Chosen Antioxidant Enzymes GPx4 and GPx8 in Human Colorectal Carcinoma: Study of the Slovak Population.

Background and Objectives: Nowadays colorectal carcinoma (CRC) is one of the most common causes of death in patients with malignant neoplasms worldwide. Our work aimed to determine the possible involvement of glutathione peroxidases 4 and 8 (GPx4 and GPx8) in this specific tumor process. Materials and Methods: The expression of GPx4 and GPx8 in 58 specimens of human colorectal cancer tissues and normal tissues was detected by the indirect immunohistochemical method under a light microscope. Statistical analysis was done by Chi-squared test. Histological findings were compared with data such as gender, age, tumor grade, histotype and lymph nodes alteration. Results: In all specimens of healthy tissue the presence of both, GPx4 and GPx8, was detected in the cytoplasm of epithelial cells. On the other hand, a positive immunohistochemical reaction against GPx4 only in 41.4% and against GPx8 only in 29.3% of human colorectal adenocarcinoma specimens were observed. Any significant difference between the presence of GPx and the age, the gender of the patient, tumor grade, histotype of cancer and the lesion of regional lymph nodes has not been detected. Conclusions: Our foundation could mean, that GPx4 and GPx8 have no important role in CRC pathogenesis, but the loss of these enzymes probably indicates a serious pathological process ongoing in the large intestine. To our knowledge, this is the first paper describing GPx8 presence in human colorectal carcinoma.

Influence of Paclitaxel and Doxorubicin Therapy of ßIII-Tubulin, Carbonic Anhydrase IX, and Survivin in Chemically Induced Breast Cancer in Female Rat.

Breast cancer is the most common cancer in females. The aim of this study was to determine the effect of paclitaxel (PTX) and doxorubicin (DOX) therapy on the ßIII-tubulin, carbonic anhydrase IX (CA IX), and survivin expression in chemically-induced rat mammary tumors. Animals with induced mammary carcinogenesis were randomly divided into treatment groups and an untreated group. The total proportion of tumors, the proportion of carcinoma in situ (CIS), and invasive carcinoma (IC) were evaluated. Protein expression in tumor tissue was determined using IHC. Statistical analysis of the data, evaluated by Fisher-exact test and unpaired t-test. Significantly increased levels of proteins in the tumor cells were confirmed using the IHC method for all studied proteins. The expression of ßIII-tubulin, CA IX, and survivin increased significantly after treatment with both cytostatics (PTX and DOX). Depending on the type of tumor, a significant increase in all proteins was observed in IC samples after PTX treatment, and CA IX expression after DOX treatment. In CIS samples, a significant increase of ßIII-tubulin and survivin expression was observed after a DOX treatment. The results suggest that ßIII-tubulin, survivin, and CA IX may be significant drug resistance markers and the clinical regulation of their activity may be an effective means of reversing this resistance.

Characterization of Glutathione Peroxidase 4 in Rat Oocytes, Preimplantation Embryos, and Selected Maternal Tissues during Early Development and Implantation.

This study aimed to describe glutathione peroxidase 4 (GPx4) in rat oocytes, preimplantation embryos, and female genital organs. After copulation, Sprague Dawley female rats were euthanized with anesthetic on the first (D1), third (D3), and fifth days of pregnancy (D5). Ovaries, oviducts, and uterine horns were removed, and oocytes and preimplantation embryos were obtained. Immunohistochemical, immunofluorescent, and Western blot methods were employed. Using immunofluorescence, we detected GPx4 in both the oocytes and preimplantation embryos. Whereas in the oocytes, GPx4 was homogeneously diffused, in the blastomeres, granules were formed, and in the blastocysts, even clusters were present mainly around the cell nuclei. Employing immunohistochemistry, we detected GPx4 inside the ovary in the corpus luteum, stroma, follicles, and blood vessels. In the oviduct, the enzyme was present in the epithelium, stroma, blood vessels, and smooth muscles. In the uterus, GPx4 was found in the endometrium, myometrium, blood vessels, and stroma. Moreover, we observed GPx4 positive granules in the uterine gland epithelium on D1 and D3 and cytoplasm of fibroblasts forming in the decidua on D5. Western blot showed the highest GPx4 levels in the uterus and the lowest levels in the ovary. Our results show that the GPx4 is necessary as early as in the preimplantation development of a new individual because we detected it in an unfertilized oocyte in a blastocyst and not only after implantation, as was previously thought.

