Natural Stilbenes: Their Role in Colorectal Cancer Prevention, DNA Methylation, and Therapy.

The resistance of colorectal cancer (CRC) to conventional therapeutic modalities, such as radiation therapy and chemotherapy, along with the associated side effects, significantly limits effective anticancer strategies. Numerous epigenetic investigations have unveiled that naturally occurring stilbenes can modify or reverse abnormal epigenetic alterations, particularly aberrant DNA methylation status, offering potential avenues for preventing or treating CRC. By modulating the activity of the DNA methylation machinery components, phytochemicals may influence the various stages of CRC carcinogenesis through multiple molecular mechanisms. Several epigenetic studies, especially preclinical research, have highlighted the effective DNA methylation modulatory effects of stilbenes with minimal adverse effects on organisms, particularly in combination therapies for CRC. However, the available preclinical and clinical data regarding the effects of commonly encountered stilbenes against CRC are currently limited. Therefore, additional epigenetic research is warranted to explore the preventive potential of these phytochemicals in CRC development and to validate their therapeutic application in the prevention and treatment of CRC. This review aims to provide an overview of selected bioactive stilbenes as potential chemopreventive agents for CRC with a focus on their modulatory mechanisms of action, especially in targeting alterations in DNA methylation machinery in CRC.

Impacts of iron on ultrastructural features of NCI-H295R cell line related to steroidogenesis.

The current study was intended to evaluate impacts of both iron (Fe) enrichment and overload (in the form of ferrous sulphate heptahydrate, FeSO4.7H2O) on ultrastructural characteristics of human adrenocarcinoma NCI-H295R cell line. Here, the NCI-H295R cells were treated with 0, 3.90, and 1000 µM FeSO4.7H2O, and consequently proceeded for purposes of ultrastructural studies. Micrographs taken under transmission electron microscope (TEM) were investigated from the qualitative and quantitative (unbiased stereological approaches) aspects, and obtained findings were compared among the three groups of the cells. The ultrastructural features related to the steroidogenic process were found to be similar between the untreated and both Fe-exposed cell populations, with conspicuous mitochondria with well-defined lamellar cristae (creating clusters of varying sizes in the regions of increased energy demands) and concentric whorls of smooth endoplasmic reticulum (SER) being the most noticeable characteristics. The precise estimates of the component (volume, surface) fractions of the nucleus, mitochondria, and lipid droplets (LDs), as well as of the nucleus/cytoplasm (N/C) ratio have revealed close similarities (P > 0.05) in all cell groups investigated. Nonetheless, the low concentration of FeSO4.7H2O exhibited beneficial action on ultrastructural organization of the NCI-H295R cells. In effect, these cells were distinguished by mitochondria with smoother surfaces and clearer outlines, higher density of thin, parallel lamellar cristae (deeply extending into the mitochondrial matrix), and more widespread distribution of fine SER tubules as compared to the control ones, all of them suggesting higher level of energy requirements and metabolic activity, and more intensive rate of steroidogenesis. Interestingly, no obvious ultrastructural modifications were observed in the NCI-H295R cells treated with high FeSO4.7H2O concentration. This finding can be linked to either an adaptive ultrastructural machinery of these cells to cope with the adverse effect of the element or to insufficient dose of FeSO4.7H2O (1000 µM) to induce ultrastructural signs of cytotoxicity. Purposefully, the results of the current study complement our previous paper dealing with impacts of FeSO4.7H2O on the NCI-H295R cell viability and steroidogenesis at the molecular level. Hence, they fill a knowledge gap considering structure-function coupling in this cellular model system upon the metal exposure. This integrated approach can enhance our understanding of the cellular responses to Fe enrichment and overload which can be helpful for individuals with reproductive health concerns.

In Vitro versus Cryo-Induced Capacitation of Bovine Spermatozoa, Part 2: Changes in the Expression Patterns of Selected Transmembrane Channels and Protein Kinase A.

