Gene expression profile of hiPSC-derived cells differentiated with growth factors, forskolin and conditioned medium from human adrenocortical cell line.

BACKGROUND: Adrenocortical carcinoma (ACC) affects approx. 2 in 1,000,000 individuals in the USA, and is more common in females than males. Adrenocortical carcinoma often presents with severe symptoms, such as abdominal pain, high blood pressure, acne, hair overgrowth, and voice deepening. OBJECTIVES: Research on ACC constitutes a large body of published data. There is an increased need for easy access to ACC-derived biological material. Moreover, there are limited numbers of human cell lines available. For this reason, we attempted to differentiate human induced pluripotent stem cells (hiPSCs) into adrenocortical-like cells to establish a new functional cell line. MATERIAL AND METHODS: We conducted a long-term differentiation process (35 and 70 days) in the presence of growth factors (GFs), forskolin and conditioned medium collected from the human adrenal carcinoma (HAC15) cell line. Then, we analyzed the gene expression profile of the differentiated cells. RESULTS: The obtained cells possess features characteristic of all 3 primary germ layers. Interestingly, the differentiated cells demonstrated an extremely high level of gene expression for those involved in endocrine processes, namely glycoprotein hormones, alpha polypeptide (CGA), insulin receptor substrate 4 (IRS4), and pancreatic progenitor cell differentiation and proliferation factor-like protein (PPDPFL). CONCLUSIONS: The results of the study indicate that we obtained progenitors derived from endoderm with some characteristics of pancreatic-like cells. The endodermal derivative differentiation is a very challenging and complicated process; thus, the results presented in this study deserve closer consideration.

The Enhanced Expression of ZWILCH Predicts Poor Survival of Adrenocortical Carcinoma Patients.

Zwilch kinetochore protein (ZWILCH) plays a key role in proper cell proliferation. The upregulation of the ZWILCH gene was observed in many types of cancers, but the association of ZWILCH with adrenocortical carcinoma (ACC) was not investigated so far. The main aim of the presented study was to verify if the enhanced level of the ZWILCH gene can be used as a diagnostic marker for ACC development and progression, as well as a predictor of survival time for ACC patients. The performed analyses included investigation of the ZWILCH expression profile in tumors with publicly available TCGA (The Cancer Genome Atlas) datasets and transcriptomic data from the Gene Expression Omnibus (GEO) database, as well as, in human biological samples of normal adrenal, adrenocortical carcinoma and in commercially available tissue microarrays. The findings demonstrate statistically significant higher ZWILCH gene expression in ACC tissue in comparison with normal adrenal glands. Furthermore, there is a strong correlation between ZWILCH upregulation and tumor mitotic rate and the probability of patient survival. The enhanced ZWILCH level is also connected with the activation of genes involved in cell proliferation and the inhibition of genes related to the immune system. This work contributes to a better understanding of the role of ZWILCH as an ACC biomarker and diagnostic tool.

Lichen Secondary Metabolites Inhibit the Wnt/ß-Catenin Pathway in Glioblastoma Cells and Improve the Anticancer Effects of Temozolomide.

Lichens are a source of secondary metabolites with significant pharmacological potential. Data regarding their possible application in glioblastoma (GBM) treatment are, however, scarce. The study aimed at analyzing the mechanism of action of six lichen secondary metabolites: atranorin, caperatic acid, physodic acid, squamatic acid, salazinic acid, and lecanoric acid using two- and three-dimensional GBM cell line models. The parallel artificial membrane permeation assay was used to predict the blood-brain barrier penetration ability of the tested compounds. Their cytotoxicity was analyzed using the MTT test on A-172, T98G, and U-138 MG cells. Flow cytometry was applied to the analysis of oxidative stress, cell cycle distribution, and apoptosis, whereas qPCR and microarrays detected the induced transcriptomic changes. Our data confirm the ability of lichen secondary metabolites to cross the blood-brain barrier and exert cytotoxicity against GBM cells. Moreover, the compounds generated oxidative stress, interfered with the cell cycle, and induced apoptosis in T98G cells. They also inhibited the Wnt/ß-catenin pathway, and this effect was even stronger in case of a co-treatment with temozolomide. Transcriptomic changes in cancer related genes induced by caperatic acid and temozolomide were the most pronounced. Lichen secondary metabolites, caperatic acid in particular, should be further analyzed as potential anti-GBM agents.

