Flow cytometric evidence of apoptosis in human pancreatic cancer xenografts treated with Sandostatin (octreotide).

BACKGROUND: The antiproliferative effect of octreotide (Sandostatin) is partly attributed to induction of apoptosis in the given tumors. In this work, apoptosis was assessed in human pancreatic carcinoma xenografts after a 4-week high-dose Sandostatin treatment. MATERIALS AND METHODS: Subcutaneously growing human pancreatic cancer xenografts (PZX-5) in immunosuppressed mice were treated with 500 micrograms/kgb.w. Sandostatin twice a day i.p. for 4 weeks. Apoptosis was evaluated by means of conventional histology, Apoptag-immunohistochemistry and flow cytometry. RESULTS: The Sandostatin-treatment resulted in a decreased tumor volume in 9 out of 16 animals. Immunohistochemical detection of apoptosis by Apoptag revealed a 75-fold increase of the positively stained tumorous nuclei (210.9 +/- 53.9 per square mm) versus nontreated tumors (2.8 +/- 0.5 per square mm). The sub-G1 fraction was 3.61 +/- 0.4% in untreated samples while it doubled after treatment (p < 0.001). CONCLUSION: A 4-week octreotide (Sandostatin) treatment induced significantly increased apoptosis in human pancreatic carcinoma xenografts evidenced by morphological studies and Apoptag-immunohistochemistry, and these results were clearly reinforced by flow cytometry.

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.

[Causes of recurrence after laparoscopic hiatal reconstruction]. / Laparoszkópos hiatus rekonstrukciók után fellépó recidívák okai.

The authors report our recurrent hiatal hernias occurred after laparoscopic hiatal reconstruction. The situation found during laparoscopic re-operations are illustrated on pictures. The show the methods against the recurrence.

Apoptotic and antiapoptotic effect of (-)deprenyl and (-)-desmethyl-deprenyl on human cell lines.

The antiapoptotic effect of (-) deprenyl on human phaeochromocytoma cells after serum deprivation has been reported by earlier. Two melanoma (M-1 and HT-2058) cell lines were used in our experiments. Serum deprivation for five days resulted in excessive number of apoptosis in the cell cultures. Very low doses of (-)-deprenyl (10(-7)-10(-13) mol) caused an approximately 2 days delay in the onset of apoptosis. At the same time, +deprenyl was ineffective. In further experiments (-)-deprenyl and (-)desmethyl-deprenyl was administered in higher doses (10(-2), 10(-3) and 10(-4) mol) to A-2058 melanoma and HT-1080 fibrosarcoma cells in culture. In these experiments no serum deprivation was applied and the treatment was started 24 hours after plating. Total eradication of the A-2058 cells was caused by 10(-2) mol (-)-deprenyl and (-)-desmethyl-deprenyl. The type of cell death appeared to be apoptosis. Sixty percent apoptotic ratio was seen 24 hours and 72 hours after 10(-3) mol (-)-desmethyl-deprenyl treteatment. The same dose of (-)-deprenyl caused 50% apoptosis an 72 h. Only (-)-desmethyl-deprenyl induced apoptosis (20%) at 24 hours, in the dose of 10(-4) mol. Interestingly (-)-deprenyl treatment resulted in 60% apoptosis.

Apoptosis, mitosis, p53, bcl(2), Ki-67 and clinical outcome in prostate carcinoma treated by androgen ablation.

A prospective study on 16 patients with advanced (stage III and IV) prostate cancer was carried out. TNM stage, general clinical status, serum PSA level, the histological type and Gleason's grade of the tumor were registered. Total androgen blockade or single-drug therapy (flutamide) was performed. On average, 4.81 months after the start of therapy rebiopsy, serum PSA determination and general clinical examination were performed. Histologic examination before and after treatment of HE-stained slides, as well as apop-tag reaction to show apoptotic cells, p53, bcl(2), and Ki-67 immunostaining. Clinical improvement manifested by regression or lack of progression was observed in 10 patients. Increase of the apoptotic index and decrease of the mitotic index was detected in these cases. Serum PSA level decreased in all patients except in 3 fatal cases. The 6 clinically nonresponders who died after the second biopsy did not show an increased apoptotic or decreased mitotic index. Ki-67 positivity correlated well with the mitotic activity. Mutant p53 expression was higher in patients in whom antiandrogen therapy was ineffective. The bcl(2) expression was a characteristic of the tumors of patients who later died. These results show that the degree of induction of apoptosis in prostate carcinoma by hormonal therapy varies from case to case. A given prostate cancer patient's response to therapy may be predicted by following apoptotic and mitotic activity, as well as Ki-67 and p53 expression in repeated biopsies.

