R-deprenyl: pharmacological spectrum of its activity.

Deprenyl has been discovered by Knoll and co-workers. The R-enantiomer of deprenyl (selegiline) is a selective and irreversible inhibitor of the B-isoform of monoamine oxidase (MAO-B) enzyme. Due to its dopamine potentiating and possible neuroprotective properties it has an established role in the treatment of parkinsonian patients. By inhibiting MAO-B enzyme, R-deprenyl decreases the formation of hydrogen peroxide, alleviating the oxidative stress also reduced by increased expression of antioxidant enzymes (superoxide dismutases and catalase) reported during chronic treatment. It was shown to prevent the detrimental effects of neurotoxins like MPTP and DSP-4. R-Deprenyl elicits neuroprotective and neuronal rescue activities in concentrations too low to inhibit MAO-B. It is extensively metabolized and some of the metabolites possess pharmacological activities, thus their contribution to neuroprotective properties was also suggested. The recently identified deprenyl-N-oxide is extensively studied in our laboratory. Effects other than neuroprotection, like influencing cell adhesion and proliferation cannot be neglected.

Cytoprotective effect of (-)-deprenyl, (-)desmethyl-deprenyl and (-)deprenyl-N-oxide on glutathione depleted A-2058 melanoma cells.

Postulated cytoprotective action of (-)-deprenyl (D), (-)-desmethyl-deprenyl (DD) and (-)-deprenyl-N-oxide (DNO) on L-buthionine-(S,R)-sulfoximine (BSO) toxicity was investigated using in vitro cultures of serum-deprived A-2058 melanoma cells. BSO (10 microM/l) decreased viable cell number and mitotic rate, while increased the apoptotic index. D and both of its metabolites, given together with BSO in the concentration of 50 microM/l, mitigated cell loss and decreased the apoptotic ratio. DD was the most effective compound in decreasing apoptotic activity, while DNO stabilized the cell number on control level and increasing the ratio of mitotic cells above the only serum-deprived control. Surveillance on mitochondrial membrane stability and antioxidant properties may play an important role in these processes.

Cell proliferation and apoptosis in stromal corneal dystrophies.

The aim of our study was to evaluate corneal cell proliferation and apoptosis in cases of granular, macular and lattice dystrophy, and to provide evidence which may help to clarify whether apoptosis is a pathogenic factor in any of these dystrophies. The study group comprised 39 eyes (from 33 patients) which had undergone penetrating keratoplasty (PK) for stromal dystrophies: these comprised 12 eyes (from 9 patients, 55.5% males) with granular dystrophy, 13 eyes (12 patients, 33.3% males) with macular dystrophy, and 14 eyes (13 patients, 61.5% males) with lattice type I dystrophy. A further 4 corneal buttons from enucleated eyes of 4 patients with choroideal melanoma served as controls. Immunocytochemical analysis of Ki67 (DNAcon Kit, DakoCytomation A/S, Glostrup, Denmark) was used for evaluation of cell proliferation. Apoptosis was detected by use of the TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labelling) assay method (Apoptag reagent, Q-Biogene, Strasbourg, France). Statistical comparisons were made using the Mann-Whitney test. No Ki67-positive cells were detected in the study-group or control corneas. In control corneas no apoptotic activity was found. In the study group the mean (normalised) apoptotic keratocyte number was 1.1+/-1.7 in granular dystrophy and 0.5+/-1.1 in lattice type I dystrophy (p = 0.36, 0.63 respectively). Compared to the controls, the difference was statistically significant only for macular dystrophy (1.6+/-1.2; p = 0.01). Keratocyte apoptosis seems to be a concomitant or pathogenic factor in macular dystrophy. However, the pathways that are triggered to result in increased apoptotic cell death remain to be clarified.

Effect of formaldehyde and resveratrol on the viability of Vero, HepG2 and MCF-7 cells.

