Chimeric caspase molecules with potent cell killing activity in apoptosis-resistant cells

Cellular defects which prevent apoptotic cell death can result in the generation of hyperproliferative disorders and can prevent the effective treatment of such diseases. The majority of cellular defects which result in apoptosis resistance lie upstream of caspase activation. We have described chimeric caspase molecules consisting of the prodomain of caspase-2 fused to the amino terminus of caspase-3, and which are tagged at the carboxyl terminus with green fluorescent protein (GFP) to allow direct visualisation of transfected cells. Here we show that these chimeric caspase molecules possess potent, rapid cell-killing activity in cell lines which display a range of defects resulting in apoptosis resistance.     

MT-4 Suppresses Resistant Ovarian Cancer Growth through Targeting Tubulin and HSP27

Objective

In this study, the anticancer mechanisms of MT-4 were examined in A2780 and multidrug-resistant NCI-ADR/res human ovarian cancer cell lines.

Methods

To evaluate the activity of MT-4, we performed in vitro cell viability and cell cycle assays and in vivo xenograft assays. Immunoblotting analysis was carried out to evaluate the effect of MT-4 on ovarian cancer. Tubulin polymerization was determined using a tubulin binding assay.

Results

MT-4 (2-Methoxy-5-[2-(3,4,5-trimethoxy-phenyl)-ethyl]-phenol), a derivative of moscatilin, can inhibit both sensitive A2780 and multidrug-resistant NCI-ADR/res cell growth and viability. MT-4 inhibited tubulin polymerization to induce G2/M arrest followed by caspase-mediated apoptosis. Further studies indicated that MT-4 is not a substrate of P-glycoprotein (p-gp). MT-4 also caused G2/M cell cycle arrest, accompanied by the upregulation of cyclin B, p-Thr161 Cdc2/p34, polo-like kinase 1 (PLK1), Aurora kinase B, and phospho-Ser10-histone H3 protein levels. In addition, we found that p38 MAPK pathway activation was involved in MT-4-induced apoptosis. Most importantly, MT-4 also decreased heat shock protein 27 expression and reduced its interaction with caspase-3, which inured cancer cells to chemotherapy resistance. Treatment of cells with SB203580 or overexpression of dominant negative (DN)-p38 or wild-type HSP27 reduced PARP cleavage caused by MT-4. MT-4 induced apoptosis through regulation of p38 and HSP27. Our xenograft models also show the in vivo efficacy of MT-4. MT-4 inhibited both A2780 and NCI-ADR/res cell growth in vitro and in vivo.

Conclusion

These findings indicate that MT-4 could be a potential lead compound for the treatment of multidrug-resistant ovarian cancer.     

Defining the Minimal Important Difference for the Visual Analogue Scale Assessing Dyspnea in Patients with Malignant Pleural Effusions

Background

The minimal important difference (MID) is essential for interpreting the results of randomised controlled trials (RCTs). Despite a number of RCTs in patients with malignant pleural effusions (MPEs) which use the visual analogue scale for dyspnea (VASD) as an outcome measure, the MID has not been established.

Methods

Patients with suspected MPE undergoing a pleural procedure recorded their baseline VASD and their post-procedure VASD (24 hours after the pleural drainage), and in parallel assessed their breathlessness on a 7 point Likert scale.

Findings

The mean decrease in VASD in patients with a MPE reporting a ‘small but just worthwhile decrease’ in their dyspnea (i.e. equivalent to the MID) was 19mm (95% CI 14-24mm). The mean drainage volume required to produce a change in VASD of 19mm was 760ml.

Interpretation

The mean MID for the VASD in patients with a MPE undergoing a pleural procedure is 19mm (95% CI 14-24mm). Thus choosing an improvement of 19mm in the VASD would be justifiable in the design and analysis of future MPE studies.     

Caffeine abolishes the mammalian G(2)/M DNA damage checkpoint by inhibiting ataxia-telangiectasia-mutated kinase activity

Recent evidence indicates that arrest of mammalian cells at the G(2)/M checkpoint involves inactivation and translocation of Cdc25C, which is mediated by phosphorylation of Cdc25C on serine 216. Data obtained with a phospho-specific antibody against serine 216 suggest that activation of the DNA damage checkpoint is accompanied by an increase in serine 216 phosphorylated Cdc25C in the nucleus after exposure of cells to gamma-radiation. Prior treatment of cells with 2 mM caffeine inhibits such a change and markedly reduces radiation-induced ataxia-telangiectasia-mutated (ATM)-dependent Chk2/Cds1 activation and phosphorylation. Chk2/Cds1 is known to localize in the nucleus and to phosphorylate Cdc25C at serine 216 in vitro. Caffeine does not inhibit Chk2/Cds1 activity directly, but rather, blocks the activation of Chk2/Cds1 by inhibiting ATM kinase activity. In vitro, ATM phosphorylates Chk2/Cds1 at threonine 68 close to the N terminus, and caffeine inhibits this phosphorylation with an IC(50) of approximately 200 microM. Using a phospho-specific antibody against threonine 68, we demonstrate that radiation-induced, ATM-dependent phosphorylation of Chk2/Cds1 at this site is caffeine-sensitive. From these results, we propose a model wherein caffeine abrogates the G(2)/M checkpoint by targeting the ATM-Chk2/Cds1 pathway; by inhibiting ATM, it prevents the serine 216 phosphorylation of Cdc25C in the nucleus. Inhibition of ATM provides a molecular explanation for the increased radiosensitivity of caffeine-treated cells.     