A specifically radiolabeled somatostatin analog with strong antitumor activity induces apoptosis and accumulates in the cytosol and the nucleus of HT29 human colon carcinoma cells.

The new heptapeptide somatostatin analog TT-232 decreases proliferation of HT-29 human colon carcinoma cells in vitro by reducing mitotic and increasing apoptotic activity. We have synthesized and characterized a specifically tritium labeled 3H-Tyr3-TT-232 (30 Ci/mmol) to investigate the effect and the fate of this antitumor peptide on human colon tumor cells. 3H-labeled TT-232 could be detected on the cell surface, on cytoplasmic membranes and also in the nucleus of HT-29 cells, 1-6 h after the administration of 0.5 and 50 microg/mL [3H]TT-232. Binding and internalization of TT-232 to human colon tumor cells at a relatively high dose provide further evidence for the existence of low-affinity somatostatin receptors in such cells, which might mediate the apoptosis-inducing effect. Our data suggest the possible use of TT-232 in the treatment of human colon tumors.

[Preparation and characterization of potential antineoplastic agents]. / Potenciális daganatellenes hatású vegyületek elóállítása és vizsgálata.

A parallel combinatorial library of over sixteen hundred compounds has been designed and synthesized for the development of new potential peptidomimetic protein tyrosine kinase (PTK) inhibitor leads that is aimed for intervening with the substrate binding site of the pp60c-src enzyme. The new structures were based on known PTK inhibitors having at least two variously substituted aromatic moieties attached by spacer groups of different length and flexibility. Eleven bis-aryl type inhibitory compounds were found in the range of 18-100 micromolar IC50 concentrations from combinations of twelve different substituents. Molecular modeling of the active compounds showed a characteristic distance of 13-14 A between the farthest sp2 carbon atoms of the two aromatic rings. Conformational analysis of several peptide substrates recently found for pp60c-src PTK [5,6,7] showed that the energy minimized conformers had the same distance between two aromatic moieties. Several compounds in the library not only showed remarkable PTK inhibitory activity but also a significant apoptosis inducing effect on HT-29 human colon tumor cells.

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.

Coexpression of p53 and tissue transglutaminase genes in human normal and pathologic adrenal tissues.

The presence of p53 and tissue transglutaminase (tTG) gene expressions was investigated in human normal and pathologic adrenal tissues with two aims (1) to determine the tissue content of p53 protein, its messenger ribonucleic acid (mRNA) and, especially, tTG mRNA which has not been previously reported and (2) to study possible differences in the coexpression of p53 and tTG in various adrenal disorders. Using Northern blot analysis, p53 and tTG mRNAs were detected in each adrenal tissue examined including 5 normal human adrenals, 6 aldosterone-producing adenomas, 3 Cushing's adenomas, 1 primary nodular adrenocortical hyperplasia causing Cushing's syndrome in an infant, 12 non-hyperfunctioning adrenocortical adenomas, and 4 adrenocortical carcinomas. The results showed a significant positive correlation between these two mRNAs in all adrenal tissues except adrenocortical carcinomas. Compared to normal adrenals, high p53 mRNA levels were observed in aldosterone-producing and Cushing's adenomas and, most markedly, in a tissue from a primary nodular adrenocortical hyperplasia. Also, Cushing's adenomas had significantly higher tTG mRNA contents. Immunohistochemistry for wild-type and mutant p53 protein showed numerous p53 positive cells with a strong nuclear staining in a tissue from a primary nodular adrenocortical hyperplasia, whereas the p53 positive cells were absent, except those with a faint nuclear staining, in all other adrenal tissues. However, all adrenal tissues showed detectable p53 contents by the more sensitive method of luminometric immunoassay (LIA). Using this method, aldosterone-producing adenomas exhibited significantly higher p53 contents than normal adrenal tissues. These observations may support potentially important roles for p53 and tTG in adrenal pathophysiology, especially in mechanisms which influence the evolution and/or progression of aldosterone-producing and Cushing's adenomas and, most probably, hyperplasias.

Apoptosis and p53 expression in human gliomas.

Twenty-five human gliomas of different histological grade and type were studied for p53 expression by immunohistochemistry and for apoptosis using ApopTag method. p53 expression (percentage of positive cells) was highest in anaplastic astrocytomas, followed by low grade astrocytomas and surprisingly in glioblastomas. Granular cytoplasmic p53 positivity appeared in 4/5 low grade oligodendroglioma and in 2/5 low grade mixed oligoastrocytomas. The means of apoptosis index in the different tumor types ranged between 0.8 and 11.5 with the highest values in anaplastic astrocytoma and glioblastomas. Although the number of cases per group were relatively low and the individual vales showed differences it seems that p53 expression is related to the biological aggressiveness of gliomas. It is also suggested that high level of apoptosis in malignant glioma could represent a p53 independent pathway.