A non-transformed (Vero) and two tumor cell lines (HepG2 and MCF-7) were treated with 10nM to 100 microM formaldehyde. Lower doses (10nM to 10 microM) enhanced the viability of the cultured cells, measured by MTT assay. Higher doses (75-100 microM) decreased viability of the cells by 50% or more. The 100 microM concentration of HCHO has been chosen for combination treatment of the three cell lines with a series of concentrations (0.2-100 microM) of resveratrol, a phytoestrogen occurring in various fruits. Resveratrol decreased the cytotoxicity of formaldehyde depending on cell line and point of time, especially in case of MCF-7 cells at 24 and 72 h, Vero cells at 24h and HepG2 cells at 48 h after treatment. Possible modes of interactions are discussed, considering the role of resveratrol in formaldehyde metabolism and also the estrogen receptor positivity of MCF-7 cells.

Effect of Avemar--a fermented wheat germ extract--on rheumatoid arthritis. Preliminary data.

OBJECTIVE: To investigate the effect of the fermented wheat germ extract (Avemar)in patients with severe rheumatoid arthritis (RA). METHODS: Fifteen female RA (Steinbrocker II-III) patients, who had unsuccessfully tried two different DMARD treatments, were enrolled in an open-label, 1-year long, pilot clinical study. DMARD and steroid therapies were recorded and continued. All patients received Avemar as additional therapy. For measurement of efficacy the Ritchie Index, the Health Assessment Questionnaire (HAQ) and the assessment of morning stiffness were applied. Patients were evaluated at baseline, 6 and 12 months. For statistical analyses the Wilcoxon test was used. RESULTS: At both 6 and 12 months, Ritchie index, HAQ and morning stiffness showed significant improvements compared with the baseline values. Dosages of steroids could be reduced in about half of the patients. No side effects of Avemar were observed. CONCLUSION: Supplementation of standard therapies with a continuous administration of Avemar is beneficial for RA patients.

Epithelial cell, keratocyte, and endothelial cell apoptosis in Fuchs' dystrophy and in pseudophakic bullous keratopathy.

PURPOSE: To elucidate the pathomechanism of Fuchs' dystrophy and pseudophakic bullous keratopathy (PBK) by examining cell apoptosis in different corneal layers. METHODS: The authors studied corneal buttons obtained from 21 eyes following central penetrating keratoplasty: 14 corneal buttons (13 patients, age 70.8+/-10.0 years) with Fuchs' dystrophy, and 7 buttons (7 patients, age 69.6+/-10.2 years) with PBK. Four buttons from enucleated eyes with choroidal melanoma served as controls. Histologic changes were examined using light microscopy with hematoxylin-eosin (HE) staining. The average numbers of apoptotic cells per field of view (125x magnification) in separate samples of the epithelial, stromal, and endothelial layers were determined using the TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling) assay. RESULTS: In 11 of the Fuchs' dystrophy corneas and 2 of the PBK corneas, apoptotic activity was detected. In the control corneas no apoptotic activity was found. Compared to the controls there was a statistically significant difference in the mean (normalized) apoptotic cell numbers for all three layers (p=0.01 in each case) in the Fuchs' dystrophy corneas, and for the stromal layer (p<0.01) in PBK corneas. The apoptotic cell numbers for the epithelial and endothelial layers of the latter were higher, but the difference was not statistically significant (p=0.07, 0.07). CONCLUSIONS: Apoptosis may play a role in the pathomechanism of Fuchs' dystrophy and in keratocyte death in corneas with PBK.

Epithelial cell, keratocyte, and endothelial cell apoptosis in Fuchs dystrophy and in pseudophakic bullous keratopathy.

