The influence of phenothiazine derivatives on doxorubicin treatment in sensitive and resistant human breast adenocarcinoma cells.

Breast cancer is commonly treated by various combinations of surgery, radiation therapy, chemotherapy and hormone therapy. Most cancers either are increasingly resistant to any initial treatment or acquire resistance to a broad spectrum of anticancer drugs over time. Combination of more than one drug or combination with multidrug resistance (MDR) modifiers will possibly support the efficiency of the applied therapy. Understanding the MDR mechanisms in malignancies is crucial for developing novel strategies for treatment. The main goal of our study was to determine the cytostatic effect of doxorubicin in combination with phenothiazine derivatives (PD; promazine and triflupromazine) in doxorubicin-sensitive (MCF-7/WT) and -resistant (MCF-7/DOX) human breast adenocarcinoma cell lines. We determined cytotoxicity of the investigated compounds (MTT assay) after 24 and 48 h. The effect of phenothiazine derivatives was evaluated and doxorubicin localization was performed using confocal microscopy. The mode of the cell death was examined by the comet assay. We also determined the expression of P-glycoprotein (P-gp), which is a membrane-associated protein responsible for the multidrug resistance.

Photooxidative action in cancer and normal cells induced by the use of photofrin in photodynamic therapy.

Photofrin-mediated PDT was applied to malignant (A549 and MCF-7) and normal (HUV-EC-C) cells. The cells were incubated for different lengths of time after PDT. The cell responses to the therapy were examined by changes in SOD activity, phototoxicity, and mode of the cell death. PDT induced dynamic changes in SOD activity. Initially, an increase in SOD activity was observed, and after 6 hours of culture it decreased to the control level. Results obtained from MTT and the comet assay indicate that PDT caused immediate cell death via apoptosis in the A549, MCF-7, and HUV-EC-C cell lines. Our studies confirm that SOD is involved in the response of both cancer and normal cells to PDT.

The influence of photodynamic therapy on apoptosis in human melanoma cell line.

Melanoma is the most severe of all skin cancers as it may grow rapidly and metastasize. The application of photodynamic therapy (PDT) opens new perspectives in treatment of this cancer. Numerous studies suggest that the exposure of tumor cells to PDT can lead to cell death via two separate processes: apoptosis or necrosis. The aim of this study was to assess in vitro photodynamic therapy which induces apoptosis in the human Beidegröm Melanoma (BM) cell line, using neutral comet assay. The cells were incubated with Photofrin II (15 microg/ml and 30 microg/ml) 4 h before and 3 h after irradiation for 5 or 10 min with the light intensity of 10 mW/cm2, using a lamp with red filter (632.8 nm). The percentage of apoptotic cells was significantly higher after PDT comparing to control cells. We observed 25% and 70% of apoptotic cells after shorter irradiation and treatment with 15 microg/ml and 30 microg/ml of Ph II, respectively. After longer irradiation, the respective values were 71.9% and 90%. The results suggest that induction of apoptosis is an important determinant of photodynamic sensitivity in the studied cell line and that some types of DNA damage are dependent on photosensitizer concentration and time of irradiation.

Effect of octreotide on proliferation of in vitro cultured thyroid medullary carcinoma cells.

In TT cells, originating from medullary carcinoma of the human thyroid, the presence of receptors for somatostatin was demonstrated at the ultrastructural level. Inhibitory effect of octreotide (a somatostatin analogue) was observed on proliferation of in vitro cultured TT cells and confirmed by evaluating levels of PCNA and Ki-67 proliferation-associated antigens and examining the extent of DNA damage using the comet assay. Our studies indicate a potential for application of somatostatin analogues to diagnosis and adjunct treatment in thyroid medullary carcinomas.

Comparison of techniques permitting to detect apoptosis in situ.

Several morphological and biochemical techniques are in use for identification of apoptotic and necrotic cells in a studied cell population. It is essential to define not only the type of cell death but also to identify the apoptotic process itself, which represents a multistage, active process, requiring activation of a molecular event cascade. In the present study, we have examined and discussed effectiveness of the selected techniques detecting apoptosis in lymphocytes exposed to incubation at an elevated temperature. The appraisal involved detection of caspase-3 active form, detection of Bcl-2, TUNEL reaction, the comet assay and electrophoresis of DNA.

Effect of calcitriol on proliferation of TT cells and on expression of calcitonin gene.

The absence of serum steroids was demonstrated to restrict proliferation of cultured TT cells (cell line originating from human thyroid medullary carcinoma) and supplementation with calcitriol was found to partially restore the proliferation. Calcitriol stimulated TT cell proliferation by augmenting expression of proliferation-associated proteins and by restricting apoptosis. Moreover, calcitriol decreased the intensity of transcription but failed to change direction of the altemate splicing of the calcitonin gene.