GPx8 Expression in Rat Oocytes, Embryos, and Female Genital Organs During Preimplantation Period of Pregnancy.

This study aimed to detect the presence of glutathione peroxidase 8 (GPx8) in rat during preimplantation period of pregnancy. Females were killed on first (D1), third (D3), and fifth (D5) day of pregnancy. The presence of GPx8 in embryos was detected under the confocal microscope, the presence of GPx8 in genital organs was confirmed immunohistochemically, and the amount of GPx8 was determined using densitometry. We found that GPx8 is dispersed in the cytoplasm of oocytes, while after fertilization, it is concentrated in granules. From 4-cell stage till blastocyst, GPx8 reaction was found in the perinuclear region. In the ovary, GPx8 was seen in granulosa-lutein cells, in plasma of blood vessels, and inside Graafian follicles. In oviduct, GPx8 was detected in the plasma and in the extracellular matrix (ECM). Moreover, epithelial cells of isthmus were positive. In uterus, GPx8 was observed in the uterine glands, in the plasma, and in ECM. On D5, the enzyme disappeared from the uterine glands and appeared in fibroblasts. Densitometry revealed that the highest amount of GPx8 was on D1 and subsequently declined. To our knowledge, this is the first paper describing GPx8 presence in the oocytes, preimplantation embryos, and female genital organs in mammals. Our results improve the understanding of antioxidant enzymes presence during pregnancy in defense against oxidative stress, which is considered to be one of the main causes of infertility.

Monoamine Oxidase B in Renal Cell Carcinoma.

BACKGROUND Studies on monoamine oxidase B (MAO-B) expression in renal cell carcinoma (RCC) are lacking. This study focused on the immunohistochemical evaluation of MAO-B in RCC. MATERIAL AND METHODS Sixty-three RCC samples were compared on basic clinical and histopathological parameters, including histopathological type and tumor grade. RCC samples were divided according to the histopathological type into 2 groups: conventional type (51 samples) and other types (12 samples). For MAO-B detection, a standard immunohistochemical procedure was employed. RESULTS In healthy kidney samples, MAO-B was detected predominantly in tubules. Fifty-two cancer tissue samples were MAO-B negative and 11 tissue samples were MAO-B low positive. Enzymes were detected only in the cytoplasm. We did not find any significant correlation between the percentage of positive MAO-B specimens and nuclear grade. Additionally, Fisher's test did not reveal any difference in numbers of positive and negative MAO-B samples between the 2 RCC types (P>0.05). CONCLUSIONS From our results, it was clear that MAO-B expression played no significant role in stimulation of renal cancer development. We found that MAO-B occurred only in 19% of kidney tumors and that the positivity of protein expression was low. Moreover, it seems that the disappearance of this enzyme in RCC is a consequence of replacement of healthy tissue by cancer cells. On the other hand, one can assume that the loss of MAO-B expression could be associated with severe pathological processes in the kidney.

The effect of R-(-)-deprenyl administration on antioxidant enzymes in rat testis.