PURPOSE: To elucidate the pathomechanism of Fuchs dystrophy and pseudophakic bullous keratopathy (PBK) by examining cell apoptosis in different corneal layers. METHODS: The authors studied corneal buttons obtained from 21 eyes following central penetrating keratoplasty: 14 corneal buttons (13 patients, age 70.8 10.0 years) with Fuchs dystrophy, and 7 buttons (7 patients, age 69.6 10.2 years) with PBK. Four buttons from enucleated eyes with choroidal melanoma served as controls. Histologic changes were examined using light microscopy with hematoxylin-eosin (HE) staining. The average numbers of apoptotic cells per field of view (125x magnification) in separate samples of the epithelial, stromal, and endothelial layers were determined using the TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling) assay. RESULTS: In 11 of the Fuchs dystrophy corneas and 2 of the PBK corneas, apoptotic activity was detected. In the control corneas no apoptotic activity was found. Compared to the controls there was a statistically significant difference in the mean (normalized) apoptotic cell numbers for all three layers (p=0.01 in each case) in the Fuchs dystrophy corneas, and for the stromal layer (p<0.01) in PBK corneas. The apoptotic cell numbers for the epithelial and endothelial layers of the latter were higher, but the difference was not statistically significant (p=0.07, 0.07). CONCLUSIONS: Apoptosis may play a role in the pathomechanism of Fuchs dystrophy and in keratocyte death in corneas with PBK. (Eur J Ophthalmol 2005; 15: #-22).

Pharmacological aspects of (-)-deprenyl.

Deprenyl, the selective irreversible inhibitor of monoamine oxidase-B (MAO-B), has been synthesised as a potential antidepressant, however, due to its dopamine potentiating capacity, became a registered drug in the treatment of Parkinson's disease. Deprenyl possesses a wide range of pharmacological activities; some of them are not related to its MAO-B inhibitory potency. Beside its dopamine potentiating effect, it renders protection against a number of dopaminergic, cholinergic and noradrenergic neurotoxins with a complex mechanism of action. By inducing antioxidant enzymes and decreasing the formation of reactive oxygen species, deprenyl is able to combat an oxidative challenge implicated as a common causative factor in neurodegenerative diseases. In a dose substantially lower than required for MAO-B inhibition (10(-9)-10(-13) M), deprenyl interferes with early apoptotic signalling events induced by various kinds of insults in cell cultures of neuroectodermal origin, thus protecting cells from apoptotic death. Deprenyl requires metabolic conversion to a hitherto unidentified metabolite to exert its antiapoptotic effect, which serves to protect the integrity of the mitochondrion by inducing transcriptional and translational changes. Pharmacokinetic and metabolism studies have revealed that deprenyl undergoes intensive first pass metabolism, and its major metabolites also possess pharmacological activities. The ratio of the parent compound and its metabolites reaching the systemic circulation and the brain are highly dependent on the routes of administration. Therefore, in the treatment of neurodegenerative diseases, reconsideration of the dosing schedule, by lowering the dose of deprenyl and choosing the most appropriate route of administration, would diminish undesired adverse effects, with unaltered neuroprotective potency.

TT-232: a somatostatin structural derivative as a potent antitumor drug candidate.

TT-232 (D-Phe-Cys-Tyr-D-Trp-Lys-Cys-Thr-NH2) has been developed as an antitumor somatostatin analog. TT-232 has no growth hormone release inhibitory effect and does not inhibit the secretion of gastric acid. This analog induces apoptosis in and exerts pronounced antiproliferative effects on various human tumors (colon, pancreas, lymphoma, leukemia, melanoma, hepatoma) cell lines. The growth of human xenografts (prostate, breast carcinoma, lymphoma, melanoma) and animal tumors (colon-26, P-388, S-180, B16, MXT) was inhibited by TT-232 (dose range: 30-750 microg/kg/day) in 54-98% of cases. Continuous long-term infusion proved to be the most effective way of administration. TT-232 combined with decarbazine or etoposide treatment enhanced the antitumor activity of these drugs on human melanoma and lymphoma xenografts, respectively. Regarding the mode of action, TT-232 activates cell cycle inhibitors via SSTR receptors, inhibits tyrosine kinases through interfering with the proliferative signaling cascades, and interacts with an intracellular receptor and an enzyme involved in glycolysis causing translocation of this enzyme to the nucleus, thus inducing apoptosis. TT-232 may be a promising candidate in the therapy of human malignancies.

Digital slide and virtual microscopy based routine and telepathology evaluation of routine gastrointestinal biopsy specimens.