Photofrin--factor of photodynamic therapy induces apoptosis and necrosis.

Photodynamic therapy (PDT) causes irreversible photodamage of tumor and other malignant tissues. The effect of reactive oxygen species generation in the presence of photofrin (HpD) was studied. The studies were performed on endothelial cell line from foetal aorta of calves and on normal fibroblasts cell line (3T3 -Balb) and also on malignant line (A431). The cells were grown in presence of photofrin at different time intervals. Time of interaction of photosensitiser with cells was very important. Short time of exposure of the cells to photofrin induced mostly apoptosis in normal cells and apoptotic or necrotic changes in malignant cells. Longer effect of these factors on cells provoked necrosis. The factors of PDT influence dynamic changes of SOD and CT activity. It was dependent on the intensity of factors. These results strongly suggest that HpD has an effect on generation of ROS, which are a signal for development of morphological changes (apoptosis or necrosis) in normal and malignant cells.

Immunocytochemical studies on pancreatic endocrine cells at early stages of development of the pig.

The studies were performed on pig embryos between 23rd and 31st day of intrauterine life. Immunocytochemical markers of neuroendocrine cells, i.e. neuron-specific enolase, chromogranin and synaptophysin as well as basal hormones, i.e. insulin, glucagon, somatostatin, pancreatic polypeptide and, additionally, serotonin and gastrin were detected in serial sections. Our studies indicate that differentiation of pancreatic endocrine cells does not take place unitemporally. At the first stage, the cells acquire the traits of neuroendocrine cells and secrete more than one hormone while final specialization toward cells secreting individual hormones take places at a later stage.

Successful full-term pregnancy in a patient three and a half years after a heart transplant.

The patient is a 28 year old woman who received a heart transplant in 1992 secondary to hypertrophic cardiomyopathy with unremarkable post-operative course. In the period immediately post transplantation the patient was on a four-drug immunosuppressive regimen which was subsequently changed to standard three-agent therapy. This therapy was continued until the patient became pregnant. In the first trimester only Cyclosporine (CsA) was used, and thereafter, the patient was continued on the previous three agent regimen. Toward the end of pregnancy a rise in systolic pressure was observed, but the child was delivered by spontaneous vaginal delivery without complications in the 38th week of pregnancy. The newborn weighed 3320 g and was in good health. A sharp fall in the newborn CsA blood levels was observed post delivery reaching zero level on the third day of life. At the present time, both mother and baby are in good health, 6 weeks after delivery.

Microfocal myocardial lesions in cardiac allograft recipients.

Endomyocardial biopsy has been widely accepted as a method for diagnosing acute heart transplant rejection. However, disperse and microfocal changes due to a smaller specificity, cause difficulty in histological differential diagnosis. The purpose of the present study was to morphologically assess microfocal lesions, to determine their relative incidence and their specificity in relation to the time of onset after cardiac transplantation and to compare these data with those concerning more extensive changes. We examined 658 samples in 152 endomyocardial biopsies obtained from 45 posttransplant patients, including 311 samples identified as containing focal lesions of varying size, and 33 as containing diffuse lesions. Samples with focal and diffuse lesions (344 samples) were submitted for further studies which revealed that microfocal changes in the transplanted heart show more enhanced polymorphism in the composition of cellular infiltrates than more extensive changes. Furthermore they are frequently seen in biopsies in the first two months after transplantation and as single changes. Necrosis and cellular infiltrates at that time are more diversified than those seen in later biopsies. In our experience differential diagnosis of microfocal myocardial lesions in cardiac allograft recipients is difficult. Potentially microfocal lesions may be a result not only of acute rejection but also a result of myocardial alterations in the course of intensive therapy both in the donor and in the recipient, cold ischemia, reperfusion, and inflammation. The morphological picture in some cases is not sufficient to make an unequivocal diagnosis. This indicates the need for improvement of diagnostic methods and criteria on the one hand, and a suitable approach to the problem on the other hand. Our experience shows that a temporary solution could be establishing of a separate classification category for these types of changes and defining them as nonspecific microfocal lesions.

Immunocytochemical studies on endocrine cells of alimentary tract of the pig in the embryonic and fetal period of life.

The study aimed at immunocytochemical analysis of alimentary tract endocrine cells between 20th day of embryonal life and 105th day of fetal life of domestic pig. In the pancreas, presence of endocrine cells was detected already in 20th day and, at the time, the cells comprised around 3/4 all cells in primordia of the organ. Starting at that time, numerous endocrine cells produced insulin and glucagon and individual cells synthesized somatostatin and pancreatic polypeptide. In the 20th day, stomach and duodenum contained single endocrine cells but hormone production was not detected until days 27 and 30. Beginning from this days, both organs manifested rapid increase in the number of gastrin-producing cells. In each of the three organs, the number of somatostatin-producing cells exhibited most extensive changes.