverse the dominant negative effect of its oncogenic mutants or to stabilize yet unidentified factors.

p53 Ser15 phosphorylation disrupts the p53-RPA70 complex and induces RPA70-mediated DNA repair in hypoxia.

Cellular stressors are known to inhibit the p53-RPA70 (replication protein A, 70 kDa subunit) complex, and RPA70 increases cellular DNA repair in cancer cells. We hypothesized that regulation of RPA70-mediated DNA repair might be responsible for the inhibition of apoptosis in hypoxic tumours. We have shown that, in cancer cells, hypoxia disrupts the p53-RPA70 complex, thereby enhancing RPA70-mediated NER (nucleotide excision repair)/NHEJ (non-homologous end-joining) repair. In normal cells, RPA70 binds to the p53-NTD (N-terminal domain), whereas this binding is disrupted in hypoxia. Phosphorylation of p53-NTD is a crucial event in dissociating both NTD-RPA70 and p53-RPA70 complexes. Serial mutations at serine and threonine residues in the NTD confirm that p53(Ser15) phosphorylation induces dissociation of the p53-RPA70 complex in hypoxia. DNA-PK (DNA-dependent protein kinase) is shown to induce p53(Ser15) phosphorylation, thus enhancing RPA70-mediated NER/NHEJ repair. Furthermore, RPA70 gene silencing induces significant increases in cellular apoptosis in the resistant hypoxic cancer cells. We have thus elucidated a novel pathway showing how DNA-PK-mediated p53(Ser15) phosphorylation dissociates the p53-RPA70 complex, thus enhancing NER/NHEJ repair, which causes resistance to apoptosis in hypoxic cancer cells. This novel finding may open new strategies in developing cancer therapeutics on the basis of the regulation of RPA70-mediated NER/NHEJ repair.

SCO2 Induces p53-Mediated Apoptosis by Thr845 Phosphorylation of ASK-1 and Dissociation of the ASK-1–Trx Complex

ABSTRACT p53 prevents cancer via cell cycle arrest, apoptosis, and the maintenance of genome stability. p53 also regulates energy-generating metabolic pathways such as oxidative phosphorylation (OXPHOS) and glycolysis via transcriptional regulation of SCO2 and TIGAR. SCO2, a cytochrome c oxidase assembly factor, is a metallochaperone which is involved in the biogenesis of cytochrome c oxidase subunit II. Here we have shown that SCO2 functions as an apoptotic protein in tumor xenografts, thus providing an alternative pathway for p53-mediated apoptosis. SCO2 increases the generation of reactive oxygen species (ROS) and induces dissociation of the protein complex between apoptosis signal-regulating kinase 1 (ASK-1) (mitogen-activated protein kinase kinase kinase [MAPKKK]) and its cellular inhibitor, the redox-active protein thioredoxin (Trx). Furthermore, SCO2 induces phosphorylation of ASK-1 at the Thr845 residue, resulting in the activation of the ASK-1 kinase pathway. The phosphorylation of ASK-1 induces the activation of mitogen-activated protein kinase kinases 4 and 7 (MAP2K4/7) and MAP2K3/6, which switches the c-Jun N-terminal protein kinase (JNK)/p38-dependent apoptotic cascades in cancer cells. Exogenous addition of the SCO2 gene to hypoxic cancer cells and hypoxic tumors induces apoptosis and causes significant regression of tumor xenografts. We have thus discovered a novel apoptotic function of SCO2, which activates the ASK-1 kinase pathway in switching “on” an alternate mode of p53-mediated apoptosis. We propose that SCO2 might possess a novel tumor suppressor function via the ROS–ASK-1 kinase pathway and thus could be an important candidate for anticancer gene therapy.

CHIP stabilizes amyloid precursor protein via proteasomal degradation and p53‐mediated trans‐repression of β‐secretase