AIMS: To evaluate a recently developed digital slide and virtual microscope system, and to compare this method with optical microscopy on routine gastrointestinal biopsy specimens in both local and remote access modes. METHODS: A fully computer controlled commercial microscope was used. The scanning program included object detection, autofocus, and image compression algorithms. The overall hard disk space for a gastric biopsy was between 30 and 50 MB and the scanning time was between 20 and 40 minutes. Haematoxylin and eosin stained routine gastric (61) and colon (42) biopsy specimens were selected, scanned, and evaluated by two specialists on an optical (OM) and virtual microscope (VM). RESULTS: The overall concordance of VM and OM with the consensus diagnosis was 95.1% and 97%, respectively. Clinically important concordance was 96.1% and 98% for VM and OM, respectively. The two methods showed concordance in 92% of cases and clinically important concordance in 94.1% of cases. The reasons for discordance were image quality (one case), interpretation difference (three cases), and insufficient clinical information (three cases). Remote evaluation of the digital slides through the Internet has the advantages of the previously used static and dynamic telepathology methods. CONCLUSIONS: Diagnostic concordance was found between OM and VM. The digital slide and the virtual microscope can be alternative techniques in the computerisation of the histology laboratory and in teleconsultation services after further evaluation of time and storage constraints.

Malignant myoepithelioma. Clinicopathological and immunohistochemical characteristics.

A malignant myoepithelioma arising in the submucosal accessory glands of the oral cavity of a 23-year-old woman is reported. The patient was very young compared to the cases in the literature, and the tumour had an unusual vestibular location. A false diagnosis of pleomorphic adenoma had been made by fine needle aspiration biopsy. The surgery comprised a wide tumour excision, but neck dissection was not indicated. The final histologic diagnosis was malignant myoepithelioma. Immunohistochemically putative myoepithelial markers were highly expressed.

Effect of a novel somatostatin analogue combined with cytotoxic drugs on human tumour xenografts and metastasis of B16 melanoma.

A novel somatostatin analogue, TT-232 (which inhibits the proliferation of various cell cultures and transplantable mouse tumours), was examined regarding its effect on human melanoma and lymphoma xenografts as a single treatment or in combination with DTIC (dacarbazine) and etoposide. TT-232 inhibited the growth of HT-18 melanoma xenografts, a dose of 5 mg kg(-1) being the most effective. Combination of 1 mg kg(-1) TT-232 with 30 or 60 mg kg(-1) DTIC (administered daily) resulted in a stronger inhibitory effect compared to TT-232 or DTIC as a single modality. Antimetastatic effect of TT-232 treatment combined with DTIC was studied using the B16 mouse melanoma muscle - lung metastasis model. The number of lung metastases of B16 melanoma could be decreased by the daily administration of 1 mg kg(-1) TT-232 or 60 mg kg(-1), but not of 30 mg kg(-1) DTIC. TT-232, combined with 30 or 60 mg kg(-1) DTIC decreased the lung metastasis number significantly lower than the control. Nearly 50% growth inhibition of HT-58 lymphoma was achieved by daily treatment with 1 mg kg(-1) TT-232. 5 mg kg(-1) etoposide, administered daily, resulted in a similar effect. The combination of 1 mg kg(-1) TT-232 and 5 mg kg(-1) etoposide was significantly more effective than TT-232 or etoposide as a single treatment. The very strong tumour growth inhibitory effect of 10 mg kg(-1) etoposide could even be increased by combination with TT-232. These experimental data suggest that TT-232 may be an effective new tool in the combination chemotherapy of malignant tumours like melanoma and lymphoma.

Pro-apoptotic and anti-apoptotic molecules affecting pathways of signal transduction.

