A Recipe for Successful Metastasis: Transition and Migratory Modes of Ovarian Cancer Cells.

One of the characteristic features of ovarian cancer is its early dissemination. Metastasis and the invasiveness of ovarian cancer are strongly dependent on the phenotypical and molecular determinants of cancer cells. Invasive cancer cells, circulating tumor cells, and cancer stem cells, which are responsible for the metastatic process, may all undergo different modes of transition, giving rise to mesenchymal, amoeboid, and redifferentiated epithelial cells. Such variability is the result of the changing needs of cancer cells, which strive to survive and colonize new organs. This would not be possible if not for the variety of migration modes adopted by the transformed cells. The most common type of metastasis in ovarian cancer is dissemination through the transcoelomic route, but transitions in ovarian cancer cells contribute greatly to hematogenous and lymphatic dissemination. This review aims to outline the transition modes of ovarian cancer cells and discuss the migratory capabilities of those cells in light of the known ovarian cancer metastasis routes.

A Potential Objective Sign of Central Sensitization: Referred Pain Elicited by Manual Gluteus Minimus Muscle Exploration is Coincident with Pathological Autonomic Response Provoked by Noxious Stimulation.

Referred pain/sensation provoked by trigger points suits the nociplastic pain criteria. There is a debate over whether trigger points are related to a peripheral phenomenon or central sensitization (CS) processes. Referred pain is considered a possible sign of CS, which occurs probably mainly due to the abnormal activity of the immune and autonomic nervous systems. To confirm abnormal autonomic reactivity within the referred pain zone of active trigger points, a new diagnostic tool, the Skorupska Protocol® (the SP test®), was applied. The test uses noxious stimulation (10 minutes of dry needling under infrared camera control) as a diagnostic tool to confirm abnormal autonomic nervous system activity. A response to the SP test® of healthy subjects with referred pain sensations provoked by latent trigger points (LTrPs) stimulation was not explored before. The study aims at examining if LTrPs can develop an autonomic response. Methods. Two groups of healthy subjects, (i) gluteus minimus LTrPs with referred pain (n = 20) and (ii) control (n = 27), were examined using the SP test®. Results. Abnormal autonomic activity within the referred pain zone was confirmed for all analyzed LTrPs subjects. 70% of control subjects had no feature of vasodilatation and others presented minor vasomotor fluctuations. The size of vasomotor reactivity within the referred pain zone was LTrPs 11.1 + 10.96% vs. control 0.8 + 0.6% (p < 0.05). Conclusions. Noxious stimulation of latent TrPs induces abnormal autonomic nervous system activity within the referred pain zone. The observed phenomenon supports the concept of central nervous system involvement in the referred pain patomechanizm.

The Role of Circulating Tumor Cells in Ovarian Cancer Dissemination.

Metastatic ovarian cancer is the main reason for treatment failures and consequent deaths. Ovarian cancer is predisposed to intraperitoneal dissemination. In comparison to the transcoelomic route, distant metastasis via lymph vessels and blood is less common. The mechanisms related to these two modes of cancer spread are poorly understood. Nevertheless, the presence of tumor cells circulating in the blood of OC patients is a well-established phenomenon confirming the significant role of lymphatic and hematogenous metastasis. Thus, the detection of CTCs may provide a minimally invasive tool for the identification of ovarian cancer, monitoring disease progression, and treatment effectiveness. This review focuses on the biology of ovarian CTCs and the role they may play in cancer diagnosis and therapy.

Physiological and Biochemical Parameters of Salinity Resistance of Three Durum Wheat Genotypes.