Biological response of adrenal carcinoma and melanoma cells to mitotane treatment.

A previous case report described an adrenal incidentaloma initially misdiagnosed as adrenocortical carcinoma (ACC), which was treated with mitotane. The final diagnosis was metastatic melanoma of unknown primary origin. However, the patient developed rapid disease progression after mitotane withdrawal, suggesting a protective role for mitotane in a non-adrenal-derived tumor. The aim of the present study was to determine the biological response of primary melanoma cells obtained from that patient, and that of other established melanoma and ACC cell lines, to mitotane treatment using a proliferation assay, flow cytometry, quantitative PCR and microarrays. Although mitotane inhibited the proliferation of both ACC and melanoma cells, its role in melanoma treatment appears to be limited. Flow cytometry analysis and transcriptomic studies indicated that the ACC cell line was highly responsive to mitotane treatment, while the primary melanoma cells showed a moderate response in vitro. Mitotane modified the activity of several key biological processes, including 'mitotic nuclear division', 'DNA repair', 'angiogenesis' and 'negative regulation of ERK1 and ERK2 cascade'. Mitotane administration led to elevated levels of DNA double-strand breaks, necrosis and apoptosis. The present study provides a comprehensive insight into the biological response of mitotane-treated cells at the molecular level. Notably, the present findings offer new knowledge on the effects of mitotane on ACC and melanoma cells.

Ionizing radiation exposure of stem cell-derived chondrocytes affects their gene and microRNA expression profiles and cytokine production.

Human induced pluripotent stem cells (hiPSCs) can be differentiated into chondrocyte-like cells. However, implantation of these cells is not without risk given that those transplanted cells may one day undergo ionizing radiation (IR) in patients who develop cancer. We aimed to evaluate the effect of IR on chondrocyte-like cells differentiated from hiPSCs by determining their gene and microRNA expression profile and proteomic analysis. Chondrocyte-like cells differentiated from hiPSCs were placed in a purpose-designed phantom to model laryngeal cancer and irradiated with 1, 2, or 3 Gy. High-throughput analyses were performed to determine the gene and microRNA expression profile based on microarrays. The composition of the medium was also analyzed. The following essential biological processes were activated in these hiPSC-derived chondrocytes after IR: "apoptotic process", "cellular response to DNA damage stimulus", and "regulation of programmed cell death". These findings show the microRNAs that are primarily responsible for controlling the genes of the biological processes described above. We also detected changes in the secretion level of specific cytokines. This study demonstrates that IR activates DNA damage response mechanisms in differentiated cells and that the level of activation is a function of the radiation dose.

Adropin Stimulates Proliferation and Inhibits Adrenocortical Steroidogenesis in the Human Adrenal Carcinoma (HAC15) Cell Line.

