Two novel aspirin analogues show selective cytotoxicity in primary chronic lymphocytic leukaemia cells that is associated with dual inhibition of Rel A and COX-2.

OBJECTIVES: Non-steroidal anti-inflammatory drugs have been shown to induce apoptosis in primary B-cell chronic lymphocytic leukaemia (CLL) cells, but the molecular mechanisms that underpin this observation have not been fully elucidated. Here, we have analysed the effect two novel aspirin analogues, 2-hydroxy benzoate zinc (2HBZ) and 4-hydroxy benzoate zinc (4HBZ), on primary CLL samples. MATERIALS AND METHODS: Cytotoxic effects of 2HBZ and 4HBZ were analysed in primary CLL cells derived from 52 patients, and normal B- and T-lymphocytes. Mechanisms of action of these agents were also elucidated. RESULTS: Both analogues induced apoptosis in a dose-dependent and time-dependent manner. Apoptosis was associated with activation of caspase-3 that could be partially abrogated by the caspase-9 inhibitor (Z-LEHD.fmk). Importantly, both agents demonstrated preferential cytotoxicity in CLL cells when compared to normal B- and T-lymphocytes. In terms of their molecular mechanisms of action, 4HBZ and 2HBZ inhibited COX-2 transcription and protein expression and this was associated with upstream inhibition of transcription factor Rel A. Co-culture of CLL cells with CD40 ligand-expressing mouse fibroblasts significantly increased COX-2 expression and inhibited spontaneous apoptosis. Importantly, the most potent analogue, 4HBZ, overcame pro-survival effects of the co-culture system and significantly repressed COX-2. Finally, elevated COX-2 expression was associated with poor prognostic subsets and increased sensitivity to 4HBZ. CONCLUSIONS: Our results demonstrate therapeutic potential of 4HBZ and are consistent with a mechanism involving suppression of Rel A nuclear translocation and inhibition of COX-2 transcription.

Sub-millimolar concentration of the novel phenol-based compound, 2-hydroxy benzoate zinc, induces apoptosis in human HT-1080 fibrosarcoma cells.

OBJECTIVES: To examine the effect of a novel phenolic-based compound, 2-hydroxy benzoate zinc (2HBZ), and acetylsalicylic acid (ASA) on human HT-1080 fibrosarcoma cells. MATERIALS AND METHODS: MTT assay was used to assess cell proliferation while different methods were used to detect apoptosis morphologically and immunohistochemically in Human HT-1080 fibrosarcoma cells. Apoptosis was determined by Annexine-V labelling, and caspase-3 activation. In addition, western blot was used to analyse p21, p53 and Bax and flow cytometry was to analyse the cell cycle. RESULTS: 2HBZ exhibited a more than 5-fold increase in cytotoxic potency when compared with ASA with mean LD50 values of 210 and 1100 lM respectively (P < 0.0001). The cytotoxic effects of 2HBZ were both time- and dosedependent with marked apoptosis being evident only after 24 h at concentrations as low as 200 mM. In contrast, ASA-induced apoptosis was observed only at concentrations in excess of 1000 mM at the same time point. Both 2HBZ and ASA induced caspase-3 activation in the cells, which confirmed that their cytotoxic effects were the result of apoptotic cell death. These findings were further confirmed by immunomorphological studies for the detection of apoptosis including haematoxylineosin, methyl green/pyronin Y staining and scanning electron microscopy. In addition, 2HBZ caused a marked increase in p21, p53 and Bax protein expressions and these effects were associated with an increase in G1 and G2 arrest of the cell cycle and a reduction in S-phase. CONCLUSIONS: These results demonstrate that the novel phenolic compound 2HBZ is a potent apoptosis-inducing agent in HT-1080 cells and warrants further investigation as a potential chemotherapeutic agent in primary cancer cell models.

Calcium salicylate-mediated apoptosis in human HT-1080 fibrosarcoma cells.