The aim of the study was to investigate the effect of R-(-)-deprenyl administration on the activity and localization of superoxide dismutases (SODs) and catalase (CAT) in rat testis. After 30 days of intraperitoneal administration of either saline (control) or R-(-)-deprenyl dissolved in saline at concentrations of 0.0025mg/kg (low dose of deprenyl, LDD) or 0.25mg/kg (high dose of deprenyl, HDD), males were killed by thiopental, and their testes were collected. We found that deprenyl administration significantly increased the activity of antioxidant enzymes, and this effect varied by dosage. LDD caused significant elevation of all monitored enzymes, but HDD did not increase the activity of SOD2. Employing immunohistochemistry, we detected enzymes predominantly in Leydig cells (SOD1, SOD2, CAT), in late spermatids and residual bodies (SOD1, SOD2), and in primary spermatocytes (SOD2). Histopathological examination did not reveal testicular damage in experimental groups compared to control. Deprenyl proved to be a potent stimulator of antioxidant enzymes in rat testes; therefore, it could be used in the therapy of male infertility. On the other hand, it is crucial to choose a proper dose, since lower dose was more competent compared to a dosage that was one hundred times higher.

The effect of R-(-)-deprenyl administration on reproductive parameters of rat males.

The aim of the study was to investigate the effect of R-(-)-deprenyl administration on the reproductive parameters of rat males. After 30 days of intraperitoneal administration of saline or 0.0025mg/kg (10(-5)mol/l) of R-(-)-deprenyl dissolved in saline, males were mated with females of the same strain. Subsequently, animals were killed by thiopental, and their blood and sperm were collected. We found that epididymis of males exposed to R-(-)-deprenyl had higher sperm count (P<0.05), and females who mated with these males gave birth to a greater number of offspring (P<0.05) compared to control. The blood of experimental animals contained higher levels of testosterone (P<0.05), FSH (P<0.01), and total antioxidants (P<0.01). We did not detect sperm DNA fragmentation in control or in experimental males. Interestingly, round spermatids were often observed inside seminiferous tubules of experimental animals, but obviously without any negative consequences on male fertility. Our findings could be verified on a sample of human male volunteers treated for infertility, because human organism tolerate higher doses of R-(-)-deprenyl, which is a selective inhibitor of monoamine oxidase B employed in our experiment and used in the therapy of Parkinson׳s disease, rather well.

MRP1 and GSTp1 expression in non-small cell lung cancer does not correlate with clinicopathological parameters: A Slovakian population study.

We detected MRP1 (multidrug resistance-associated protein 1) and GSTp1 (glutathione-S-transferase p1) protein expression in samples of non-small cell lung cancer (NSCLC) and our results were compared to basic clinicopathological parameters. The indirect immunohistochemical method was used for localization of monitored proteins. A total of 135 tissue samples of NSCLC were characterized according to histopathological type of tumor. Next, we compared our results with basic clinicopathological parameters (histopathological type of tumor, tumor grade and TNM stage of disease). In MRP1 and GSTp1 positive tumor cells, strong brown cytoplasmic immunostaining was visible. In our set of samples 71% showed MRP1 positivity, while according to histopathological type the squamous cell carcinoma reached the highest level of positivity (76%). Our GSTp1 results showed that similarly to MRP1, 70% of samples were GSTp1 positive. According to histopathological type the adenocarcinoma samples showed the highest GSTp1 expression (77%). For precise statistical evaluation the Kruskal-Wallis, Chi-square and Mann-Whitney tests were used. We did not find any statistically significant correlations between MRP1 and clinicopathological parameters. In the group of GSTp1, by Mann-Whitney test we found a statistically significant correlation between GSTp1 and histological grade (p=0.025) in adenocarcinoma samples. As this was only one group of statistically significant correlation we wanted to confirm this finding. For this we applied also Chi-square test which revealed no statistically significant dependence (p=0.077). No statistically significant relation was seen in the coexpression of both proteins (p=0.753). Despite this, the majority of samples simultaneously expressed MRP1 and GSTp1 proteins. In conclusion, our results show that MRP1 and GSTp1 proteins represent independent prognostic features in NSCLC. Nevertheless, the clinical outcome in individual patients is often difficult to predict. Identification of the factors that characterize the resistant cases would permit immediate treatment of the patients with alternative therapeutic approaches.

Investigation of tumour supressor protein p53 in renal cell carcinoma patients.

