Interplay of reactive oxygen species,intracellular Ca2+ and mitochondrial homeostasis in the apoptosis of prostate cancer cells by deoxypodophyllotoxin

The limited treatment option for recurrent prostate cancer and the eventual resistance to conventional chemotherapy drugs has fueled continued interest in finding new anti‐neoplastic agents of natural product origin. We previously reported anti‐proliferative activity of deoxypodophyllotoxin (DPT) on human prostate cancer cells. Using the PC‐3 cell model of human prostate cancer, the present study reveals that DPT induced apoptosis via a caspase‐3‐dependent pathway that is activated due to dysregulated mitochondrial function. DPT‐treated cells showed accumulation of the reactive oxygen species (ROS), intracellular Ca surge, increased mitochondrial membrane potential (MMP, ΔΨ_m), Bax protein translocation to mitochondria and cytochrome c release to the cytoplasm. This resulted in caspase‐3 activation, which in turn induced apoptosis. The antioxidant N‐acetylcysteine (NAC) reduced ROS accumulation, MMP and Ca surge, on the other hand the Ca²⁺ chelator BAPTA inhibited the Ca overload and MMP without affecting the increase of ROS, indicating that the generation of ROS occurred prior to Ca²⁺ flux. This suggested that both ROS and Ca signaling play roles in the increased MMP via Ca‐dependent and/or ‐independent mechanisms, since ΔΨ_m elevation was reversed by NAC and BAPTA. This study provides the first evidence for the involvement of both ROS‐ and Ca‐activated signals in the disruption of mitochondrial homeostasis and the precedence of ROS production over the failure of Ca²⁺ flux homeostasis. J. Cell. Biochem. 114: 1124–1134, 2013. © 2012 Wiley Periodicals, Inc.     

Metaphase arrest and delay in cell cycle kinetics of root apical meristems and mouse bone marrow cells treated with leaf aqueous extract of Clerodendrum viscosum Vent

Objectives

To study cell cycle delay and metaphase arresting activity of leaf aqueous extract of Clerodendrum viscosum Vent. (LAECV) in root apical meristems and mouse bone marrow cells.

Materials and methods

Cell cycle delay and metaphase arresting activities of LAECV were analysed, in root apical meristems of onion and wheat, and in mouse bone marrow cells, by scoring mitotic index, metaphase frequency and transition of cells from metaphase to anaphase. Colchicine was used as the standard metaphase arresting drug. Phytochemicals present in LAECV were detected and their phytotoxic activity was evaluated by analysing green‐gram (Vigna radiata) seedling's root growth retardation and branch root swelling phenomenon.

Results

LAECV treatment resulted in dose‐dependent root growth retardation of green‐gram seedling root length (P < 0.01) and half maximal growth inhibitory concentration (IC₅₀) of LAECV was 0.87 mg/ml at 144 h. In onion and wheat root meristem cells the mitotic index decreased, metaphase frequency increased and transition from metaphase to anaphase reduced. Experimentation with mouse bone marrow cells indicated that LAECV induced metaphase arrest (164.3% increase in arrested metaphases per 300 mg/kg body weight, over 2.5 h). Phytochemicals like carbohydrates, glycosides, saponins, terpenoids, triterpenoids, tannins and trace amounts of alkaloids were detected in LAECV.

Conclusion

It may be said that LAECV contains mitostatic and metaphase arresting components that are able to induce significant metaphase arrest in root apical meristems and also in mouse bone marrow cells.

     

Selective induction of apoptosis in various cancer cells irrespective of drug sensitivity through a copper chelate, copper N-(2 hydroxy acetophenone) glycinate: crucial involvement of glutathione

Drug induced toxicity and drug resistance are the major impediments to successful application of cancer chemotherapy. Therefore, selective targeting of the key biochemical events of the malignant cells may have a great therapeutic potential in specifically kill the cancer cells. We have evaluated in vitro the cytotoxic efficacy of a previously reported copper complex viz. copper N-(2-hydroxy acetophenone) glycinate (CuNG) on different drug sensitive and resistant cancer cell lines by MTT, annexin V positivity and caspase 3 activation assays. We have also investigated the underlying signalling events in CuNG mediated apoptosis of cancer cells by Western blotting technique. We have found that CuNG preferentially induces apoptosis to malignant cells irrespective of drug sensitivity and spares the normal cells. Our studies disclose that CuNG causes cellular redox imbalance in cancer cells through depletion of intracellular GSH level. CuNG mediated depletion of intracellular GSH level induces mitochondrial superoxide generation, which detaches cyto C from mitochondrial membrane through lipid peroxidation. The detached cyto C then release into the extra mitochondrial milieu in Bax mediated pathway where CuNG facilitates the binding of Bax through dissociation of hexokinase II from mitochondrial membrane. The present study opens the possibility of developing effective chemotherapeutic drugs by synthesizing numerous chemical compounds capable of targeting cellular redox environment and thus specifically kills cancer cells of broad spectrum.     

Polymorphism in the Prolactin Promoter and Its Association with Growth Traits in Chickens

The pituitary hormone prolactin has a wide variety of functions involving growth, behavioral, and ovarian activities in chickens. The objectives of the present study were to identify polymorphisms in the prolactin promoter and estimate their effects on growth traits in White Leghorn chickens. Among 28 haplotypes found, the h1 haplotype was predominant. Body weight at 16 and 64 weeks and age at sexual maturity were significantly associated with haplotype combinations (P < 0.05). The h1/h1 haplogroup showed the highest body weight at 16 weeks of age, and h1/h7 was the highest at 64 weeks. The lowest age at sexual maturity was found in birds with the h1/h6 haplotype combination, and mRNA expression of prolactin was lowest in h1/h4 birds and highest in h1/h5 birds. The prolactin promoter was highly polymorphic and had significant associations with growth traits in White Leghorn chickens.     