Modulation of endothelial nitric oxide synthase by fibronectin

Nitric oxide (NO) produced by the action of endothelial nitric oxide synthase (eNOS) plays an important role in the regulation of vascular tone, cell survival, and angiogenesis. Interaction of endothelial cells (ECs) with a fibronectin (FN) rich matrix is important in the regulation of EC function and survival during angiogenesis. The present study was carried out to examine if FN can regulate eNOS and thereby NO levels in ECs. The activity and the levels of mRNA and protein of eNOS were significantly low in HUVECs maintained in culture on FN. Inhibition of p38 MAPK and blocking the interaction of FN with α₅β₁ integrin using antibody caused the reversal of the FN effect. Immunoblot analysis of Ser/Thr phosphorylation of purified eNOS suggested that FN downregulates post-translational phosphorylation of eNOS at Ser residues. These results suggest that FN negatively modulates eNOS in an α₅β₁ integrin-p38 MAPK-dependent pathway.     

Preservation of boar semen at 18 degrees C induces lipid peroxidation and apoptosis like changes in spermatozoa

Boar sperm functions, lipid peroxidation status, mitochondrial membrane potential (DeltaPsi(m)) and membrane permeability (apoptosis like features) were assessed during liquid preservation. Four ejaculates each from four Hampshire boars were extended with Beltsville Thawing Solution and preserved at 18 degrees C. At 0, 24, 48, 72 and 96 h of storage, each ejaculate was examined for sperm functions, lipid peroxidation, DeltaPsi(m), and membrane permeability. The lipid peroxidation status of the sperm was assessed based on the malonaldehyde (MDA) levels. Detection of DeltaPsi(m) was done using 3,3'-dihexyloxacarbocyanine iodide [DiOC(6)(3)]/propidium iodide (PI) assay and Yo-pro-1/PI assay was used to detect change in plasma membrane permeability. The sperm motility, viability and acrosomal integrity declined significantly (p<0.05) from 0 to 96 h of preservation. At the start of the preservation, the MDA levels (nM/10(9) sperm) were low in sperm (99.83+/-2.69) and seminal plasma (191.98+/-11.58), which gradually increased up to the 96 h of storage. Highest negative correlation (r value) was observed between MDA levels and sperm motility (-0.97), live percent (-0.97), acrosomal integrity (-0.97) and hypo-osmotic sperm swelling test (HOSST) positive sperm percentage (-0.98). Strong positive correlation was observed between HOSST positive sperm percentage and intact acrosome percentage (r=0.98). There was a significant (p<0.05) increase in the sperm cells with low DeltaPsi(m) from 0 to 96 h of preservation. Before preservation, 14.85+/-4.66% of sperm cells of the ejaculate showed low mitochondrial membrane potential, whereas after 96 h of preservation, this proposition of cells increased up to 32.00+/-6.25%. The apoptotic sperm population was 8.33+/-2.31% in fresh semen, while this population was 25.19+/-4.25% at 96 h of preservation and the difference was significant (p<0.05). The findings of the present study revealed that liquid preservation of boar semen at 18 degrees C induces lipid peroxidation, decrease mitochondrial membrane potential and increase the plasma membrane permeability.     

A Novel Approach to Flurbiprofen Pulsatile Colonic Release: Formulation and Pharmacokinetics of Double-Compression-Coated Mini-Tablets

A significant plan is executed in the present study to study the effect of double-compression coating on flurbiprofen core mini-tablets to achieve the pulsatile colonic delivery to deliver the drug at a specific time as per the patho-physiological need of the disease that results in improved therapeutic efficacy. In this study, pulsatile double-compression-coated tablets were prepared based on time-controlled hydroxypropyl methylcellulose K100M inner compression coat and pH-sensitive Eudragit S100 outer compression coat. Then, the tablets were evaluated for both physical evaluation and drug-release studies, and to prove these results, in vivo pharmacokinetic studies in human volunteers were conducted. From the in vitro drug-release studies, F6 tablets were considered as the best formulation, which retarded the drug release in the stomach and small intestine (3.42 ± 0.12% in 5 h) and progressively released to the colon (99.78 ± 0.74% in 24 h). The release process followed zero-order release kinetics, and from the stability studies, similarity factor between dissolution data before and after storage was found to be 88.86. From the pharmacokinetic evaluation, core mini-tablets producing peak plasma concentration (C_max) was 14,677.51 ± 12.16 ng/ml at 3 h T_max and pulsatile colonic tablets showed C_max = 12,374.67 ± 16.72 ng/ml at 12 h T_max. The area under the curve for the mini and pulsatile tablets was 41,238.52 and 72,369.24 ng-h/ml, and the mean resident time was 3.43 and 10.61 h, respectively. In conclusion, development of double-compression-coated tablets is a promising way to achieve the pulsatile colonic release of flurbiprofen.KEY WORDS: core mini-tablets, double-compression coating, inner compression coat, outer compression coat, similarity factor     

Root carboxylate exudation capacity under phosphorus stress does not improve grain yield in green gram

Key message

Genetic variability in carboxylate exudation capacity along with improved root traits was a key mechanism for P-efficient green gram genotype to cope with P-stress but it did not increase grain yield.