Salicylates are novel biologically active compounds that exhibit multiple therapeutic activities. The anti-cancer effectiveness of calcium salicylate has been investigated on human HT-1080 fibrosarcoma cell lines at relatively low concentrations (predominantly 0.4 mM) compared to those previously reported. Although low calcium salicylate concentrations did not retard tumour growth progression significantly, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and time-lapse assays, its cytotoxic characteristics were proven to be prominent by various morphological and immunocytological techniques. The results here demonstrate evidence for approximately 25% apoptosis after treatment with calcium salicylate, which up-regulatd the expression of p53, p21 and Bax, and down-regulated Bcl-2 in HT-1080 cells.

The historical analysis of aspirin discovery, its relation to the willow tree and antiproliferative and anticancer potential.

For several millennia, the willow tree and salicin have been associated with salicylic acid, the key precursor molecule that has contributed to the discovery of acetylsalicylic acid, traded as aspirin. These molecules have been shown to possess phyto- and chemotherapeutic activities as analgesic drugs. In recent decades, aspirin has become the focus of extensive investigation into antiproliferative and anticancer activities. The historical steps that led to the discovery of aspirin, and its antiproliferative and anticancer potential are highlighted in this review.

The effect of ZD1839 (Iressa), an epidermal growth factor receptor tyrosine kinase inhibitor, in combination with cisplatin, on apoptosis in SCC-15 cells.

The aim of this study was to determine the effect of ZD1839 on growth and apoptosis in SCC-15 (a human head and neck cancer cell line) lone, or in combination with cisplatin. High expression of the epidermal growth factor receptor has been implicated in the development of squamous cell carcinomas of head and neck. ZD1839 ('Iressa') is an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor that blocks signal transduction pathways implicated in proliferation and survival of cancer cells, and other host-dependent processes promoting cancer growth. Here, growth arrest was observed with 3.64 microm ZD1839. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (sMTT) viability assay revealed a significant decrease (P < 0.001) in the percentage of surviving cells upon treatment with ZD1839 and cisplatin compared with cisplatin or ZD1839 on their own. Combined therapy of 3.64 microm ZD1839 for 24 h, prior to administration of 100 microm cisplatin, significantly (P < 0.001) and additively increased the cytotoxicity effect of cisplatin. p53-independent apoptosis was seen with cisplatin treatment, a novel finding. These data support the use of ZD1839 in anti-cancer therapy, and particularly in combination therapy. Cisplatin may induce p53-independent apoptosis. Over-expression of Bcl-2 in head and neck squamous cell carcinoma tumour cell lines is unlikely to be a general mechanism to protect these cells from apoptosis.

Renal tubular cell necrosis and apoptosis in transplanted kidneys.

Renal wedge biopsies were taken from donors' kidneys immediately at the end of cold ischaemia and 30 min after transplantation in 11 cases. Five renal grafts showed immediate and six showed delayed renal function clinically. The ratio of apoptotic and necrotic renal tubular cells and Ki67 activity was determined in both biopsies. Necrotic and apoptotic as well as proliferating renal tubular cells were seen in all samples. Both apoptotic and proliferative activity was decreased in samples taken 30 min after transplantation in cases of immediate renal function compared to the samples taken before transplantation. This phenomenon was not observed in cases of delayed renal function.

Correlation between the clonogenic initial slope and the response of polykaryon-forming units: the behavior of strains defective in XRCC5 and ATM and the heritability of small variations in radioresponse.

The polykaryon-forming unit (PFU) assay measures the survival of multiple cycles of DNA synthesis after exposure to ionizing radiation, and it is known that there is a strong correlation between the slope of the PFU dose-response curve and the clonogenic initial slope. This suggests that DNA lesions expressed in clonogens are also important in PFU. Cells having a mutation in XRCC5 (also known as Ku80; strain xrs-6) and ATM (strain AT5BIVA) were hypersensitive in the PFU assay and in clonogens, while a strain of xrs-6 cells transfected with hamster wild-type XRCC5 cDNA displayed wild-type resistance in both assays. These data suggest that the DNA double-strand break (DSB) is an important lesion in PFU, although the relative radioresistance of PFU compared to clonogens indicates differential DSB toxicity. We propose that this results from the absence of cytokinesis-related loss of DNA fragments. Small variations in the radioresponse of PFU were observed between CHO K1 cell substrains, such that the xrs parental substrain RR-CHOK1 (carrying wild-type XRCC5) was more sensitive than an independent K1 substrain (E-CHOK1). Somatic hybridization showed that this variation is heritable and that the resistant E phenotype is dominant. In RR-CHOK1 cells there was a biphasic PFU radioresponse, which suggests that there may be transient expression at a locus selectively affecting PFU sensitivity.