Sir-2.1 modulates 'calorie-restriction-mediated' prevention of neurodegeneration in Caenorhabditis elegans: implications for Parkinson's disease

The phenomenon of aging is known to modulate many disease conditions including neurodegenerative ailments like Parkinson’s disease (PD) which is characterized by selective loss of dopaminergic neurons. Recent studies have reported on such effects, as calorie restriction, in modulating aging in living systems. We reason that PD, being an age-associated neurodegenerative disease might be modulated by interventions like calorie restriction. In the present study we employed the transgenic Caenorhabditis elegans model (P_dat-1::GFP) expressing green fluorescence protein (GFP) specifically in eight dopaminergic (DA) neurons. Selective degeneration of dopaminergic neurons was induced by treatment of worms with 6-hydroxy dopamine (6-OHDA), a selective catecholaminergic neurotoxin, followed by studies on effect of calorie restriction on the neurodegeneration. Employing confocal microscopy of the dopaminergic neurons and HPLC analysis of dopamine levels in the nematodes, we found that calorie restriction has a preventive effect on dopaminergic neurodegeneration in the worm model. We further studied the role of sirtuin, sir-2.1, in modulating such an effect. Studies employing RNAi induced gene silencing of nematode sir-2.1, revealed that presence of Sir-2.1 is necessary for achieving the protective effect of calorie restriction on dopaminergic neurodegeneration.Our studies provide evidence that calorie restriction affords, an sir-2.1 mediated, protection against the dopaminergic neurodegeneration, that might have implications for neurodegenerative Parkinson’s disease.     

Accessory cholera enterotoxin, Ace, from Vibrio cholerae: structure, unfolding, and virstatin binding

Vibrio cholerae accessory cholera enterotoxin (Ace) is the third toxin, along with cholera toxin (CT) and zonula occludens toxin (Zot), that causes the endemic disease cholera. Structural characterization of Ace has been restricted because of the limited production of this toxic protein by V. cholerae. We have cloned, overexpressed, and purified Ace from V. cholerae strain O395 in Escherichia coli to homogeneity and determined its biological activity. The unfolding of the purified protein was investigated using circular dichroism and intrinsic tryptophan fluorescence. Because Ace is predominantly a hydrophobic protein, the degree of exposure of hydrophobic regions was identified from the spectral changes of the environment-sensitive fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) that quenches the fluorescence of tryptophan residues of Ace in a concentration-dependent manner. Results showed that bis-ANS binds one monomeric unit of Ace with a 1:1 stoichiometry and a K' of 0.72 μM. Ace exists as a dimer, with higher oligomeric forms appearing upon glutaraldehyde cross-linking. This study also reports the binding of virstatin, a small molecule that inhibits virulence regulation in V. cholerae, to Ace. The binding constant (K=9×10(4) M(-1)) and the standard free energy change (ΔG°=-12 kcal mol(-1)) of Ace-virstatin interaction have been evaluated by the fluorescence quenching method. The binding does not affect the oligomeric status of Ace. A cell viability assay of the antibacterial activity of Ace has been performed using various microbial strains. A homology model of Ace, consistent with the experimental results, has been constructed.     

Allometric shape vector projection: a new method for the identification of allometric shape characters and trajectories applied to the human astragalus (talus)

The surface morphology of the human astragalus (talus) is difficult to represent accurately using landmarks as it is essentially globular in shape. Advances in laser scanning technology allow fast and accurate capture of bone surface morphology. However, methodologies to utilise these new accurate 3D data have not been fully developed. The present study uses canonical sampling of whole surface morphology attained through laser scanning and for the first time applies the technique to analysis of bone morphology. We introduce a new technique for identifying allometric shape characters in whole bone surface morphology. In a sample of adult human astragalus the new technique is successful in identifying and isolating intra-specific allometric shape characters in a bone which typically lacks landmarks and has, consequently, proved difficult to analyse using traditional 3D morphometric methods.     

Associations between novel polymorphisms at the 5'-UTR region of the prolactin gene and egg production and quality in chickens

The objective was to characterize polymorphisms at the 5′-UTR region of the prolactin gene, and determine their association with egg production and egg quality traits in White Leghorn chickens. The study was conducted on four strains of White Leghorn chickens, namely IWH, IWI, IWK, and layer control. Overall, there were three alleles (designated A, B, and C) and five genotypes, with genotypic frequencies of 0.09, 0.75, 0.07, 0.02, and 0.07 for AA, AB, AC, BB, and BC, respectively. There were significant differences among genotypes for egg production up to 52 and 64 wk of age, with maximal egg yields for genotypes AA and AC (144.5 ± 5.06 and 143.2 ± 4.67 eggs, respectively). Furthermore, there were significant differences among genotypes for egg quality traits, including egg weight and Haugh unit at 40 wk of age, Haugh unit at 52 wk, and yolk color index and Haugh unit at 64 wk. Birds with AA or AC genotypes had the best egg quality traits. On the contrary, these genotypes had the lowest prolactin expression, whereas this expression was highest in birds with the BB genotype. In conclusion, polymorphisms at the 5′-UTR of prolactin gene were significantly associated with egg production and egg quality traits in White Leghorn chickens.