Abstract

This study evaluates genotypic variability in green gram for total root carbon exudation under low phosphorus (P) using ¹⁴C and its relationship with root exuded carboxylates, growth and yield potential in contrasting genotypes. Forty-four genotypes grown hydroponically with low (2 μM) and sufficient (100 μM) P concentrations were exposed to ¹⁴CO₂ to screen for total root carbon exudation. Contrasting genotypes were employed to study carboxylate exudation and their performance in soil at two P levels. Based on relative ¹⁴C exudation and biomass, genotypes were categorized. Carboxylic acids were measured in exudates and root apices of contrasting genotypes belonging to efficient and inefficient categories. Oxalic and citric acids were released into the medium under low-P. PDM-139 (efficient) was highly efficient in carboxylate exudation as compared to ML-818 (inefficient). In low soil P, the reduction in biomass was higher in ML-818 as compared to PDM-139. Total leaf area and photosynthetic rate averaged for genotypes increased by 71 and 41 %, respectively, with P fertilization. Significantly, higher root surface area and volume were observed in PDM-139 under low soil P. Though the grain yield was higher in ML-818, the total plant biomass was significantly higher in PDM-139 indicating improved P uptake and its efficient translation into biomass. The higher carboxylate exudation capacity and improved root traits in the later genotype might be the possible adaptive mechanisms to cope with P-stress. However, it is not necessary that higher root exudation would result in higher grain yield.     

Long-term stability in biomass and production of terpene indole alkaloids by hairy root culture of Rauvolfia serpentina and cost approximation to endorse commercial realism

The effect of 6 years of cultivation and use of table-sugar (TS) on the biomass/terpene alkaloid productivities and rol gene expression were studied in a hairy root (HR) clone of Rauvolfia serpentina. The media cost could be reduced >94 % by replacing sucrose (SUC) with TS—an unexplored avenue for HR cultivation. The overall productivities increased over long-term cultivation with sugar proving superior to SUC for biomass (24.4 ± 2.11 g/l DW after 40 days to 17.31 % higher) and reserpine (0.094 ± 0.008 % DW after 60 days to 193.8 % more) production. The latter however revealed comparatively better yields concerning ajmaline (0.507 ± 0.048 % DW after 60 days to 61.98 % higher) and yohimbine (0.628 ± 0.062 % DW after 60 days to 38.32 % higher), respectively. PCR amplification of rol genes confirmed long-term expression stability.     

Flavone-resistant Leishmania donovani Overexpresses LdMRP2 Transporter in the Parasite and Activates Host MRP2 on Macrophages to Circumvent the Flavone-mediated Cell Death

In parasites, ATP-binding cassette (ABC) transporters represent an important family of proteins related to drug resistance and other biological activities. Resistance of leishmanial parasites to therapeutic drugs continues to escalate in developing countries, and in many instances, it is due to overexpressed ABC efflux pumps. Progressively adapted baicalein (BLN)-resistant parasites (pB²⁵R) show overexpression of a novel ABC transporter, which was classified as ABCC2 or Leishmania donovani multidrug resistance protein 2 (LdMRP2). The protein is primarily localized in the flagellar pocket region and in internal vesicles. Overexpressed LdABCC2 confers substantial BLN resistance to the parasites by rapid drug efflux. The BLN-resistant promastigotes when transformed into amastigotes in macrophage cells cannot be cured by treatment of macrophages with BLN. Amastigote resistance is concomitant with the overexpression of macrophage MRP2 transporter. Reporter analysis and site-directed mutagenesis assays demonstrated that antioxidant response element 1 is activated upon infection. The expression of this phase II detoxifying gene is regulated by NFE2-related factor 2 (Nrf2)-mediated antioxidant response element activation. In view of the fact that the signaling pathway of phosphoinositol 3-kinase controls microfilament rearrangement and translocation of actin-associated proteins, the current study correlates with the intricate pathway of phosphoinositol 3-kinase-mediated nuclear translocation of Nrf2, which activates MRP2 expression in macrophages upon infection by the parasites. In contrast, phalloidin, an agent that prevents depolymerization of actin filaments, inhibits Nrf2 translocation and Mrp2 gene activation by pB²⁵R infection. Taken together, these results provide insight into the mechanisms by which resistant clinical isolates of L. donovani induce intracellular events relevant to drug resistance.