METHOTREXATE AND MYOTREXATE INDUCE APOPTOSIS IN HUMAN MYOMA FIBROBLASTS (T hES CELL LINE) VIA MITOCHONDRIAL PATHWAY.

Uterine leiomyomas (fibroids) are the most common benign tumors in women of reproductive age. Although the local application of low doses of methotrexate (MTX) is used as an effective treatment of the myomas, myotrexate could be a promising new drug. This study investigated the cytotoxic and apoptotic effects of both MTX and myotrexate in human fibroblasts derived from the uterine fibroids (T hES cell line). The myotrexate adduct is an aqueous solution of MTX and L-arginine. Cells were treated with a graded concentrations of both MTX and myothrexate (0.1-16 µM) for 24 h. The cytotoxicity was assayed by MTT test, apoptosis was evaluated by Annexin V-FITC assay and their possible role in apoptosis was determined by immnu- flourescence. Both MTX and myotrexate induced apoptosis in T hES cells in a dose dependent manner (p < 0.001). Myotrexate significantly increased the percentage of AnnexinV positive cells, BAX/Bcl-2 ratio and subsequent caspase-3 activation compared to the MTX treated cells (p < 0.05). Both MTX or myotrexate treatment showed a diffuse staining of cytochrome c indicating its release from mitochondria to the cytosol, suggesting that their mechanisms of action most likely involves the mitochondrial apoptotic pathway.

Inhibitory role of monovalent ions on rat brain cortex adenylyl cyclase activity.

Adenylyl cyclases, comprise of a large family of enzymes that catalyze synthesis of the cyclic AMP from ATP. The aim of our study was to determine the effect of monovalent ions on both basal, stimulated adenylate cyclase EC 4.6.1.1 (AC) activity and C unit of AC and on GTPase active G-protein in the synaptic membranes of rat brain cortex. The effect of ion concentration from 30 to 200 mM (1 mM MgCl2) showed dose-dependent and significant inhibition of the basal AC activity, stimulated and unstimulated C unit activity. Stimulation of AC with 5 µM GTPγS in the presence of 50-200 mM of tested salts showed inhibitory effect on the AC activity. From our results it could be postulated that the investigated monovalent ions exert inhibitory effect on the AC complex activity by affecting the intermolecular interaction of the activated α subunit of G/F protein and the C unit of AC complex an inhibitory influence of tested monovalent ions on these molecular interaction.

Prognostic significance of co-overexpression of bcl-2 and c-erbB-2/neu in uterine cervix carcinomas and premalignant lesions.

AIM: To establish the role of co-overexpression of bcl-2 and cerbB- 2/neu protooncogenes in uterine cervix carcinogenesis, we examined 138 tissue samples of low grade cervical squamous intraepithelial lesions (SIL), high grade SIL, cervical carcinoma in situ and cervical invasive carcinoma, stage IA-IIA (study group) and 36 samples without SIL or malignancy (control group). METHODS: The expression of bcl-2 and c-erbB-2/neu was detected immunohistochemically using a monoclonal antibody. Fisher's exact test (p less than 0.05) was used to assess statistical significance. RESULTS: Co-overexpression of Bcl-2 and c-erbB-2/neu was found to increase in direct relation to the grade of the squamose intraepitelial lesions (SIL) of cervix. Statistically significant difference was found in the frequency of co-overexpression in patients with high grade SIL (12/22, p=0.006), cervical carcinoma in situ (10/22, p=0.018) and cervical invasive carcinoma (12/26, p=0.012), in relation to the control group. High sensitivity was of great diagnostic significance for the detection of these types of changes in the uterine cervix. CONCLUSION: On the basis of high predictive values it can be said that in patients with co-overexpression of bcl-2 and c-erbB-2/neu overexpression there is a great possibility that they have premalignant or malignant changes in the uterine cervix. However, a more extensive series of samples and additional tests are required to establish the prognostic significance of these oncogenes in cervical carcinogenesis.

Ovarian endometroid adenocarcinoma in pregnancy.