The area of farming lands affected by increasing soil salinity is growing significantly worldwide. For this reason, breeding works are conducted to improve the salinity tolerance of important crop species. The goal of the present study was to indicate physiological or biochemical parameters characterizing three durum wheat accessions with various tolerance to salinity. The study was carried out on germinating seeds and mature plants of a Polish SMH87 line, an Australian cultivar 'Tamaroi' (salt-sensitive), and the BC5Nax2 line (salt-tolerant) exposed to 0-150 mM NaCl. Germination parameters, electrolyte leakage (EL), and salt susceptibility index were determined in the germinating caryopses, whereas photosynthetic parameters, carbohydrate and phenolic content, antioxidant activity as well as yield were measured in fully developed plants. The parameters that most differentiated the examined accessions in the germination phase were the percentage of germinating seeds (PGS) and germination vigor (Vi). In the fully developed plants, parameters included whether the plants had the maximum efficiency of the water-splitting reaction on the donor side of photosystem II (PSII)-Fv/F0, energy dissipation from PSII-DIo/CSm, and the content of photosynthetic pigments and hydrogen peroxide, which differentiated studied genotypes in terms of salinity tolerance degree. Salinity has a negative impact on grain yield by reducing the number of seeds per spike and the mass of one thousand seeds (MTS), which can be used as the most suitable parameter for determining tolerance to salinity stress. The most salt-tolerant BC5Nax2 line was characterized by the highest PGS, and Vi for NaCl concentration of 100-150 mM, content of chlorophyll a, b, carotenoids, and also MTS at all applied salt concentrations as compared with the other accessions. The most salt-sensitive cv. 'Tamaroi' demonstrated higher H2O2 concentration which proves considerable oxidative damage caused by salinity stress. Mentioned parameters can be helpful for breeders in the selection of genotypes the most resistant to this stress.

Photosynthetic efficiency, growth and secondary metabolism of common buckwheat (Fagopyrum esculentum Moench) in different controlled-environment production systems.

Light-emitting diodes (LEDs) and high-pressure sodium lamps (HPS) are among the most commonly used light sources for plant cultivation. The objective of this study was to evaluate the effect of two controlled-environment production systems differing in light sources on growth, photosynthetic activity, and secondary metabolism of common buckwheat. We hypothesized that LED light with the majority of red and blue waves would increase physiological and biochemical parameters compared to sunlight supplemented with HPS lamps. The experiment was performed in a phytotronic chamber (LEDs) and in a greenhouse (solar radiation supplemented with HPS lamps as a control). The effects were analyzed at the flowering phase with biometric measurements, leaf chlorophyll index, the kinetics of chlorophyll a fluorescence, content of soluble carbohydrates and phenolics in the leaves. Applied LED light decreased the biomass but stimulated the production of phenolics compared to control plants. In control plants, a positive correlation between flavonoid content and energy dissipation from photosystem II (DIo/CSm) was found, while in plants under LEDs total pool of phenolic content correlated with this parameter and the quantum yield of electron transport (φ Ro and ψ Ro) was lower than that of the control, probably affecting buckwheat biomass.

Physiological and Biochemical Response to Fusarium culmorum Infection in Three Durum Wheat Genotypes at Seedling and Full Anthesis Stage.

Fusarium culmorum is a worldwide, soil-borne plant pathogen. It causes diseases of cereals, reduces their yield, and fills the grain with toxins. The main direction of modern breeding is to select wheat genotypes the most resistant to Fusarium diseases. This study uses seedlings and plants at the anthesis stage to analyze total soluble carbohydrates, total and cell-wall bound phenolics, chlorophyll content, antioxidant activity, hydrogen peroxide content, mycotoxin accumulation, visual symptoms of the disease, and Fusarium head blight index (FHBi). These results determine the resistance of three durum wheat accessions. We identify physiological or biochemical markers of durum wheat resistance to F. culmorum. Our results confirm correlations between FHBi and mycotoxin accumulation in the grain, which results in grain yield decrease. The degree of spike infection (FHBi) may indicate accumulation mainly of deoxynivalenol and nivalenol in the grain. High catalase activity in the infected leaves could be considered a biochemical marker of durum sensitivity to this fungus. These findings allowed us to formulate a strategy for rapid evaluation of the disease severity and the selection of plants with higher level, or resistance to F. culmorum infection.

A rapid method for protein staining in polyacrylamide gels using water saturated with chloroform.

Polyacrylamide gel electrophoresis, followed by an appropriate staining, is a popular and useful analytical procedure for protein identification and characterization. The aim of this study was to develop a method for protein visualization in polyacrylamide gels that would be alternative to Coomassie blue or silver staining. The proposed method is simple, fast and inexpensive. The optimized protocol for protein staining and visualization takes as little as 6 minutes and utilizes deionized water and chloroform. Fluorescence of proteins is induced by UV light and can be detected with a standard transilluminator.

Changes in the Flower and Leaf Proteome of Common Buckwheat (Fagopyrum esculentum Moench) under High Temperature.

