Maria Wołuń-Cholewa

Nucleases isolated from Chelidonium majus L. milky sap can induce apoptosis in human cervical carcinoma HeLa cells but not in Chinese Hamster Ovary CHO cells.

Milky sap isolated from Chelidonium majus L. (Greater Celandine) serves as a rich source of various biologically active substances such as alkaloids, flavonoids and phenolic acids. Previous research showed that the activity of Ch. majus milky sap may depend also on the presence of biologically active proteins. The goal of this study was to evaluate the biological effect of two nucleases isolated from Ch. majus milk sap, CMN1 of 20 kDa and CMN2 of 36 kDa, on HeLa and CHO tumour cell lines. Both studied nucleases together with other proteins in the sap of the plant are involved in stress and defence reactions against different pathogens. After 48 h incubation of CMN1 and CMN2 only with HeLa cells, the dependence between the number of apoptotic lesions and the concentration of applied nuclease was observed. The highest proapoptotic activity was induced by 13.3 ng/ml concentration of CMN2 collected in May (62 +/- 3% HeLa cells were apoptotic). Moreover, the proportion of necrotic cells in all concentrations of the nucleases and both cell lines was relatively low (1-8 +/- 0.5%). In summary, results of this study show that purified nucleases CMN1 and CMN2 isolated from Ch. majus milky sap exhibit apoptotic activity in HeLa tumour cell line, but not in CHO cells, without inflammatory reaction.

Cytotoxic activity of proteins isolated from extracts of Corydalis cava tubers in human cervical carcinoma HeLa cells

BackgroundCorydalis cava Schweigg. & Koerte, the plant of numerous pharmacological activities, together with the studied earlier by our group Chelidonium majus L. (Greater Celandine), belong to the family Papaveraceae. The plant grows in Central and South Europe and produces the sizeable subterraneous tubers, empty inside, which are extremely resistant to various pathogen attacks. The Corydalis sp. tubers are a rich source of many biologically active substances, with the extensive use in European and Asian folk medicine. They have analgetic, sedating, narcotic, anti-inflammatory, anti-allergic and anti-tumour activities. On the other hand, there is no information about possible biological activities of proteins contained in Corydalis cava tubers.MethodsNucleolytic proteins were isolated from the tubers of C. cava by separation on a heparin column and tested for DNase activity. Protein fractions showing nucleolytic activity were tested for cytotoxic activity in human cervical carcinoma HeLa cells. Cultures of HeLa cells were conducted in the presence of three protein concentrations: 42, 83 and 167 ng/ml during 48 h. Viability of cell cultures was appraised using XTT colorimetric test. Protein fractions were separated and protein bands were excised and sent for identification by mass spectrometry (LC-ESI-MS/MS).ResultsThe studied protein fractions showed an inhibiting effect on mitochondrial activity of HeLa cells, depending on the administered dose of proteins. The most pronounced effect was obtained with the highest concentration of the protein (167 ng/ml) - 43.45 ± 3% mitochondrial activity of HeLa cells were inhibited. Mass spectrometry results for the proteins of applied fractions showed that they contained plant defense- and pathogenesis-related (PR) proteins.ConclusionsThe cytotoxic effect of studied proteins toward HeLa cell line cells has been evident and dependent on increasing dose of the protein. The present study, most probably, represents the first investigations on the effect of purified PR proteins from tuber extracts of a pharmacologically active plant on cell lines.

An Efficient 3D Cell Culture Method on Biomimetic Nanostructured Grids

Current techniques of in vitro cell cultures are able to mimic the in vivo environment only to a limited extent, as they enable cells to grow only in two dimensions. Therefore cell culture approaches should rely on scaffolds that provide support comparable to the extracellular matrix. Here we demonstrate the advantages of novel nanostructured three-dimensional grids fabricated using electro-spinning technique, as scaffolds for cultures of neoplastic cells. The results of the study show that the fibers allow for a dynamic growth of HeLa cells, which form multi-layer structures of symmetrical and spherical character. This indicates that the applied scaffolds are nontoxic and allow proper flow of oxygen, nutrients, and growth factors. In addition, grids have been proven to be useful in in situ examination of cells ultrastructure.