BACKGROUND: Ovarian cancer is very rare in pregnancy. It is mainly of epithelial origin, low grade and low malignant potential. CASE REPORT: We presented a patient in which ultrasound confirmed the presence of clearly limited tumor in the left ovary when she was eight weeks pregnant. The results of 4D Color Doppler showed a central type of tumor vascularisation with resistance index (IR) less than 0.5. The Consultancy Board in Gynecology of the Institute for Oncology and Radiology of Serbia decided to remove the patient's left adnexa and intensively monitor the pregnancy period. The operation (Adnexectomia lateralis sinistra) was performed at 18th week of gestation. Histopathological analysis showed adenocarcinoma invasivum, endometroid well-differentiated type (histological grade I, nuclear grade 2). In 37th week of gestation, the patient gave birth to a male child by cesarean section. In the next 3 years the patient had no subjective interference, laboratory tests and ultrasonographic findings were normal. CONCLUSION: Ovarian cancer in pregnancy is usually asymptomatic and diagnosed during routine clinical and ultrasound examination. The color Doppler technique have particular importance in the diagnosis of pathological blood supply in tumors and in indication of malignant ovarian mass.

Cytosolic pro-apoptotic SPIKE induces mitochondrial apoptosis in cancer.

Proteins of the BCL-2 family are important regulators of apoptosis. The BCL-2 family includes three main subgroups: the anti-apoptotic group, such as BCL-2, BCL-XL, BCL-W, and MCL-1; multi-domain pro-apoptotic BAX, BAK; and pro-apoptotic "BH3-only" BIK, PUMA, NOXA, BID, BAD, and SPIKE. SPIKE, a rare pro-apoptotic protein, is highly conserved throughout the evolution, including Caenorhabditis elegans, whose expression is downregulated in certain tumors, including kidney, lung, and breast. In the literature, SPIKE was proposed to interact with BAP31 and prevent BCL-XL from binding to BAP31. Here, we utilized the Position Weight Matrix method to identify SPIKE to be a BH3-only pro-apoptotic protein mainly localized in the cytosol of all cancer cell lines tested. Overexpression of SPIKE weakly induced apoptosis in comparison to the known BH3-only pro-apoptotic protein BIK. SPIKE promoted mitochondrial cytochrome c release, the activation of caspase 3, and the caspase cleavage of caspase's downstream substrates BAP31 and p130CAS. Although the informatics analysis of SPIKE implicates this protein as a member of the BH3-only BCL-2 subfamily, its role in apoptosis remains to be elucidated.

[Histochemical and immunohistochemical analysis of ruptured atherosclerotic abdominal aortic aneurysm wall].

BACKGROUND/AIM: The main complication of the atherosclerotic abdominal aortic aneurysm (AAA) is her rupture that begins with lesion in intima and rupture. The purpose of this work was to determine immunocytochemical and morphofunctional characteristics of the cells in aortic wall in ruptured atherosclerotic abdominal aortic aneurysm. METHOD: During the course of this study, 20 samples of atherosclerotic AAA were analyzed, all of them obtained during authopsy. The samples were fixed in 4% formalin and embedded in paraffin. Sections of 5 microm thickness were stained histochemically (of Heidenhain azan stain and Periodic acid Schiff--PAS stain) and immunocytochemically using a DAKO LSAB+/HRP technique to identify alpha-smooth muscle actin (alpha-SMA), vimentin, myosin heavy chains (MHC), desmin, S-100 protein, CD45 and CD68 (DAKO specification). RESULTS: The results of our study showed that ruptured atherosclerotic AAA is characterized by a complete absence of endothelial cells, the disruption of basal membrane and internal elastic lamina, as well as a presence of the remains of hypocellular complicated atherosclerotic lesion in intima. On the plaque margins, as well as in the media, smooth muscle cells (SMCs) are present, which express a alpha-SMA and vimentin (but without MHC or desmin expression), as well as leukocyte infiltration, and a large number of foam cells. Some of the foam cells show a CD68- immunoreactivity, while the others show vimentin- and S-100 protein-immunoreactivity. Media is thinned out with a disorganized elastic lamellas, while adventitia is characterized by inflammatory inflitrate (infection). CONCLUSION: Rupture of aneurysm occurs from the primary intimal disruption, which spreads into thinned out media and adventitia. Rupture is caused by unstable atherom, hypocellularity, loss of contractile characteristics of smooth muscle cells in intima and media, neovascularization of the media, as well as by the activity of the macrophages in the lesion.