Laparoscopic fenestration of symptomatic solid spleen cyst with harmonic scalpel instrument.

The treatment of two operated solid splenic cysts has been reported by authors. Laparoscopic cyst fenestration has been demonstrated to be a useful alternative method to open surgery. The aim of the authors was to analyse the use of Ultracision Harmonic Scalpel in two spleen preserving procedures. Cysts were located in the superior and the anterior-inferior pole of the spleen. Cyst wall not covered by spleen tissues was removed, drain was left in the abdomen. In the demonstrating spleen cyst operation the advantages of HS instrument was the clean operating field, correct coagulation of cyst wall, short hospital stay. Authors believe that this new technology will make it easier and more desirable for surgeons to fenestrate symptomatic spleen cysts.

Laparoscopic hiatal reconstruction and use of harmonic scalpel instrument.

The method of laparoscopic repair of hiatal hernia is accepted in surgery. Usually associated with Nissen fundoplication which is the most commonly performed antireflux operation. Within a ten-year period authors have done 90 antireflux operations, 52 was laparoscopic procedures. (30 operations for GERD, 10 for hiatal hernia, 12 for the combination of both.) A patient was submitted to operation with large hiatal hernia. He was treated laparoscopic way with success and good results. The Ultracision Harmonic Scalpel instrument helped their operation in many ways, and provided correct bloodless preparation of cardiac region. The authors demonstrate our procedure and the use of the Harmonic Scalpel. Their patient were completely pleased with the results after discharge, they are still under regular control.

A tumor-selective somatostatin analog (TT-232) with strong in vitro and in vivo antitumor activity.

We report a series of new in vitro and in vivo data proving the selective antitumor activity of our somatostatin structural derivative, TT-232. In vitro, it inhibited the proliferation of 20 different human tumor cell lines in the range of 50-95% and induced a very strong apoptosis. In vivo TT-232 was effective on transplanted animal tumors (Colon 26, B16 melanoma, and S180 sarcoma) and on human tumor xenografts. Treatment of MDA-MB-231 human breast cancer xenografted in mice with low submaximal doses of TT-232 [0.25 and 0.5 mg/kg of body weight (b.w.)] caused an average 80% decrease in the tumor volume resulting in 30% tumor-free animals surviving for longer than 200 days. Treatment of prostate tumor (PC-3) xenografted animals with 20 mg/kg of b.w. of TT-232 for 3 weeks resulted in 60% decrease in tumor volume and 100% survival even after 60 days, while 80% of nontreated animals perished. We have demonstrated that TT-232 did not bind to the membrane preparation of rat pituitary and cortex and had no antisecretory activity. TT-232 was not toxic at a dose of 120 mg/kg of b.w. in mice. Long-term incubation (24 h) of tumor cells with TT-232 caused significant inhibition of tyrosine kinases in good correlation with the apoptosis-inducing effect. The level of p53 or KU86 did not change following TT-232 treatment, suggesting a p53-independent apoptotic effect. Preincubation of human breast cancer cells (MDA-MB-453) with TT-232 for 2 h decreased the growth factor receptor autophosphorylation. All of these data suggest that TT-232 is a promising and selective antitumor agent.

Tyrphostin induces non-apoptotic programmed cell death in colon tumor cells.

The programmed cell death inducing effect of the EGF receptor tyrosine kinase inhibitor alpha-cyano-3,4-dihydroxycinnamthioamide (AG213) was investigated in vitro on HT-29 human colon tumor. AG213 at concentrations between 45 to 450 microM blocks the proliferation of HT-29 cells. Morphological findings suggest that the selective tyrosine kinase inhibitor AG213 induces Clarke III type (non-lysosomal vesiculate cytoplasmic) programmed cell death; unlike ATP analog non-selective tyrosine kinase inhibitors like Genistein which were found to induce apoptosis. Cycloheximide and Actinomycin-D reduced the effect of AG213 pointing to the fact that protein and RNA synthesis are also needed for this form of cell death. Acid phosphatase activity was found in the Golgi and in the newly formed intracytoplasmic vacuoles 3 hours after AG213 treatment which disappeared by 6 hours. The induction of Clarke III cell death by tyrosine kinase inhibitors may open a new modality to selective killing of tumor cells.