BACKGROUND: Investigation of p53 immunoreactivity in formalin-fixed paraffin-embedded tissues of normal renal tissue and renal cell carcinoma with respect to histopathologic subtype and nuclear grade of RCC. METHODS: 42 tissue sections of RCC and 5 samples of normal renal tissue were stained for p53 expression using immunohistochemical assay. The results were analyzed in relation to nuclear grade and histopathologic subtype. RESULTS: In total, p53 expression was found to be 4 to 5 times higher (30.8%) in other types of RCC than in the clear-cell type of RCC (6.9%). Further, there was no statistically significant difference in p53 overexpression among the histopathologic subtypes (P>0.05, P=0.063). No association was found between the expression of p53 and nuclear grade (P>0.05, P=0.17). Interestingly, our study also showed weak cytoplasmic positivity in renal tubular epithelium. CONCLUSION: Our findings suggest that p53 might play an important role in tumour development or progression and it might be used as a new predictor and therapeutic target for RCC.

Relation between expression pattern of wild-type p53 and multidrug resistance proteins in human nephroblastomas.

One of the best characterized resistance mechanisms of human cancer is multidrug resistance (MDR) mediated by P-glycoprotein (Pgp/MDR1) and multidrug-resistant related protein (MRP1). In addition to Pgp/MDR1 and MRP1, p53 inactivation or mutation might play a relevant role in therapeutic failure. This study involved 25 children (17 girls and 8 boys) aged 7 months to 10 years treated for unilateral Wilms' tumor. 25 tissue samples of Wilms' tumor and 5 samples of normal human kidneys were obtained from the Department of Pathological Anatomy, Jessenius Faculty of Medicine in Martin, Slovak Republic. We used an indirect immunohistochemical method to determine expression of Pgp/MDR1, MRP1 and wild-type p53 in 25 tissue samples of nephroblastoma. The minority of nephroblastoma specimens showed positivity for both MDR proteins, as well as for wild-type p53. 24% of tissue samples revealed positive results for Pgp/MDR1, 48% for MRP1 and 8% for wild-type p53. Furthermore, our study showed a statistically significant difference between p53 and MRP1 protein expression (p<0.01), but not between p53 and Pgp/MDR1 (p>0.05). No correlation was found between the expression of both multidrug resistance proteins (Pgp/MDR1 and MRP1) and the expression of wild-type p53. Immunohistochemical detection of the expression of MDR proteins and wild-type p53 at the time of diagnosis might assist in choosing specific chemotherapeutics to improve prognosis and therapy.

Comparison of expression pattern of monoamine oxidase A with histopathologic subtypes and tumour grade of renal cell carcinoma.

BACKGROUND: Studies of the biochemical properties of MAO-A (monoamine oxidase) are numerous, but the information about determination of MAO-A in human normal and tumour renal tissue is limited. Our objectives in the present study were to determine the localization of MAO-A in normal kidney and level of expression of this protein in tumour kidney. MATERIAL/METHODS: Enzyme immunohistochemical method was chosen for detection of MAO-A in 63 clinical samples of all histopathological types of RCC (renal cell carcinoma). Our results were compared to basic clinical and histopathological parameters such as histopathological type and tumour grade. We also compared MAO-A expression between normal and tumour tissue samples. RESULTS: We confirmed the elevated expression of MAO-A in high-grade tumours of renal cell carcinoma specimens. The percentage of MAO-positive samples progressively increased from 9% in grade 2 to 45% in grade 3. We also noted high levels of MAO-A immunoreactivity in epithelial cells of proximal tubules in normal renal tissue. MAO-A was absent or very low in epithelial cells of distal tubules and glomerular capsule, as well as in endothelial cells of renal vessels. CONCLUSIONS: Taken together, our results and findings of other studies show that MAO-A expression in high-grade tumours may have a direct role in maintaining a dedifferentiated phenotype and promoting aggressive behaviour. The ability of clorgyline (an MAO-A inhibitor) to counteract oncogenic pathways and promote differentiation suggests that MAO-A inhibitors, which have been used for many years in clinical practise for treating neurological disorders, could be therapeutic options for advanced stages of tumours.