Selection of reliable reference genes in eutopic and ectopic endometrium for quantitative expression studies.

PURPOSE Physiological changes during menstrual cycle cause the endometrium and endometriosis to develop specific kind of tissues, especially in regard to the gene expression profiles, which may include also housekeeping genes, commonly used as reference genes (RGs) in quantitative studies. Reverse transcription, followed by quantitative polymerase chain reaction (RT-qPCR) is the most precise and commonly used method in gene expression studies. In order to reduce effects of technical approaches and biological variability of genes expression level, the studies often employ RGs in experimental data normalization. However, the expression of RGs is not always stable and depends on several variables. Thus, the selection of appropriate RG is one of the most significant steps to obtain reliable results in RT-qPCR-based methods. MATERIAL AND METHODS With the usage of RT-qPCR, we researched the expression of seven genes (ACTB, B2M, G6PD, GAPD, GUSB, HPRT and PPIA) as reliable reference genes in eutopic and ectopic endometrial tissue specimens obtained during standard surgery of women of reproductive age. Stability of expression level was analyzed by the most universal MS Excel plug-ins including: geNorm, NormFinder and BestKeeper. The descriptive statistics were evaluated using Statistica software. RESULTS The distribution of threshold (Ct) values was not equal. We identified genes with higher expression level (referring to Ct values) such as ACTB and B2M, medium e.g., GAPD and low expression level, e.g., G6PD and HPRT. We demonstrated that the stability of the analyzed reference genes was not homogenous, and different algorithms pointed to PPIA, GAPD and B2M as the most stable ones in eutopic and ectopic endometrium. On the contrary to these, GUSB and G6PD were the most unstable ones. CONCLUSIONS In RT-qPCR-based analyses of gene expression level in eutopic and ectopic endometrium, we strongly recommend that a minimum of two reference genes are to be used and we determined that the most suitable seem to be PPIA and GAPD.

Photodiagnosis and photodynamic therapy of endometriotic epithelial cells using 5-aminolevulinic acid and steroids

BACKGROUND The photodynamic diagnosis and therapy represent relatively new methods used, i.a., in the detection of some preneoplastic and neoplastic conditions. They are based on selective accumulation of photosensitizers in the altered cells, which can be identified by fluorescence of the sensitizers and, using light of an appropriate wavelength, can be eliminated. Currently, investigations continue on application of the methods in diagnosis and therapy of endometriosis, one of the most prevalent causes of a reduced fertility in women. METHODS In this study protoporphyrin IX, a photosensitizer derived from 5-aminolevulinic acid, was used to locate and destroy endometrial epithelium. Material for the investigations involved primary epithelial cells, isolated from 15 normal endometria and 15 ovarian endometriotic epithelia. Taking into account the cyclical hormonal alterations, which affect endometrial cells in individual phases of the menstrual cycle, experiments were conducted on accumulation of the photosensitizer and photodestruction of the cells preceded by their hormonal stimulation (17β-estradiol and progesterone). RESULTS AND CONCLUSION It was found that following 48 h stimulation with 17β-estradiol and/or progesterone a significantly augmented synthesis of protoporphyrin IX can be obtained in cells of endometrial epithelium as compared to the normal epithelium. Moreover, the endometriotic epithelial cells were most effectively eliminated following 48 h prestimulation with progesteron alone. The obtained result permits to assume that photodynamic diagnosis and photodynamic therapy of endometrial epithelium should be performed in the secretory phase of endometrium in order to optimise their results.

5-Aminolevulinic Acid–Mediated Photodynamic Therapy of Human Endometriotic Primary Epithelial Cells