Common buckwheat (Fagopyrum esculentum Moench), a pseudocereal crop, produces a large number of flowers, but this does not guarantee high seed yields. This species demonstrates strong abortion of flowers and embryos. High temperatures during the generative growth phase result in an increase in the degeneration of embryo sacs. The aim of this study was to investigate proteomic changes in flowers and leaves of two common buckwheat accessions with different degrees of heat tolerance, Panda and PA15. Two-dimensional gel electrophoresis and mass spectrometry techniques were used to analyze the proteome profiles. Analyses were conducted for flower buds, open flowers capable of fertilization, and wilted flowers, as well as donor leaves, i.e., those growing closest to the inflorescences. High temperature up-regulated the expression of 182 proteins. The proteomic response to heat stress differed between the accessions and among their organs. In the Panda accession, we observed a change in abundance of 17, 13, 28, and 11 proteins, in buds, open and wilted flowers, and leaves, respectively. However, in the PA15 accession there were 34, 21, 63, and 21 such proteins, respectively. Fifteen heat-affected proteins were common to both accessions. The indole-3-glycerol phosphate synthase chloroplastic-like isoform X2 accumulated in the open flowers of the heat-sensitive cultivar Panda in response to high temperature, and may be a candidate protein as a marker of heat sensitivity in buckwheat plants.

Reducing Flower Competition for Assimilates by Half Results in Higher Yield of Fagopyrum esculentum.

Despite abundant flowering throughout the season, common buckwheat develops a very low number of kernels probably due to competition for assimilates. We hypothesized that plants with a shorter flowering period may give a higher seed yield. To verify the hypothesis, we studied nutrient stress in vitro and in planta and analyzed different embryological and yield parameters, including hormone profile in the flowers. In vitro cultivated flowers on media with strongly reduced nutrient content demonstrated a drastic increase in degenerated embryo sacs. In in planta experiments, where 50% or 75% of flowers or all lateral ramifications were removed, the reduction of the flower competition by half turned out to be the most promising treatment for improving yield. This treatment increased the frequency of properly developed embryo sacs, the average number of mature seeds per plant, and their mass. Strong seed compensation under 50% inflorescence removal could result from increased production of salicylic and jasmonic acid that both favor more effective pollinator attraction. Plants in single-shoot cultivation finished their vegetation earlier, and they demonstrated greater single seed mass per plant than in control. This result suggests that plants of common buckwheat with shorter blooming period could deliver higher seed yield.

The Study of the Expression of CGB1 and CGB2 in Human Cancer Tissues.

Human chorionic gonadotropin (hCG) is a well-known hormone produced by the trophoblast during pregnancy as well as by both trophoblastic and non-trophoblastic tumors. hCG is built from two subunits: α (hCGα) and ß (hCGß). The hormone-specific ß subunit is encoded by six allelic genes: CGB3, CGB5, CGB6, CGB7, CGB8, and CGB9, mapped to the 19q13.32 locus. This gene cluster also encompasses the CGB1 and CGB2 genes, which were originally considered to be pseudogenes, but as documented by several studies are transcriptionally active. Even though the protein products of these genes have not yet been identified, based on The Cancer Genome Atlas (TCGA) database analysis we showed that the mutual presence of CGB1 and CGB2 transcripts is a characteristic feature of cancers of different origin, including bladder urothelial carcinoma, cervical squamous cell carcinoma, esophageal carcinoma, head and neck squamous cell carcinoma, ovarian serous cystadenocarcinoma, lung squamous cell carcinoma, pancreatic adenocarcinoma, rectum adenocacinoma, testis germ cell tumors, thymoma, uterine corpus endometrial carcinoma and uterine carcinosarcoma.

Regulation of human chorionic gonadotropin beta subunit expression in ovarian cancer.