In patient with Alzheimers disease (AD), deposition of amyloid‐beta Aβ, a proteolytic cleavage of amyloid precursor protein (APP) by β‐secretase/BACE1, forms senile plaque in the brain. BACE1 activation is caused due to oxidative stresses and dysfunction of ubiquitin–proteasome system (UPS), which is linked to p53 inactivation. As partial suppression of BACE1 attenuates Aβ generation and AD‐related pathology, it might be an ideal target for AD treatment. We have shown that both in neurons and in HEK‐APP cells, BACE1 is a new substrate of E3‐ligase CHIP and an inverse relation exists between CHIP and BACE1 level. CHIP inhibits ectopic BACE1 level by promoting its ubiquitination and proteasomal degradation, thus reducing APP processing; it stabilizes APP in neurons, thus reducing Aβ. CHIPUbox domain physically interacts with BACE1; however, both U‐box and TPR domain are essential for ubiquitination and degradation of BACE1. Further, BACE1 is a downstream target of p53 and overexpression of p53 decreases BACE1 level. In HEK‐APP cells, CHIP is shown to negatively regulate BACE1 promoter through stabilization of p53s DNA‐binding conformation and its binding upon 5′ UTR element (+127 to +150). We have thus discovered that CHIP regulates p53‐mediated trans‐repression of BACE1 at both transcriptional and post‐translational level. We propose that a CHIP–BACE1–p53 feedback loop might control APP stabilization, which could further be utilized for new therapeutic intervention in AD.

Stress response of a p53 homologue in the radioresistant Sf9 insect cells.

Purpose: To investigate homology and stress response of p53 (a 53 kDa tumor suppressor protein) orthologue in Sf9 Lepidopteran insect cell line that exhibits very high radioresistance. Materials and methods: Western immunoblotting, immunoprecipitation, degenerate RT-PCR (reverse transcription-polymerase chain reaction), electrophoretic gel mobility shift assay, flow cytometry and immuno-fluorescence microscopy were used for characterizing structural and functional features of Sfp53 (Spodoptera frugiperda p53) in γ-irradiated or etoposide-treated Sf9 insect and BMG-1 (brain malignant glioma) human cells. Cells were pre-treated with caffeine for inhibiting ATM/ATR (ataxia-telangiectasia mutated protein/ATM and Rad-3-related protein) activation, wherever required. Results: A 47–49 kDa protein band was observed with antibodies against three different epitopes, demonstrating conservation of respective domains in Sfp53. Immunoprecipitation also yielded similar-sized protein. Degenerate RT-PCR resulted in product of same size in both cell lines. Similar gel mobility shift of p53-binding oligonucleotide with BMG-1 and Sf9 cell lysates indicated analogous transcriptional activity of Sfp53. Constitutive Sfp53 level was higher than hp53 (human p53) and showed primarily cytoplasmic localization. Radiation-induced accumulation was considerably less in Sf9 even as an analogous ATM/ATR-dependent nuclear translocation was observed following γ-irradiation and etoposide. Conclusions: A smaller-sized Sfp53 orthologue shows highly conserved native structure with DNA-binding, N-terminus and C-terminus domains, and has analogous p53 transcriptional activity. While its nuclear translocation and ATM/ATR dependence were similar to hp53, the cytoplasmic localization and subdued accumulation following γ-irradiation indicate functional differences from human cells.

A proximal tissue-specific module and a distal negative regulatory module control apolipoprotein(a) gene transcription

The apo(a) [apolipoprotein(a)] gene is responsible for variations in plasma lipoprotein(a), high levels of which are a risk factor for atherosclerosis and myocardial infarction. The apo(a) promoter stimulates the expression of reporter genes in HepG2 cells, but not in HeLa cells. In the present study, we demonstrate that the 1.4 kb apo(a) promoter comprises two composite regulatory regions: a distal negative regulatory module (positions -1432 to -716) and a proximal tissue-specific module (-716 to -616). The distal negative regulatory module contains two strong negative regulatory regions [polymorphic PNR (pentanucleotide repeat region) and NREbeta (negative regulatory element beta)], which sandwich the postive regulatory region PREbeta (positive regulatory element beta). The PNR was shown to bind to transcription factors in a tissue-specific manner, whereas the ubiquitous transcription factors hepatocyte nuclear factor 3alpha and GATA binding protein 4 bound to NREbeta to repress gene transcription. The proximal tissue-specific module contains two regulatory elements: an activating region (PREalpha) that activates transcription in HepG2 cells, and NREalpha, which is responsible for repressing the apo(a) gene in HeLa cells. NREalpha binds to a HeLa-specific repressor. These multiple regulatory elements might work co-operatively to finely regulate apo(a) gene expression. Although the tissue-specific module is required for apo(a) gene activation and repression in a tissue-specific manner, the combinatorial interplay of the distal and proximal regulators might define the complex pathway(s) of apo(a) gene regulation.