Antiproliferative effect of diallyl disulfide (DADS) on prostate cancer cell line LNCaP

Garlic has been used throughout the world to treat coughs, toothache, earache, dandruff, hypertension, hysteria, diarrhoea, dysentery, diptheria, vaginitis and many other conditions. Garlic contains a complex mixture of oil and water-soluble organosulfur compounds. Diallyl disulfide (DADS), an oil-soluble constituent of garlic seems to be effective in reducing tumour cells originating from colon, lung and skin. Hence our present study focuses on the dose-dependent effect of DADS on an androgen-dependent prostate cancer cell line. Various concentrations of DADS ranging from 25 to 100 microM were given to LNCaP cells and the activity of lactate dehydrogenase (LDH) prostatic acid phosphatase (PAcP) and the level of prostate specific antigen were studied. DADS reduced the secretory activity of LNCaP cells with the gradual increase in dosage. DADS was found to act as a good antiproliferative agent, which was confirmed by proliferation assay. DADS also induced apoptosis and nuclear segmentation in the higher doses.     

Quercetin regulates insulin like growth factor signaling and induces intrinsic and extrinsic pathway mediated apoptosis in androgen independent prostate cancer cells (PC-3)

Progression of prostate cancer is facilitated by growth factors that activate critical signaling cascades thereby promote prostate cancer cell growth, survival, and migration. To investigate the effect of quercetin on insulin-like growth factor signaling and apoptosis in androgen independent prostate cancer cells (PC-3), IGF-IR, PI-3K, p-Akt, Akt, cyclin D1, Bad, cytochrome c, PARP, caspases-9 and 10 protein levels were assessed by western blot analysis. Mitochondrial membrane potency was detected by rhodamine-123 staining. Quercetin induced caspase-3 activity assay was performed for activation of apoptosis. Further, RT-PCR was also performed for Bad, IGF-I, II, IR, and IGFBP-3 mRNA expression. Quercetin significantly increases the proapoptotic mRNA levels of Bad, IGFBP-3 and protein levels of Bad, cytochrome C, cleaved caspase-9, caspase-10, cleaved PARP and caspase-3 activity in PC-3 cells. IGF-IRβ, PI3K, p-Akt, and cyclin D1 protein expression and mRNA levels of IGF-I, II and IGF-IR were decreased significantly. Further, treatment with PI3K inhibitor (LY294002) and quercetin showed decreased p-Akt levels. Apoptosis is confirmed by loss of mitochondrial membrane potential in quercetin treated PC-3 cells. This study suggests that quercetin decreases the survival of androgen independent prostate cancer cells by modulating the expression of insulin-like growth factors (IGF) system components, signaling molecules and induces apoptosis, which could be very useful for the androgen independent prostate cancer treatment.     

Novel intestinal splice variants of RNA-binding protein CUGBP2: isoform-specific effects on mitotic catastrophe

CUG triplet repeat-binding protein 2 (CUGBP2) is a RNA-binding protein that regulates mRNA translation and modulates apoptosis. Here, we report the identification of two splice variants (termed variants 2 and 3) in cultured human intestinal epithelial cells and in mouse gastrointestinal tract. The variants are generated from alternative upstream promoters resulting in the inclusion of additional NH(2)-terminal residues. Although variant 2 is the predominant isoform in normal intestine, its expression is reduced, whereas variant 1 is overexpressed following gamma-irradiation. All three variants bind cyclooxygenase-2 (COX-2) mRNA. However, only variant 1 inhibits the translation of the endogenous COX-2 mRNA and a chimeric luciferase mRNA containing the COX-2 3'untranslated region. Furthermore, whereas variant 1 is predominantly nuclear, variants 2 and 3 are predominantly cytoplasmic. These data imply that the additional amino acids affect CUGBP2 function. Previous studies have demonstrated that variant 1 induces intestinal epithelial cells to undergo apoptosis. However, in contrast to variant 1, the two novel variants do not affect proliferation or apoptosis of HCT116 cells. In addition, only variant 1 induced G(2)/M cell cycle arrest, which was overcome by prostaglandin E(2). Moreover, variant 1 increased cellular levels of phosphorylated p53 and Bax and decreased Bcl2. Caspase-3 and -9 were also activated, suggesting the initiation of the intrinsic apoptotic pathway. Furthermore, increased phosphorylation of checkpoint kinase (Chk)1 and Chk2 kinases and increased nuclear localization of Cdc2 and cyclin B1 suggested that cells were in mitotic transition. Taken together, these data demonstrate that cells expressing CUGBP2 variant 1 undergo apoptosis during mitosis, suggesting mitotic catastrophe.     

Purification, cloning, and DNA sequence analysis of a chitinase from an overproducing mutant of Streptomyces peucetius defective in daunorubicin biosynthesis

Extracellular chitinases of Streptomyces peucetius and a chitinase overproducing mutant, SPVI, were purified to homogeneity by ion exchange and gel filtration chromatography. The purified enzyme has a molecular mass of 42 kDa on SDS-PAGE, and the N-terminal amino acid sequence of the protein from the wild type showed homology to catalytic domains (Domain IV) of several other Streptomyces chitinases such as S. lividans 66, S. coelicolor A3(2), S. plicatus, and S. thermoviolaceus OPC-520. Purified SPVI chitinase cross-reacted to anti-chitinase antibodies of wild-type S. peucetius chitinase. A genomic library of SPVI constructed in E. coli using lambda DASH II was probed with chiC of S. lividans 66 to screen for the chitinase gene. A 2.7 kb fragment containing the chitinase gene was subcloned from a lambda DASH II clone, and sequenced. The deduced protein had a molecular mass of 68 kDa, and showed domain organization similar to that of S. lividans 66 chiC. The N-terminal amino acid sequence of the purified S. peucetius chitinase matched with the N-terminus of the catalytic domain, indicating the proteolytic processing of 68 kDa chitinase precursor protein to 42 kDa mature chitinase containing the catalytic domain only. A putative chiR sequence of a two-component regulatory system was found upstream of the chiC sequence.     