PURPOSE: Expression of human chorionic gonadotropin beta subunit by cancers is extensively documented, yet regulation of the multiple genes that can code for this protein is poorly understood. The aim of the study was to examine the mechanisms regulating CGB gene expression in ovarian cancer. METHODS: Expression of CGB genes and SP1, SP3, TFAP2A transcription factor genes was evaluated by RT-qPCR. The methylation status of CGB genes promoter regions was examined by methylation-specific PCR. RESULTS: mRNA arising from multiple CGB genes was detected in both ovarian control and malignant tissues. However, expression of CGB3-9 genes was shown to be significantly higher in malignant than healthy ovarian tissues. CGB1 and CGB2 transcripts were shown to be present in 20% of ovarian cancers, but were not detected in any of the control samples. Malignant tissues were characterized by DNA demethylation of CGB promoter regions. In ovarian cancer CGB expression positively correlated with TFAP2A transcripts level and expression of TFAP2A transcription factor was significantly higher in cancer than in control tissues. In contrast SP3 expression level was significantly lower in ovarian tumours than in control ovarian tissue. CONCLUSIONS: In ovarian cancers increased expression of human chorionic gonadotropin beta subunit is associated with demethylation of CGB promoter regions. CGB3-9 expression level strongly correlates with expression of the TFAP2A transcription factor. Presence of mRNA arising from CGB1 and CGB2 genes appears to be a unique feature of a subset of ovarian cancers.

hCG - related molecules and their measurement.

Measurements of human chorionic gonadotropin (hCG) synthesized by trophoblast cells is a powerful tool of pregnancy monitoring. It was showed that similarly to pregnancy also trophoblastic and nontrophoblastic malignancies produce variety of hCG molecules. In urine and serum of both pregnant women and tumors patients a fifteen various forms of hCG, such as: regular hCG, hyperglycosylated hCG and predominant hyperglycosylated hCG free ß, were identified. These forms might be useful in order to recognize between physiological and pathological pregnancies as well as cancers. Even the presence of these different hormone variants is well documented the commercially available biochemical tests detecting hCG failed to identified and distinguish among these forms. Especially hard is to identify glycan chains linked to heterodimer. Thus, a detailed analysis of hCG-related molecules produced during physiological and pathological condition, together with a new tests development are needed.

Human chorionic gonadotropin ß subunit affects the expression of apoptosis-regulating factors in ovarian cancer.

Expression of human chorionic gonadotropin, especially its free ß subunit (hCGß) were shown to play an important role in cancer growth, invasion and metastasis. It is postulated that hCGß is one of the factors determining cancer cell survival. To test this hypothesis, we applied two models: an in vitro model of ovarian cancer using OVCAR-3 and SKOV-3 cell lines transfected with the CGB5 gene and an in vivo model of ovarian cancer tissues. The material was tested against changes in expression level of genes encoding factors involved in apoptosis: BCL2, BAX and BIRC5. Overexpression of hCGß was found to cause a decrease in expression of the analyzed genes in the transfected cells compared with the control cells. In ovarian cancer tissues, high expression of CGB was related to significantly lower BCL2 but higher BAX and BIRC5 transcript levels. Moreover, a low BCL2/BAX ratio, characteristic of advanced stages of ovarian cancer, was revealed. Since tumors were discriminated by a significantly lower LHCGR level than the level noted in healthy fallopian tubes and ovaries, it may be stated that the effect of hCGß on changes in the expression of apoptosis-regulating agents observed in ovarian cancer is LHCGR-independent. The results of the study suggest that the biological effects evoked by hCGß are related to apoptosis suppression.

CGB and GNRH1 expression analysis as a method of tumor cells metastatic spread detection in patients with gynecological malignances.

BACKGROUND: Metastasis is a common feature of many advanced stage cancers and metastatic spread is thought to be responsible for cancer progression. Most cancer cells are localized in the primary tumor and only a small population of circulating tumor cells (CTC) has metastatic potential. CTC amount reflects the aggressiveness of tumors, therefore their detection can be used to determine the prognosis and treatment of cancer patients.The aim of this study was to evaluate human chorionic gonadotropin beta subunit (CGB) and gonadoliberin type 1 (GNRH1) expression as markers of tumor cells circulating in peripheral blood of gynecological cancer patients, indicating the metastatic spread of tumor. METHODS: CGB and GNRH1 expression level in tumor tissue and blood of cancer patients was assessed by real-time RT-PCR. The data was analyzed using the Mann-Whitney U and Spearman tests. In order to distinguish populations with homogeneous genes' expression the maximal likelihood method for one- and multiplied normal distribution was used. RESULT: Real time RT-PCR results revealed CGB and GNRH1 genes activity in both tumor tissue and blood of gynecological cancers patients. While the expression of both genes characterized all examined tumor tissues, in case of blood analysis, the transcripts of GNRH1 were found in all cancer patients while CGB were present in 93% of patients. CGB and GNRH1 activity was detected also in control group, which consisted of tissue lacking cancerous changes and blood of healthy volunteers. The log-transformation of raw data fitted to multiplied normal distribution model showed that CGB and GNRH1 expression is heterogeneous and more than one population can be distinguished within defined groups.Based on CGB gene activity a critical value indicating the presence of cancer cells in studied blood was distinguished. In case of GNRH1 this value was not established since the results of the gene expression in blood of cancer patients and healthy volunteers were overlapping. However one subpopulation consists of cancer patient with much higher GNRH1 expression than in control group was found. CONCLUSIONS: Assessment of CGB and GNRH1 expression level in cancer patients' blood may be useful for indicating metastatic spread of tumor cells.