Association between polymorphisms of XRCC1, p53 and MDR1 genes, the expression of their protein products and prognostic significance in human breast cancer.

BACKGROUND: This study aimed to examine the relationship between XRCC1, p53 and MDR1 protein, along with polymorphisms of their genes and their prognostic values in breast cancer. The following clinical and pathological parameters were evaluated: histopathological type of tumor, grade, stage, Her2/neu expression, ER, PR positivity and involvement of regional lymph nodes. MATERIAL/METHODS: Expression of proteins was determined in 39 samples of breast cancer by immunohistochemistry. Nucleotide polymorphisms were analyzed by PCR-RFLP. For statistical analysis, chi-square test (Yates), Fisher's exact test, and correlation test were used to analyze the data. RESULTS: The highest protein expression was immunohistochemically found in MDR1 protein, with 54% of samples testing positive. In addition, the evaluation of MDR1 expression revealed higher positive immunoreactivity in lobular (LIC) and other types of tumor in comparison to ductal (DIC) type. The expression of p53 and XRCC1 protein was equal, but lower compared to MDR1, both testing positive in 36% of all tissue samples. Comparison of XRCC1 protein and histopathological type of tumor revealed that DIC and LIC types were mostly XRCC1-negative, while other types, papillary and mucinous were more likely to be XRCC1-positive. Interestingly, when evaluating LIC samples separately, a negative correlation between the Her2/neu and expression of XRCC1 was detected. Apparently, all Her2/neu-positive samples were XRCC1-negative (6/86%). The correlation test indicated a negative correlation between Her2/neu-positive samples and XRCC1-negative specimens (r = 1, p < 0.05). Statistical analysis did not reveal a correlation of p53 expression with clinical and pathological parameters. Similarly, no statistically significant difference was found between the tested polymorphisms and protein expression. CONCLUSIONS: We did not find statistically significant correlation between tested polymorphisms and their protein expression.

Prognostic significance of p53 protein and X-ray repair cross-complementing protein 1 in non-small cell lung cancer.

OBJECTIVES: p53 and XRCC1 protein expression were evaluated in 54 samples of nonsmall cell lung cancer. PATIENTS AND METHODS: The immunohistochemical method was used for detection of the monitored proteins. Tissue samples were divided according to histopathological tumor type. The results were then compared with basic clinical and histopathological parameters (histopathological type, nuclear grade and TNM tumor stage IA, IB). RESULTS: Statistically significant correlations were found between histopathological type and p53 expression, since P < 0.05 (P = 0.015). Comparing p53 expression with grade resulted in a strong positive correlation (P < 0.0396, R2 = 0.9223). The percentage of p53-positive tumors progressively increased from 0% in grade 1 to 75% in grade 4. No correlation was found between p53 expression and tumor stage. In case of XRCC1, the highest level was found in squamous cell lung carcinoma, where 71% of samples was positive. In case of large cell carcinoma samples, it was 67%, and in adenocarcinoma 52% of samples showed XRCC1 immunoreactivity. No statistically significant correlation was found between histopathological type, grade or early stage (IA, IB) of non-small cell lung cancer and expression of XRCC1 protein profile without neoadjuvant therapy. CONCLUSIONS: We found a statistically significant correlation between p53 expression and histopathological tumor type. It is possible that stabilized p53 protein plays an important role in the development of squamous and large cell carcinoma. Our findings also suggest that p53 expression cumulates with the dedifferentiation of cancer cells. It is possible that the expression of XRCC1 is not fixed and could be changed by the status of cancer cells and in relation to therapy. Relevant data about pre- versus post-chemotherapy and XRCC1 expression are needed to evaluate the influence of XRCC1 on drug resistance.