OBJECTIVE Despite progress in medicine, appropriate diagnosis and treatment of endometriosis poses a serious problem. For this reason, in in-vitro experiments were performed on a potential method of employing photodynamic therapy (PDT) of endometriosis using 5-aminolevulinic acid (ALA). BACKGROUND DATA The exogenous application of ALA induces the accumulation of protoporphyrin IX, a natural and effective photosensitizer used in photodynamic diagnosis and therapy. MATERIALS AND METHODS To this end primary epithelial cells were isolated from endometriotic foci, preincubated with various ALA concentrations, and then exposed to light energy (a bulb or laser) for 8 min; 24 and 48 h later cell lesions were evaluated using fluorescent staining. RESULTS When bulb illumination was used, after 48 h cells were found that had disturbed chromatin concentration and fragmentation. Illumination with a laser beam resulted in strong induction of apoptosis 24 h post-exposure. With both types of illumination the number of necrotic cells was insignificant. Staining with rhodamine 123 demonstrated the presence within the endometriotic foci of epithelial cells that were resistant to ALA-induced photodynamic therapy. CONCLUSION The effects of PDT on primary epithelial endometriotic cells may prove useful in designing a phototherapeutic procedure for the detection and treatment of endometriosis.

Analysis of cytosine-adenine repeats in P1 promoter region of IGF-1 gene in peripheral blood cells and cervical tissue samples of females with cervical intraepithelial lesions and squamous cervical cancer.

High oncogenic risk human papillomaviruses (HPVs) are closely associated with cancer of the cervix. However, HPV infection alone may not be sufficient to cause cervical cancer, and other factors or cofactors may have a cumulative effect on the risk of progression from cervical HPV infection to cancer. The present study investigates the cytosine-adenine (CA) repeat polymorphism in the P1 promoter region of the insulin-like growth factor-1 (IGF-1) gene among cervical precancerous and cancer patients and healthy control females. The association between these polymorphisms, tissue and blood serum levels of IGF-1, and cervical cancer risk and progression is evaluated. The material for analysis consisted of blood cells and postoperative tissues from patients diagnosed with low-grade squamous intraepithelial lesions (L-SILs), high-grade squamous intraepithelial lesions (H-SILs) and invasive cervical cancer (ICC). A polymerase chain reaction amplification and the sequencing of DNA were used for the identification of (CA)n repeats in the IGF-1 P1 region and detection of HPV DNA. The blood serum concentration of IGF was determined by enzyme-linked immunosorbent assay. The identification of the IGF-1 protein in the cervical tissues was performed by immunohistochemical analysis. The range of the length of the CA repeats in the study DNA was 11 to 21. However, the most common allele length and genotype in the control and study patients from serum and tissues was 19 CA repeats and a homozygous genotype of CA19/19. Statistically significant differences in the concentration of IGF-1 in the blood serum were observed between H-SILs and controls, only (p=0.047). However, the concentration of IGF-1 in the group of females with CA19/19, CA19<19 and CA19>19 was significantly higher in the group of patients with H-SIL (P=0.041) and ICC (P=0.048) in comparison with the control group. An association was detected between CA repeat length <19 and/or >19, IGF concentration in blood serum and tissues and the development of cervical cancer.

Trichrome Mallory's stain may indicate differential rates of RNA synthesis in eutopic and ectopic endometrium.

Mallorys triple staining is a histochemical technique used mainly for analysing connective tissues and glands and other tissues. We have described the differences in the nuclear staining between eutopic and ectopic endometrium as well as endometrial hyperplasia and adenocarcinoma using the Mallorys method. The ultrastructural differences between eutopic and ectopic endometrium have been detected. In normal and hyperplastic endometrium the presence of stromal cell nuclei with an increased affinity to aniline blue has been observed. The affinity has disappeared after digestion of tissues with RNase. In cases of endometriosis, independently of cell types, the nuclei have shown affinity to orange G. Similar effects in adenocarcinoma have been noted. The ultrastructural studies have shown that in normal endometrium the stroma contained cells with euchromatic and low electron density cell nuclei. In endometriosis heterochromatic cell nuclei present both in the stroma and within glands have been detected. The results indicate that the Mallorys technique may be a useful tool for recognizing the differences between eutopic and ectopic endometrium. The affinity for aniline blue in normal and hyperplastic endometrium occurs most likely due to increased RNA synthesis. Based on Mallorys staining a similarity between hyperplasia and unchanged endometrium in contrast to similar results of the staining obtained in cases of adenocarcinoma and endometriosis may be suggested.

