Julia K. Bar

Epidermal Growth Factor Receptor and c-erbB-2 Oncoproteins in Tissue and Tumor Effusion Cells of Histopathologically Different Ovarian Neoplasms

The overexpression of two growth factor receptors – epidermal growth factor receptor (EGFR) and c-erbB-2 – was evaluated immunohistochemically in malignant and benign ovarian neoplasms, considering the stage of the disease and histology of tumors. The comparison of EGFR and c-erbB-2 reactivity in tissue sections and respective cyst and/or ascitic fluid cells was also performed. c-erbB-2 expression was detected in 44.4% of ovarian carcinomas, and in benign neoplasms there was no evidence of its staining, while EGFR reactivity was found both in malignant (58.7%) and benign (50%) tumors. Significant heterogeneity of staining was observed, however, the relationship between EGFR and c-erbB-2 expression in tissue sections and cyst and/or ascitic fluid cells in individual patients was evident. The expression of both growth factor receptors was not correlated with histopathological subtypes of ovarian neoplasms. The c-erbB-2 oncoprotein was detected more frequently in III/IV than in I/II stages according to the criteria of the International Federation of Gynecology and Obstetrics (FIGO) and the EGFR expression was independent of the clinical advancement of the disease. The coexpression of c-erbB-2 and EGFR was shown in 32% of ovarian carcinomas, and it dominated in cases with FIGO stages III/IV. Our results indicate that the increase of the EGFR expression appears to be associated with early stages of ovarian tumorigenesis, and the enhancement of c-erbB-2 reactivity may cooperate with EGFR activation in the development and progression of ovarian carcinomas.

Expression of p53 protein phosphorylated at serine 20 and serine 392 in malignant and benign ovarian neoplasms: correlation with clinicopathological parameters of tumors.

Introduction: The modification of p53 protein by phosphorylation plays an important role in its stabilization and the regulation of its biological properties. The study investigated the expression of p53 protein phosphorylated at serine 20 (Ser20) and Ser392 and the association between clinicopathological parameters of ovarian neoplasms with respect to p53 protein overexpression. Methods: p53 protein expression was evaluated on tissues from malignant and benign ovarian tumors. Protein expression was measured in a subset of the specimens using immunohistochemistry. Results: The correlation between p53 protein overexpression and p53-Ser392 phosphorylation was found in ovarian carcinomas (P = 0.001, r = +0.27). In the total group of ovarian carcinomas, significant differences were observed in p53 protein overexpression between well (G1) and poor (G3) tumor grades (P = 0.005) and between serous and endometrioid types of tumor (P = 0.04), whereas p53-Ser20 phosphorylation was associated with advanced International Federation of Gynecology and Obstetrics stage (P = 0.004) and high tumor grade (P = 0.02). In p53-positive ovarian carcinomas, p53-Ser392 phosphorylation was associated with advanced tumor stage (P = 0.02) and high tumor grade (P = 0.049). p53-Ser20 phosphorylation was associated with low tumor grade of p53-positive ovarian carcinomas (P = 0.02) and with high tumor grade of p53-negative ovarian carcinomas (P = 0.02). Conclusions: These results revealed that p53 phosphorylation at Ser20 and Ser392 is an early event in ovarian tumor development. The authors suggest that the expression of p53 protein phosphorylated at Ser20 and Ser392 in ovarian carcinomas determines their individual clinical features depending on p53 protein status and may be useful biological biomarkers characterizing their behavior.

nm23 expression in tissue sections and tumor effusion cells of ovarian neoplasms

The genetic changes involved in the metastatic process of ovarian epithelial cancer remain undetermined. The expression of nm23, a putative metastasis‐suppressor gene product, was assessed immunohistochemically in malignant and benign ovarian neoplasms, considering histology of tumors and clinical advancement of disease. Comparison of nm23 protein content in tissue sections and respective cyst and/or ascitic fluid cells was also performed. Significant heterogeneity of nm23 immuno‐staining was observed, and no correlation with histological subtype of ovarian carcinoma was found. Expression of nm23 was higher in carcinomas compared with benign tumors. A significant trend to have a higher nm23 reactivity in ascitic fluid cells vs. primary tumors was observed. Our results indicate that the increase of nm23 reactivity is activated in the early stages of the disease and that the progression of ovarian carcinoma is accompanied by overexpression of nm23 protein. Our observations did not confirm the postulated role of nm23 as a suppressor gene in ovarian cancer.