Relation between expression pattern of p53 and survivin in cutaneous basal cell carcinomas.

BACKGROUND: The tumor suppressor gene p53 is a key regulator of cell division and/or apoptosis. Survivin is a multifunctional member of the inhibitor of apoptosis family. Survivin and p53 represent diametrically opposed signals that influence the apoptotic pathway. MATERIAL/METHODS: To determine the role of p53 and survivin in basal cell carcinoma (BCC), we evaluated the expression pattern of both proteins with regard to the percentage of positively immunostained tumor cells, the intensity of staining, and subcellular localization among 31 subjects with BCC. RESULTS: Overexpression of p53 protein was found in 28 of 31 cases (90.3%), whereas survivin accumulation was seen in 27 (87.1%). For p53, moderate and/or strong immunoreactivity was seen in 20 of 28 cases (71.4%), and 26 of 28 cases (92.9%) showed more than 25% reactive tumor cells. Nuclear p53 staining was detected in 23 of 28 cases (82.1%), whereas combined nuclear and cytoplasmic localization was found in only 5 of 28 cases (17.9%). Survivin revealed mild intensity of immunoreaction in 22 of 27 cases (71%), and 25 of 27 cases (92.6%) showed less than 25% labeled tumor cells. Combined nuclear and cytoplasmic survivin localization was present in 26 of 27 cases (96.3%). Statistically significant differences were detected in the assessed expression parameters between those proteins. CONCLUSIONS: Our results suggest that overexpression of wild type p53 protein may suppress the expression of survivin and its antiapoptotic activity in BCC cells.

Impact of 2 doses of clorgyline on the rat preimplantation embryo development and the monoamine levels in urine.

We have evaluated the impact of chronic administration of clorgyline, a potent monoamine oxidase A inhibitor and a former antidepressant, on the preimplantation embryo development in Wistar rats. Females were injected intraperitoneally daily for 30 days with saline (control animals), or with a low-dose clorgyline (LDC, 0.1 mg/kg per d) or with a high-dose clorgyline (HDC, 1 mg/kg per d). Embryos were isolated on day 5 of pregnancy and urine was collected by puncture of the urinary bladder. The number of embryos per female did not differ between experimental groups and control, but we have recorded a decreased number of embryos in HDC group compared to LDC (P < .05). We have found that LDC significantly reduced the presence of healthy embryos and increased the presence of the degenerated embryos (P < .001). The administration of the LDC resulted in the lowest cell number in blastocysts. We have observed significantly increased serotonin levels in HDC group compared to both control (P < .05) and LDC animals (P < .01). Norepinephrine (NE) levels in both experimental groups were significantly elevated compared to controls. Dopamine levels did not differ between groups (P > .05). We speculate that lesser negative effect of HDC compared to LDC on the preimplantation embryo development could be the consequence of the lower NE levels and/or elevated serotonin levels. Potential mechanisms mediating clorgyline-induced impaired preimplantation embryo development are proposed.

Histomorphological changes in murine fibrosarcoma after hypericin-based photodynamic therapy.

Histomorphological changes in murine fibrosarcoma after photodynamic therapy (PDT) based on the natural photosensitizer hypericin were evaluated. C3H/DiSn mice were inoculated with fibrosarcoma G5:1:13 cells. When the tumour reached a volume of 40-80 mm(3) the mice were intraperitoneally injected with hypericin, either in a single dose (5 mg/kg; 1 or 6 h before laser irradiation) or two fractionated doses (2.5 mg/kg; 6 and 1 h before irradiation with laser light; 532 nm, 70 mW/cm(2), 168 J/cm(2)). All groups of PDT-treated animals with single and fractionated hypericin dosing presented primary vascular reactions including vascular dilatation, congestion, thrombosis and oedema. Two hours after PDT there were necrotic changes with small, rather focal appearance. One day after therapy the necrotic areas were enhanced, often affecting a complete superficial layer of tumour tissue. Necrotic areas were accompanied with inflammation and haemorrhages.