Microsatellite polymorphism in the P1 promoter region of the IGF‑1 gene is associated with endometrial cancer

Endometrial carcinoma (EC) is the most common type of gynecological malignancy. Studies have demonstrated that the insulin growth factor (IGF) pathway is implicated in the development of endometrial tumors and that the serum levels of IGF-1 are affected by estrogen. Most EC cells with high microsatellite instability (MSI-H) accumulate mutations at a microsatellite sequence in the IGF-1 gene. The present study investigated the CA repeat polymorphism in the P1 promoter region of the IGF-1 gene among Caucasian females with endometrial hyperplasia, EC and healthy control subjects, whose blood serum and surgical tissue specimens were analyzed. Differences or correlations between the analyzed parameters [serum levels of IGF-1 and IGF binding protein (IGFBP)-1 and IGFBP-3 as well as estrogens among the polymorphisms] were verified using the χ2, Mann-Whitney U, Kruskal-Wallis or Spearmans rank correlation tests. A PCR amplification and DNA sequencing analysis was used for identification of (CA)n repeats in the P1 region of IGF-1. ELISA was used to determine the blood serum levels of IGF-1, IGFBP-1, IGFBP-3 and estrogens. Furthermore, IGF-1 was assessed in endometrial tissues by immunohistochemical analysis. The present study indicated no statistically significant differences between serum levels of IGF-1, IGFBP-1, IGFBP-3 and estrone, estriol and estradiol in the control and study groups. A significant correlation was identified between the IGF-1 levels and estrone levels in the MSI-H polymorphism (r=−0.41, P=0.012) as well as a highly negative correlation between IGF-1 levels and the estradiol levels in the MSI-H polymorphism (r=−0.6, P=0.002). Genotypes without the 19 CA allele were predominantly found in EC. Furthermore, statistical analysis indicated that the number of IGF-1-expressing cells was significantly elevated in MSI-H type 18-20 (P= 0.0072), MSI-L type 19-20 (P=0.025) and microsatellite-stable MSS type 19-19 (P=0.024) compared with those in the MSI-H 20-20 genotype. The present study suggested that it is rather likely that the polymorphisms in the IGF-1 promoter are associated with EC in Caucasian females with regard to its development. In the present study, polymorphisms of the IGF-1 promoter may have been introduced during the genesis of EC and contributed to it by leading to aberrant expression of IGF-1.

Studies on function of P-glycoprotein in photodynamic therapy of endometriosis.

OBJECTIVE The aim of the present study was to examine whether the effects of endometriosis-targeted photodynamic therapy (PDT), dependent on 5-aminolevulinic acid (ALA), rely on the presence of P-glycoprotein (P-gp), which is regarded as constituting one of the causes of multidrug resistance phenomenon. BACKGROUND The significance of the undertaken studies reflects the complete absence of reports related to the modulation of P-gp activity and efficacy of PDT in patients with endometriosis. MATERIALS AND METHODS Tissue samples of normal endometria were obtained from eight women after hysterectomy who were diagnosed with cervical intra-epithelial neoplasia. Fragments of ovarian endometriosis were obtained from 15 women. Epithelial cells were isolated from the material and in in vitro conditions were preincubated with P-gp blocker-verapamil-before ALA-PDT. The cytotoxicity was evaluated using the XTT test, allowing us to estimate cell growth inhibition. Statistical analysis of the results involved the nonparametric Wilcoxon paired rank test and the Mann-Whitney U-test using the Statistica v5 software (p < 0.05). In parallel, P-gp presence in the analyzed material was evaluated using immunohistochemistry. RESULTS In normal endometrial epithelium, verapamil was shown to intensify phototoxic effects at 2 and 4 mmol/L ALA (p < 0.05). In endometriotic epithelium, such intensification was noted in all examined concentrations of ALA (p < 0.001). Moreover, the ectopic epithelial cells were more sensitive than eutopic epithelial cells to PDT upon ALA alone, as well as after preincubation with verapamil. Immunohistohemical analysis allowed us to demonstrate the absence of glycoprotein P in normal endometrium. In endometriosis, P-gp was localised in both the epithelium and the stroma of the examined material. CONCLUSION Phototoxic effects could be amplified in epithelial cells of endometriotic foci by appropriate action of verapamil and 5-aminolevulinic acid.