Oxidative alterations induced in vitro by the photodynamic reaction in doxorubicin-sensitive (LoVo) and -resistant (LoVoDX) colon adenocarcinoma cells

In photodynamic therapy (PDT) a tumor-selective photosensitizer is administered and then activated by exposure to a light source of appropriate wavelength. Multidrug resistance (MDR) is largely caused by the drug efflux from the tumor cell by means of P-glycoprotein, resulting in reduced efficacy of the anticancer therapy. This study deals with photodynamic therapy with Photofrin® (Ph) on colon cancer cell lines (doxorubicin-sensitive and -resistant). The cells were treated with 15 and 30 μg/mL Ph and then irradiated by a light dose of 3 or 6 J/cm2 (632.8 nm). After irradiation the cells were incubated for 0, 3 or 18 h. Crucial factors of oxidative stress (thiobarbituric acid reactive substances [TBARS], protein damage, thiazolyl blue tetrazolium bromide [MTT] assay), changes in cytosolic superoxide dismutase (SOD1) activity after photodynamic reaction (PDR), and the intracellular accumulation of photosensitizers in the cells were examined. Moreover, the expressions of glutathione S-transferase (GST)-pi, a marker protein for photochemical toxicity, and secretory phospholipase A2, a prognostic and diagnostic marker for colon cancers, were determined. After PDR, increases in SOD1 activity and the level of TBARS were observed in both cell lines. The level of protein-associated –SH groups decreased after PDR. Both cell lines demonstrated stronger GST-pi and PLA2 expression after PDR, especially after 18 h of incubation. The increasing level of reactive oxygen species following the oxidation of sulfhydryl cell groups and lipid peroxidation influence the activity of many transporters and enzymes. The changes in SOD1 activity show that photodynamic action generates oxidative stress in treated cells. Our study presents that PDR caused oxidative alterations in both examined colon adenocarcinoma cell lines. However, the MDR cells reacted more slowly and all oxidative changes occurred in the delay.

Expression and Mutation of p53 in Tumor Effusion Cells of Patients with Ovarian Carcinoma: Response to Cisplatin-Based Chemotherapy

p53 alterations are considered as one of the most important factors responsible for drug resistance in ovarian carcinomas, although the relationship between p53 gene status and response to cisplatin-based chemotherapy in ovarian cancer patients remains unclear. The aim of the study was to evaluate the relationship between p53 protein accumulation, p53 gene mutation and response to cisplatin-based chemotherapy in patients with ovarian carcinoma considering conventional clinicopathological parameters. Tissue sections and corresponding cyst and/or ascitic fluid cells from 79 patients with epithelial ovarian cancer were analyzed immunohistochemically for p53 expression. The PCR-SSCP analysis was performed in 25 cases and the results were compared with immunohistochemical data. It was demonstrated that p53 expression reaching approximately 50% of positive cells in immunostaining was usually associated with PCR-amplified exons showing abnormal migration and suspected for mutation. p53 gene changes were not correlated with histological structure, grade of differentiation or residual tumor after cytoreductive surgery, despite being detected more frequently in III/IV than in II FIGO stages and in patients with residual disease above 2 cm. A significant correlation between p53 accumulation and p53 gene alteration and poor response to cisplatin-based chemotherapy was shown. The overall survival time of patients decreased with an increase in p53 protein expression. A strong p53 expression especially accompanied by p53 changes detectable by PCR-SSCP analysis appears to be a good indicator of the resistance to cisplatin-based chemotherapy. The association between strong p53 overexpression and shorter overall survival time was also revealed.

Combination of therapy with 5-fluorouracil and cisplatin with electroporation in human ovarian carcinoma model in vitro

High electric field, applied to plasma membrane, affects organization of the lipid molecules, generating transient hydrophilic electropores. The application of the cell membrane electroporation in combination with cytotoxic drugs could increase the drug transport into cells. This approach is known as electrochemotherapy (ECT). Our work shows new data concerning the influence of electrochemical reaction with cisplatin or with 5-fluorouracil (5-FU) on cancer ovarian cells resistant to standard therapy with cisplatin, in comparison to ECT effect on human primary fibroblasts. We investigated the effect of electroporation and electrochemotherapy with 5-FU and cisplatin on human ovarian clear-cell carcinoma cell line (OvBH-1) and epithelial ovarian carcinoma cell line (SKOV-3) - both resistant to cisplatin typically used in ovarian cancers. As control cells, human gingival fibroblasts (HGFs) from primary culture were used. Electropermeabilization efficiency was determined by FACS analysis with iodide propidium. Efficiency of electrochemotherapy was evaluated with viability assay. The cytotoxic effect was dependent on the electroporation parameters and on drug concentration. Electroporation alone only insignificantly decreased cells proliferation in OvBH-1 line; SKOV-3 line was more sensitive to the electrical field. Electrochemotherapy with cisplatin and 5-FU showed promising effects on both ovarian cell lines with recovery of normal cells revealed after 72 hours.