Modes of cell death in the hypopharyngeal gland of the honey bee (Apis mellifera l).

Different modes of cell death have been revealed in the regressing hypopharyngeal glands of worker honey bees. The hypopharyngeal gland, which is well developed in young nursing bees to produce protein for larval food, was seen to regress naturally in foraging adult worker bees. A range of techniques including histology, cytochemistry, in situ TUNEL, Annexin V and Comet assays indicated that cells within the gland demonstrate progressive symptoms of apoptosis, necrosis and a vacuolar form of programmed cell death. The latter mode of cell death did not display chromatin margination, but was accompanied by an enhanced level of autophagic and hydrolytic activity in which a cytosolic source of acid phosphatase became manifest in the extra-cisternal spaces. Normal and annexin-positive cells were found to occur in the younger nursing bees, whilst necrosis and an aberrant vacuolar type of apoptosis predominated in the older foraging bees. The relevance of these results to the classification of programmed cell death is discussed.

Apoptosis is a mode of cell death in the polykaryon-forming unit assay.

In the polykaryon-forming unit (PFU) assay, which defines cell survival as the ability to form a cytochalasin-induced polykaryon of predetermined ploidy, the mode of PFU deletion is not known. Incubation of L5178Y-S PFU in cytochalasin resulted in polyploidy (> or =32C) and most polykaryons (>75%) ultimately underwent apoptosis, detected using chromatin condensation and externalised phosphatidylserine. However, large polykaryons carrying terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL)-labelled DNA strand breaks were not observed, presumably due to rapid loss of DNA. Gamma irradiation of PFU prior to cytochalasin exposure caused a reduction in the frequency of highly polyploid cells (>16C), consistent with either a supra-induction of apoptosis or a reduction in the ability of PFU to reach high ploidies. We conclude that L5178Y-S PFU are deleted by apoptosis.

Apoptotic and non-apoptotic modes of programmed cell death in MCF-7 human breast carcinoma cells.

Apoptosis is a specific mode of programmed cell death (PCD), recognized by characteristic morphological and molecular changes. Here we present evidence for a non-apoptotic type of PCD in human MCF-7 breast carcinoma cells. We used TNF-alpha and tyrphostin AG213 to induce apoptotic and non-apoptotic cell death respectively in vitro. Microscopic and immunohistochemical studies, together with DNA analysis and flow cytometric analysis of p53 and bcl-2 oncogene expression, revealed some novel characteristics of non-apoptotic cell death. We show here for the first time some of the biochemical features of an experimentally induced non-apoptotic PCD and emphasize the distinct biochemical events leading to apoptotic and non-apoptotic PCD.

Histochemical characterization of cell death in honeybee larvae midgut after treatment with Paenibacillus larvae, Amitraz and Oxytetracycline.

A number of techniques were employed to assess cell death induced in honeybee larvae midgut after per os inoculation of bacterium Paenibacillus larvae var. larvae, the causative agent of American foulbrood disease, and separately with acaricide Amitraz and antibiotic Oxytetracycline. In honeybee larvae exposed to Amitraz, which demonstrates both necrosis and apoptosis, cell death was found in 82% of midgut columnar and in 50% of regenerative epithelial cells, 24 h after treatment. Cell death reduced to 36% in the epithelial cells, 48 h after treatment. In Oxytetracycline-treated larvae, cell death was identified in 40% of midgut epithelial cells, 24 h after inoculation and increased to 55% over the next 24 h. In Paenibacillus -infected larvae, all midgut epithelial cells died. Using ApopTag (Oncor) to label the multiple DNA ends generated by DNA fragmentation showed programmed cell death in 49% of columnar midgut cells 24 h after Amitraz application. Cell death was reduced to 9% over the next 24 h. Our data indicate that cell death could be identified and quantified in situ, using TUNEL techniques. This study also shows that the acaricide Amitraz is a trigger for programmed cell death in the midgut epithelial cells of honeybee larvae, unlike Paenibacillus which induces necrosis only. The data show that immunohistochemical methods are useful for studying in situ tissue pathology, and indicate possibilities for monitoring the effects of infective and chemical environmental stressors on cell death in honeybee larvae tissue.