Since the molecular similarities and differences among physiological capacitation and cryocapacitation have not been studied in detail, this study was designed to assess the gene and protein expression levels of the Cation channel of sperm (CatSper) 1 and 2, sodium bicarbonate (Na+/HCO3−) cotransporter (NBC) and protein kinase A (PKA) in un-capacitated (control), in vitro capacitated (CAP) and cryopreserved (CRYO) bovine spermatozoa. All samples were subjected to motility evaluation using the computer assisted sperm analysis and chlortetracycline (CTC) assay for the assessment of the capacitation patterns. Furthermore, quantitative reverse transcription PCR (qRT-PCR) and Western blots were used to monitor the expression patterns of the selected capacitation markers. The results showed a significant reduction in the gene and protein expression levels of CatSper1 and 2 in the CRYO group when compared to the CAP group (p < 0.0001). In the case of NBC, the results were not significantly different or were inconclusive. While a non-significant down-regulation of PKA was found in the CRYO group, a significant reduction in the expression of the PKA protein was found in frozen-thawed spermatozoa in comparison to the CAP group (p < 0.05). In conclusion, we may hypothesize that while in vitro capacitated and cryopreserved spermatozoa exhibit CTC-patterns consistent with capacitation events, the molecular machinery underlying CTC-positivity may be different.

Human adrenocortical carcinoma cell line (NCI-H295R): An in vitro screening model for the assessment of endocrine disruptors' actions on steroidogenesis with an emphasis on cell ultrastructural features.

Cell lines as an in vitro model for xenobiotic screening and toxicity studies provide a very important tool in the field of scientific research at the level of molecular pathways and gene expression. Good cell culture practice and intracellular characterization, as well as physiological properties of the cell line are of critical importance for in vitro reproductive toxicity testing of various endocrine-disrupting chemicals. The NCI-H295R, human adrenocarcinoma cell line, is the most widely used in vitro cellular system to study the human adrenal steroidogenic pathway at the level of hormone production and gene expression, as it expresses genes that encode for all the key enzymes for steroidogenesis. In this review, we aim to highlight the information considering the origin, development, physiological and ultrastructural characteristics of the NCI-H295R cell line. The review also creates a broad overview of the cell line usage in various range of studies related to the steroidogenesis issues. To our best knowledge, the paper provides the first report of quantitative data (ex novo) from stereological estimates of component (volume, surface) densities of nuclei, mitochondria, and lipid droplets of the NCI-H295R cells. Such ultrastructural measurements can be valuable in the assessment of underlying mechanisms of changes in the cell steroid hormone production induced by the action of diverse endocrine disruptors. Thus, they can significantly contribute to complexity of structure-function relationships in association with steroidogenesis.

Copper affects steroidogenesis and viability of human adrenocortical carcinoma (NCI-H295R) cell line in vitro.

Copper is an environmental risk factor, which has various effects on reproductive endocrinology. In this study human adrenocortical carcinoma (NCI-H295R) cell line was used as an in vitro biological model to study the effect of copper sulfate (CuSO4.5H2O) on steroidogenesis and cytotoxicity. The cell cultures were exposed to different concentrations (3.90, 62.50, 250, 500, 1000 µM) of CuSO4.5H2O and compared to control group (medium without CuSO4.5H2O). Cell viability was measured by the metabolic activity assay. Quantification of sexual steroid production directly from the medium was performed by ELISA assay. Following 48 h culture of NCI-H295R cell line in the presence of CuSO4.5H2O a dose-dependent depletion of progesterone release was observed even at the lower concentrations of CuSO4.5H2O. The lowest levels of progesterone were detected in groups with the higher doses (≥ 250 µM) of CuSO4.5H2O, which elicited significant cytotoxic action. Testosterone production decreased significantly, and this decline was more prominent in comparison to that of progesterone. The lowest release of testosterone was recorded at 1000 µM of CuSO4.5H2O. The cytotoxic effect of CuSO4.5H2O was evident at all concentrations used in the study. The presented data suggest that copper has detrimental effects on sexual steroid hormones and consecutively on reproductive physiology.

DNA methylation as mechanism of apoptotic resistance development in endometrial cancer patients.

DNA methylation is a significant epigenetic modification which plays a key role in regulation of gene expression and influences functional changes in endometrial tissue. Aberrant DNA methylation changes result in deregulation of important apoptotic proteins during endometrial carcinogenesis and apoptosis resistance development. Evading apoptosis is still a major problem in the successful treatment of endometrial cancer patients. The aim of our study was to examine the promoter DNA methylation changes in 22 apoptosis-associated genes in endometrioid endometrial cancer patients, precancerous lesions and healthy tissue from various normal menstrual cycle phases using a unique pre-designed methylation platform. We observed as the first a significant difference in promoter DNA methylation status in genes: BCL2L11 (p < 0.001), CIDEB (p < 0.03) and GADD45A (p < 0.05) during endometrial carcinogenesis and BIK gene (p < 0.03) in different phases of normal menstrual cycle. The results of our study indicate that deregulation of mitochondrial apoptotic pathway can considerably contributes to the apoptosis resistance development and may be helpful in identifying of new potent biomarkers in endometrial cancer.