The role of p53 protein and MMP-2 tumor/stromal cells expression on progressive growth of ovarian neoplasms.

The aim of our study was to evaluate p53 gene/protein status and MMP-2 expression in respect to ovarian tumors progress to define the role of these markers in the metastasis of ovarian carcinomas. MMP-2 and p53 alterations were evaluated on 80 malignant, 30 benign ovarian tumors, and 62 metastatic lesions by using HRM method for mutations in p53 gene and by using RT-PCR for mRNA MMP-2 level. Our data indicate that parallel expression of MMP-2 epithelial/stromal cells and p53 may enhance cells invasion and metastasis in ovarian carcinoma.

Relation between ovarian carcinoma-associated antigens in tumor tissue and detached cyst fluid cells of patients with ovarian neoplasms.

Aims The expression and potential diagnostic value of ovarian carcinoma-associated antigens were estimated in different types of epithelial ovarian neoplasms. The comparison of antigenic expression was performed on solid tumor tissues and loose cyst fluid cells in individual cases of malignant and benign ovarian neoplasms. Methods All studies were performed using monoclonal antibodies (mAbs) against ovarian carcinoma-associated antigens (OC125, OV-TL3, OV632, 10B, 8C) by 3-step peroxidase-antiperoxidase test. Results All ovarian carcinoma-associated antigens were detected in most serous and endometrioid carcinomas. In mucinous carcinomas as well as in benign ovarian neoplasms these antigens were present only in some cases. Significant inter- and intratumoral immunological heterogeneity was evident; however, the antigens detectable in tissue sections were also found in detached cyst fluid cells. Conclusions Our results show that mAb show OV-TL3 is the best marker for endometrioid carcinomas and confirmed that mAbs OV632, OC125 and OV-TL3 could be good complementary markers for differentiating malignant and benign lesions in the ovary. The percentage content of all ovarian carcinoma-associated antigens in solid tumors and respective cyst fluid cells was comparable.

Relationship between p53 and c-erbB-2 Overexpression in Tissue Sections and Cyst Fluid Cells of Patients with Ovarian Cancer

The expression of p53 protein and overexpression of c-erbB-2 oncoprotein was examined immunohistochemically and compared on frozen tissue sections and cyst fluid cells in patients with epithelial malignant and benign ovarian neoplasms. p53 was detected in 52.6% of carcinomas and c-erbB-2 expression was identified in 47.3% of cases. The relationship between p53 and c-erbB-2 overexpression in tissue sections and detached cyst fluid cells was evident. Moreover, a significant association between the presence of p53 and overexpression of c-erbB-2 proteins in tumor tissue sections and loose cyst fluid cells in individual patients was demonstrated. In tissue sections and loose cyst fluid cells of benign ovarian tumors no overexpression of p53 and c-erbB-2 proteins was found.

Bronchial hyper-responsiveness, subepithelial fibrosis, and transforming growth factor-β1 expression in patients with long-standing and recently diagnosed asthma

Introduction:Chronic inflammation in asthmatic airways leads to bronchial hyper-responsiveness (BHR) and the development of structural changes. Important features of remodeling include the formation of subepithelial fibrosis due to increased collagen deposition in the reticular basement membrane. Transforming growth factor (TGF)-β might be a central mediator of tissue fibrosis and remodeling.Materials and Methods:Immunohistochemistry was used to measure collagen III deposition and TGF-β1 expression in biopsies from patients with long-standing asthma treated with inhaled corticosteroids, patients with recently diagnosed asthma, and control subjects. Computer-assisted image analysis was used to evaluate total basement membrane (TBM) thickness.Results:Asthmatics, particularly those with long-standing asthma, had thicker TBMs than healthy subjects. Collagen III deposition was comparable in the studied groups. BHR was not correlated with features of mucosal inflammation and was lower in steroid-treated patients with long-standing asthma than in subjects with newly diagnosed asthma untreated with steroids. Epithelial TGF-β1 expression negatively correlated with collagen III deposition and TBM thickness.Conclusions:The study showed that TBM thickness, but not collagen III deposition, could be a differentiating marker of asthmatics of different disease duration and treatment. The lack of correlation between BHR and features of mucosal inflammation suggests the complexity of BHR development. Corticosteroids can reduce BHR in asthmatics, but it seems to be less effective in reducing subepithelial fibrosis. The role of epithelial TGF-β1 needs to be further investigated since the possibility that it plays a protective and anti-inflammatory role in asthmatic airways cannot be excluded.