Isolation and characterization of stable mutants ofStreptomyces peucetius defective in daunorubicin biosynthesis

Daunorubicin and its derivative doxorubicin are antitumour anthracycline antibiotics produced byStreptomyces peucetius. In this study we report isolation of stable mutants ofS. peucetius blocked in different steps of the daunorubicin biosynthesis pathway. Mutants were screened on the basis of colony colour since producer strains are distinctively coloured on agar plates. Different mutants showed accumulation of aklaviketone, ε-rhodomycinone, maggiemycin or 13-dihydrocarminomycin in their culture filtrates. These results indicate that the mutations in these isolates affect steps catalysed bydnrE (mutants SPAK and SPMAG),dnrS (SPFS and SPRHO) anddoxA (SPDHC) gene products.     

Characterization of the redox components of transplasma membrane electron transport system from Leishmania donovani promastigotes

An investigation has been made of the points of coupling of four nonpermeable electron acceptors e.g., alpha-lipoic acid (ALA), 5,5'-dithiobis (2-nitroaniline-N-sulphonic acid) (DTNS), 1,2-naphthoquinone-4-sulphonic acid (NQSA) and ferricyanide which are mainly reduced via an interaction with the redox sites present in the plasma membrane of Leishmania donovani promastigotes. ALA, DTNS, NQSA and ferricyanide reduction and part of O2 reduction is shown to take place on the exoplasmic face of the cell, for it is affected by external pH and agents that react with the external surface. Redox enzymes of the transplasma membrane electron transport system orderly transfer electron from one redox carrier to the next with the molecular oxygen as the final electron acceptor. The redox carriers mediate the transfer of electrons from metabolically generated reductant to nonpermeable electron acceptors and oxygen. At a pH of 6.4, respiration of Leishmania cells on glucose substrate shut down almost completely upon addition of an uncoupler FCCP and K+-ionophore valinomycin. The most pronounced effects on O2 uptake were obtained by treatment with antimycin A, 2-heptadecyl-4-hydroxyquinone-N-oxide, paracholoromercuribenzene sulphonic acid and trifluoperazine. Relatively smaller effects were obtained by treatment with potassium cyanide. Inhibition observed with respect to the reduction of the electron acceptors ALA, DTNS, NQSA and ferricyanide was not similar in most cases. The redox chain appears to be branched at several points and it is suggested that this redox chain incorporate iron-sulphur center, b-cytochromes, cyanide insensitive oxygen redox site, Na+ and K+ channel, capsaicin inhibited energy coupling site and trifluoperazine inhibited energy linked P-type ATPase. We analyzed the influence of ionic composition of the medium on reduction of electron acceptors in Leishmania donovani promastigotes. Our data suggest that K+ have some role for ALA reduction and Na+ for ferricyanide reduction. No significant effects were found with DTNS and NQSA reduction when Na+ or K+ was omitted from the medium. Stimulation of ALA, DTNS, NQSA and ferricyanide reduction was obtained by omitting Cl- from the medium. We propose that this redox system may be an energy source for control of membrane function in Leishmania cells.     

Water-stress-induced heat tolerance in geranium leaf tissues: A possible linkage through stress proteins?

Evidence is accumulating in favor of a linkage at the cellular level between various abiotic stresses. We conducted a study to evaluate the effect of water stress on the heat tolerance of zonal geraniums, Pelargonium × hortorum cv. Evening Glow. Water stress was imposed by withholding irrigation until pots reached 30% (by weight) of well‐watered controls, and by maintaining the pot weight by additions of water for another 7 days. Leaf xylem water potential (XWP, MPa), relative water content (RWC. %), and heat‐stress tolerance (HST; LT₅₀, defined as the temperature causing half‐maximal % injury based on electrolyte leakage) were measured in control, stressed, and recovered plants. Proteins were extracted from the leaves following the above treatments, and SDS‐PAGE and immunoblotting were performed by using standard procedures. Immunoblots were probed with antibodies to dehydrin and 70‐kDa heat shock cognate (HSC70) proteins. Data indicate that XWP and RWC, respectively, were −0.378 MPa and 92.3% for control plants and −0.804 MPa and 78.6% for stressed plants. Water‐stressed plants exhibited a significant increase in HST compared to control (LT₅₀ of 55°C vs 51°C). Water‐stress‐induced HST was not due to heat acclimation (leaf warming in stressed plants). Data also indicate that water‐stress treatment did not increase freezing tolerance of geranium leaves. Increased HST was associated with the accumulation of several heat‐stable, dehydrin proteins (25–60 kDa), and both cytosolic and ER luminal (BiP) HSC70 proteins. Leaf XWP, RWC, and HST reversed to control levels concomitant with the disappearance/reduction of dehydrins and HSC70 proteins in water‐stress‐relieved plants. The possibility of a cellular linkage between water stress and heat‐stress tolerance is discussed.     

Dynamic Ligand Discrimination in the Notch Signaling Pathway

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