In situ localization of heat-shock and histone proteins in honey-bee (Apis mellifera l.) larvae infected with Paenibacillus larvae.

The immunohistochemical localization of the heat shock proteins (Hsp70 and Hsp90) and histone protein in healthy and Paenibacillus larvae infected honeybee (Apis mellifera L.) larvae has been studied. Hsp70 was found in the nuclei and the cytoplasm of infected midgut, salivary gland cells and haemocytes, but not in uninfected larvae. Hsp90 was localized in both infected and uninfected cells. Exposed histone proteins were localized in the nuclei of dying uninfected cells undergoing programmed cell death. The distribution of histone protein in uninfected cells of midgut, salivary gland, and other tissues was nuclear and indicative of normal programmed cell death at levels between 1 and 5%. After applying histone protein antibodies to P. larvae infected honeybee larvae, the DAB based reaction product was located in the nuclei or immediate surroundings of all larval cells. The Hsp70, Hsp90 and histone protein distribution patterns are discussed in relation to the morphological, cytochemical and immunocytochemical characteristics of programmed cell death and pathological necrosis. Results produced by methyl green-pyronin staining confirm an elevation of RNA levels in normal programmed cell death and a reduced staining for RNA in necrotic infected cells.

Mint prevents shamma-induced carcinogenesis in hamster cheek pouch.

Shamma, a complex mixture of powdered tobacco, slaked lime, ash, oils, spices and other additives, has been linked to oral cancer in Saudi Arabia. Shamma varies in colour and odour due to the nature of the additives which characterize different brands. Using the Ames Salmonella assay, a chloroform extract of a brand named 'white shamma' (WSH) was found to be mutagenic, while that of a brand called 'brown shamma' (BSH), which is known to contain mint as a flavouring agent, was found to be non-mutagenic. Using HPLC, a mutagenic and a non-mutagenic fraction were isolated from the extract of BSH. The non-mutagenic fraction of BSH was found to neutralize the genotoxic effect of the mutagenic fraction when the two were recombined. A chloroform extract of mint showing no mutagenic activity in the Ames assay effectively inhibited the mutagenicity of carcinogens/mutagens like benzo[a]-pyrene, aflatoxin B1, methylmethane sulfonate and extract of WSH. A carcinogenicity assay designed to test the effects of WSH and BSH in the hamster cheek pouch model showed that the former was tumorigenic, while the latter was not. However, when crushed leaves of mint were mixed with powdered WSH (in 1:1 proportion), the tumorigenic effect of the latter was abolished. These data strongly suggested that mint has a chemopreventive effect against shamma-induced carcinogenesis, which could be due to its antimutagenic properties.

A new method to localize acid phosphatase using the confocal laser-scanning microscope.

The aim of the study was to work out a technique for the detection of acid phosphatase enzyme activity by confocal laser-scanning microscope using the histochemical acid phosphatase detection method (after Barka and Anderson 1962, modified by Bowen and Lewis 1985) routinely used for light microscopy. The density and the distribution of enzyme reaction product is dependent on the incubation time, as shown by different confocal images or ELISA reader. The inhibition of the enzyme activity with metal ions shows the same profile known from the literature. This staining method seems to be useful to demonstrate subcellular distribution of the enzyme in the lysosomes and in the Golgi apparatus.

Histopathological and histochemical changes in honeybee larvae (Apis mellifera L.) after infection with Bacillus larvae, the causative agent of American foulbrood disease.

Morphological, histochemical and cytochemical changes were examined in honeybee larvae after infection with the bacterium Bacillus larvae. The results indicate cell necrosis in the midgut epithelium accompanied by increasing cell vacuolization and nuclear pyknosis following per os inoculation with B. larvae. Many autolysosomes were positive for acid phosphatase. Non-vacuolar acid phosphatase activity was also found in lysed cell compartments. No such activity was found in regenerative epithelial cells. Degradation of haemocytes, salivary glands and other tissues was also observed. Histochemical analyses after per cutaneous inoculation with B. larvae of three- and five-day-old honeybee larvae show intense non-vacuolar acid phosphatase activity followed by disintegration of infected salivary glands, epithelial cell cytoplasm and haemocytes.