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

Mallory's 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 Mallory's 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 Mallory's 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 Mallory's 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.

Reduction of human chorionic gonadotropin beta subunit expression by modified U1 snRNA caused apoptosis in cervical cancer cells.

BACKGROUND: Secretion of human chorionic gonadotropin, especially its beta subunit by malignant trophoblastic tumors and varieties of tumors of different origin is now well documented; however the role of hCG in tumorogenesis is still unknown. RESULTS: This study documents the molecular presence of human chorionic gonadotropin beta subunit in uterine cervix cancer tissues and investigates a novel technique to reduce hCGbeta levels based on expression of a modified U1 snRNA as a method to study the hormone's role in biology of human cervical cancer cells cultured in vitro. The property of U1 snRNA to block the accumulation of specific RNA transcript when it binds to its donor sequence within the 3' terminal exon was used. The first 10 nucleotides of the human U1 snRNA gene, which normally binds to the 5'ss in pre-mRNA were replaced by a sequence complementary to a 10-nt segment in the terminal exon of the hCGbeta mRNA. Three different 5' end-mutated U1 snRNA expression plasmids were tested, each targeting a different sequence in the hCGbeta mRNA, and we found each one blocked the expression of hCGbeta in HeLa cells, a cervix carcinoma cell line, as shown by immunohistochemistry and qRT-PCR. Reduction of hCGbeta levels resulted in a significantly increased apoptosis rate with almost 90% of cells transfected with modified anti-hCGbeta U1 snRNAs showing morphological changes characteristic of the apoptotic process. CONCLUSION: These data suggest that human chorionic gonadotropin beta subunit may act as a tumor growth-stimulating factor.

Localization of the DAZ gene expression in seminiferous tubules of patients with spermatogenic disorders.

The research on the expression and mutations of DAZ and its homologues in human and other mammals suggests that protein products of these genes can mainly affect development of germinal cells. The aim of the present study was to analyze the expression of the DAZ gene in seminiferous tubules of six men with spermatogenic disorders (hypospermatogenesis and spermatogenic arrest). The results based on the RT-PCR IS technique demonstrated that the DAZ product was present only in some seminiferous tubules and the fluorescence intensity was different within individual tubules. The most intense fluorescence characterised the spermatogonia, especially these organised in small groups inside separate tubules. In the patients with spermatogenic arrest at the spermatocyte stage, the DAZ gene transcripts were also found in primary spermatocytes. However, the fluorescence intensity of primary spermatocytes, except the fluorescence of the spermatocytes localised upon the lumen, was weaker than the fluorescence of spermatogonia. The results of our study showed that DAZ gene activity seems to correspond to the proliferative activity of stem cells of germinal epithelium.

Localization of human chorionic gonadotropin beta subunit transcripts in ovarian cancer tissue.

Recent studies demonstrated that besides placenta and malignant trophoblastic tumors, hCG and especially its beta-subunit is secreted by a varieties of tumors of different origin. The aim of the present investigation was to determine the expression pattern of human chorionic gonadotropin gene in ovarian cancer tissue. The study included 8 patients with epithelial ovarian carcinoma. The expression and distribution of hCGbeta mRNA was assessed by in situ RT-PCR method. The semi-quantitative assessment was performed using computer image analysis. Transformation of the images into the pseudocolour scale showed a clear difference in fluorescence intensity among individual cancer cells. The intensity of ISRT-PCR products corresponding with expression level of hCGbeta demonstrated that its production by individual cancer cells is different. In all studied specimens of the ovarian carcinoma tissue, cancer cells characterized by the presence of active hCGbeta gene were found, whereas noncancerous tissue demonstrated lack of the gene expression. Thus, the study clearly shows that the expression of hCGbeta is the feature of ovarian cancer tissue.