Selective inhibition of the "false" proliferative signals via targeting tyrosine kinases resulting in the induction of apoptosis by depletion of the "survival factors" is one of the most studied and widely accepted concepts of modern chemotherapy. We have synthesized a series of potent tyrosine kinase inhibitors and tested these compounds for apoptosis induction. Some of the tyrosine kinase inhibitors caused either apoptotic or cytoplasmic vacuolar cell death in various tumor cell cultures. The somatostatin analogue oligopeptide TT-232, which indirectly inhibits tyrosine kinases, exerted a dose-dependent apoptosis-inducing effect. The tumor growth-inhibitory effect of TT-232 and some tyrosine kinase inhibitors has also been proven by in vivo experiments, using human tumor xenografts. On the other hand, a dose-dependent pro- or anti-apoptotic activity of (-)-deprenyl has been shown in melanoma cell cultures, the lower doses inhibiting and the higher doses inducing apoptosis. Various metabolites of (-)-deprenyl are responsible for these actions. The effect of (-)-deprenyl is connected with depolarization of mitochondrial membranes. The kinase inhibitors act on the growth factor receptor signaling pathways (survival factor pathways) and initiate the caspase cascade. The key enzyme for the action of both pro-apoptotic and anti-apoptotic compounds is caspase 3.

Changes in apoptotic and mitotic index, bcl2, and p53 expression in rectum carcinomas after short-term cytostatic treatment.

Twenty patients with rectal adenocarcinoma were endoscopically biopsied and given short-term cytostatic therapy [5-fluorouracil (5-FU) (600 mg/m(2)) and Ca-folinate (60 mg/m(2)) for 2 days]. Seven days later, the tumor was resected or a second biopsy was performed. Apoptotic and mitotic indices as well as (mutant) p53 and bcl(2) expression were determined in the tumor tissue before and after the short-term chemotherapy. The patients were treated thereafter with long-term, intermittent 5-FU administration and followed up clinically for 7-26 months. Six patients showed progression of the disease and died, whereas 14 improved or showed no tumor progression. Significant increase of the apoptotic index and nonsignificant decrease of the mitotic index after the short-term cytostatic treatment were seen in the tumor tissue of responder cases. Nonresponders showed no change in both mitotic activity and apoptotic activity. Both survivors and deceased showed high mutant p53 expression, and the changes after short-term 5-FU treatment were not significant. Expression of bcl(2) was present in only 5 cases, with the postchemotherapy changes being not significant. These findings suggest that apoptotic response to short-term cytostatic therapy may be an additional predictive factor in rectal adenocarcinoma.

Apoptosis in prostate carcinomas after short-term treatment with decapeptyl.

Altogether, 40 patients with advanced prostate cancer received the LH-RH analog, Decapeptyl (D), as monotherapy for 1 month. The dose of D was 3.75 mg/month, intramuscularly in microcapsules. Needle biopsy was taken from the prostate of 20 patients at 24 hours, of 10 patients at 7 days, and of 10 patients at 30 days after the injection. The biopsy samples were investigated for mitotic and apoptotic indices as well as expression of p53 and Ki67 in the tumor cells. The effect of the LH-RH analog was manifested in very low and even absent mitotic activity in all points in time. The expression of mutant p53 was high (above 80%) in all 1-day and 7-day samples and in 8 of 10 samples taken 30 days after the start of the therapy. In 2 cases, the ratio of p53-expressing tumor cells was 2% and 5%. At day 30, the previously high ratio of Ki67-positive tumor cells decreased to 2-10% in 8 cases and showed focally higher values (70-90%) in 2 cases. Extremely high (90-100%) apoptosis could be registered in scattered foci of the tumor tissue in all samples taken 1 and 7 days after D injection. The other parts of the tumor tissue showed lower (1-5%) apoptotic activity. At day 30 after the injection, the same phenomenon was observed in 4 cases, but in 6 cases the apoptotic ratio became diffusely low (1-2%). Focal increase of apoptosis observed in the 1- and 7-day samples may be attributed to the direct effect of the LH-RH analog on tumor cells. The findings at day 30 indicate a considerable decrease in proliferating activity and, in the majority of cases, also in apoptotic activity, reflecting the effect of D on the pituitary-gonadal axis.

Antitumor effect of lysine-isopeptides.