Improved cytochemical methods for demonstrating cell death using LR White as an embedding medium.

A series of techniques based on LR White resin are described, which permit the use of an anti-histone antibody for the in situ localization of DNA fragmentation characteristic of apoptosis at both the light and the electron microscope level. The methods, applied to an untreated squamous carcinoma of the pharynx, allow direct comparison of light microscopic localization of exposed nucleosomal histones using 3,3'-diaminobenzidine (DAB) and silver-enhanced techniques with a colloidal gold-based anti-histone technique at the electron microscope level. Parallel histochemical localization of acid phosphatase activity is also presented.

A comparison of the anti-histone and Apop-Tag technique for demonstrating apoptosis with option for silver enhancement.

A novel immunocytochemical method is presented for the qualitative detection of DNA fragmentation in apoptosis. Anti-histone antibody is employed to localize exposed nucleosomal histones (H1, H2a, H2b, H3 and H4) rather than tagging the cut ends of fragmenting DNA as in conventional technique. The method was tested on squamous cell carcinoma of the larynx routinely fixed in formaldehyde and embedded in paraffin wax and compared with results obtained employing Apop-Tag kit (Oncor).

Pharmacological importance of stereochemical resolution of enantiomeric drugs.

Drug enantiomers have identical properties in an achiral environment, but should be considered as different chemical compounds. This is because they often differ considerably in potency, pharmacological activity and pharmacokinetic profile, since the modules with which they interact in biological systems are also optically active. Within biological systems, the metabolism of one isomer may be via a different pathway or occur at a different rate from that of the other isomer. Preferential binding of one isomer to plasma proteins may cause differences in circulating free drug and hence alter concentrations at active sites. Interactions of both isomers may differ at the active sites through which pharmacological action is mediated. Actions and levels of activity of the stereoisomers in vivo may also differ. All the pharmacological activity may reside in a single enantiomer, whereas several possibilities exist for the other enantiomer-- it may be inactive, have a qualitatively different effect, an antagonistic effect or produce greater toxicity. Two isomers may have nearly identical qualitative pharmacological activity, qualitatively similar pharmacological activity but quantitatively different potency, or qualitatively different pharmacological activity. To avoid adverse effects and optimise the therapeutic value of enantiomeric drugs, it is necessary that methods for the resolution of racemates be evolved and devolved to determine isomeric purity, establish the effectiveness of isomers of the drug, and detect the presence of an enantiomer with lower therapeutic activity and undesirable adverse effects. Even if a drug is given as a pure enantiomer, methods to discriminate between enantiomers are required because racemisation can occur both in vitro and in vivo. Methods developed for resolution of drug enantiomers should facilitate routine testing of single isomers and their metabolites, studies of pharmacological, toxicological and clinical effectiveness, routine analysis of racemates, pure enantiomers or intermediates in manufacturing processes, and investigation of the potential for inversion of an enantiopure drug substance during the early stages of drug development and therapeutic drug monitoring.

Programmed cell death in the honey-bee (Apis mellifera L.) larvae midgut.

The histochemical and cytochemical localization of acid phosphatase has been used in an attempt to map the sites of cellular lysis and death. Reaction product was found both in the brush border of the midgut epithelium and in the basal membrane. Vacuolar acid phosphatase activity was found in the regenerative epithelial cells. Extra-cisternal reaction product was associated with the endoplasmic reticulum which was dilated in lysed areas of the cytoplasm. Free acid and alkaline phosphatase activity was found in the basal area of the midgut epithelial cells and the former also occurred in the haemocoel. In the tracheoblastic cells only vacuolar acid phosphatase activity was seen. Chromatin aggregates were distributed throughout the nucleus and the nuclear envelope showed some infolding. Certain mature epithelial cells proved positive for anti-histone associated DNA fragmentation indicative of programmed cell death.