Isopeptides (ϵ-peptides) of lysine, with a given Mw and low polydispersity (10-400 units), were synthesized to study the relationship between their chemical structure and biological effect. The designed compounds were of high purity, low polydispersity and high stereochemical purity. The effect of the compounds was tested on a human erythroleukemia cell line (K-562) and on four transplantable mouse tumors (L1210 lymphoid leukemia, P38 macrophage derived tumor, Ehrlich ascites carcinoma, Lewis lung tumor /LLT/). In case of the L1210 and P388 tumors and the Ehrlich carcinoma, survival of the animals was used as an indicator of the effect. In case of the Lewis lung tumor, the number and size of metastases in the lung and/or liver of treated and untreated mice were used as indicators. The polymers of polymerisation degree 80-120 (Mw 10.2-15.4 KD) showed the strongest antiproliferative effect both on K562 cells and the tumors growing in vivo. This effect was manifest with a significantly higher survival rate as compared to the control (L1210, P38, Ehrlich ascites), furthermore, by a decrease in the number and size of liver and lung metastases (LLT).

Changes in apoptosis, mitosis, Her-2, p53 and Bcl2 expression in breast carcinomas after short-term tamoxifen treatment.

The short-term (7 days) effect of tamoxifen on apoptosis and mitosis index, p53, Bcl2 and Her-2/neu/c-erb2 expression in invasive ductal mammary carcinoma was studied histologically in the diagnostic biopsy and surgically removed tumor tissue of 10 patients. Following tamoxifen treatment expression of HER-2 and p53 decreased but Bcl2 remained unchanged. Mitotic activity decreased slightly, but not significantly. Apoptotic activity increased in six cases in the second sample compared to the values measured of the first biopsy. These changes may be attributed to the effect of antiestrogen therapy.

bcl2, p53 and bax in thyroid tumors and their relation to apoptosis.

Twenty five thyroid tumors were investigated by immunohistochemistry for the presence of apoptosis, expression of p53, bax and bcl2. In nine adenomas (average age 40) the rate of p53 positive cells was 5.7%, while it was 13.3% for bax and 41.7% for bcl2 in the tumor cells. In six follicular carcinomas (average age 56) p53 positivity was obtained for 71.1%, while bax and bcl2 positivity constituted 3.4% and 2.5%, respectively. In ten papillary adenocarcinomas (average age 42) p53 was positive in 39.6%, but bax and bcl2 were positive in 3.4% and 3% of the tumor cells, respectively. Almost no signs of spontaneous apoptosis were found in any of the cases. Determination of p53, bax and bcl2 ratio in thyroid tumors may contribute to the differentiation between follicular adenomas and carcinomas.

Repeated biopsies in evaluation of therapeutic effects in prostate carcinoma.

BACKGROUND: Apoptosis is one of the major events following total androgen blockade (TAB). The aim of this study was to determine the predictive value of some histological parameters including apoptosis and gene products which influence apoptosis, based on repeated biopsies taken from the same patients. METHODS: At the time of diagnosis by needle biopsy TNM stage, serum PSA, Gleason's grade, apoptotic and mitotic index, Ki67, p53, and bcl(2) expression were investigated in 60 prostate carcinoma patients. Antiandrogen therapy supplemented with surgical or chemical castration was administered. Serum PSA-test and needle biopsy were repeated 13-14 weeks after starting the therapy, simultaneously with determination of the apoptotic and mitotic index, Ki67, p53, and bcl(2) expression. RESULTS: Forty-seven patients were alive at the end of the study, 13 patients died. Decrease in mitotic, increase in apoptotic index predicted favourable long-term response to antiandrogen therapy. Lower Ki67 and (mutant) p53 expression in the first and also in the second biopsy pointed to favourable effect of antiandrogen treatment. Since the ratio between Ki67 and apoptotic index strongly decreased in the survivors upon therapy, changes in Ki67/apoptosis ratio is recommended as a histologically detectable predictive factor. bcl(2) expression did not show significant correlation with the outcome of the disease. CONCLUSIONS: Histological evaluation of mitotic and apoptotic index, Ki67, and p53 expression in repeated biopsies contributes to predicting the value of the actual treatment and may be useful to institute alterations in therapy.