Adropin is a multifunctional peptide hormone encoded by the ENHO (energy homeostasis associated) gene. It plays a role in mechanisms related to increased adiposity, insulin resistance, as well as glucose, and lipid metabolism. The low adropin levels are strongly associated with obesity independent insulin resistance. On the other hand, overexpression or exogenous administration of adropin improves glucose homeostasis. The multidirectional, adropin-related effects associated with the regulation of metabolism in humans also appear to be attributable to the effects of this peptide on the activity of various elements of the endocrine system including adrenal cortex. Therefore, the main purpose of the present study was to investigate the effect of adropin on proliferation and secretory activity in the human HAC15 adrenal carcinoma cell line. In this study, we obtained several highly interesting findings. First, GPR19, the main candidate sensitizer of adrenocortical cells to adropin, was expressed in HAC15 cells. Moreover, GPR19 expression was relatively stable and not regulated by ACTH, forskolin, or adropin itself. Our findings also suggest that adropin has the capacity to decrease expression levels of steroidogenic genes such as steroidogenic acute regulatory protein (StAR) and CYP11A1, which then led to a statistically significant inhibition in cortisol and aldosterone biosynthesis and secretion. Based on whole transcriptome study and research involving transforming growth factor (TGF)-ß type I receptor kinase inhibitor we demonstrated that attenuation of steroidogenesis caused by adropin is mediated by the TGF-ß signaling pathway likely to act through transactivation mechanism. We found that HAC15 cells treated with adropin presented significantly higher proliferation levels than untreated cells. Using specific intracellular inhibitors, we showed that adropin stimulate proliferation via ERK1/2 and AKT dependent signaling pathways. We have also demonstrated that expression of GPR19 is elevated in adrenocortical carcinoma in relation to normal adrenal glands. High level of GPR19 expression in adrenocortical carcinoma may constitute a negative prognostic factor of disease progression.

Analysis of Transcriptome, Selected Intracellular Signaling Pathways, Proliferation and Apoptosis of LNCaP Cells Exposed to High Leptin Concentrations.

Leptin, the first discovered adipokine, has been connected to various physiological and pathophysiological processes, including cancerogenesis. Increasing evidence confirms its influence on prostate cancer cells. However, studies on the effects of leptin on the proliferation and apoptosis of the androgen-sensitive LNCaP line of prostate cancer cells brought conflicting results. Therefore, we performed studies on the effects of high LEP concentration (1 × 10-6 M) on gene expression profile, change of selected signaling pathways, proliferation and apoptosis of LNCaP cells. RTCA (real-time cell analyzer) revealed inhibitory effect of LEP on cell proliferation, but lower LEP concentrations (10-8 and 10-10 M) did not affect cell division. Moreover, flow cytometry with a specific antibody for Cleaved PARP-1, an apoptosis marker, confirmed the activation of apoptosis in leptin-exposed LNCaP line of prostate cancer cells. Within 24 h LEP (10-6 M) increases expression of 297 genes and decreases expression of 119 genes. Differentially expressed genes (DEGs) were subjected to functional annotation and clusterization using the DAVID bioinformatics tools. Most ontological groups are associated with proliferation and apoptosis (seven groups), immune response (six) and extracellular matrix (two). These results were confirmed by the Gene Set Enrichment Analysis (GSEA). The leptin's effect on apoptosis stimulation was also confirmed using Pathview library. These results were also confirmed by qPCR method. The results of Western Blot analysis (exposure to LEP 10 min, 1, 2, 4 and 24 h) suggest (after 24 h) decrease of p38 MAPK, p44-42 mitogen-activated protein kinase and Bcl-2 phosphorylated at threonine 56. Moreover, exposure of LNCaP cells to LEP significantly stimulates the secretion of matrix metallopeptidase 7 (MMP7). Obtained results suggest activation of apoptotic processes in LNCaP cells cultured at high LEP concentration. At the same time, this activation is accompanied by inhibition of proliferation of the tested cells.

Liquid Biopsy in Oligometastatic Prostate Cancer-A Biologist's Point of View.

Prostate cancer (PCa) is the main cause of cancer-related mortality in males and the diagnosis, treatment, and care of these patients places a great burden on healthcare systems globally. Clinically, PCa is highly heterogeneous, ranging from indolent tumors to highly aggressive disease. In many cases treatment-generally either radiotherapy (RT) or surgery-can be curative. Several key genetic and demographic factors such as age, family history, genetic susceptibility, and race are associated with a high incidence of PCa. While our understanding of PCa, which is mainly based on the tools of molecular biology-has improved dramatically in recent years, efforts to better understand this complex disease have led to the identification of a new type of PCa-oligometastatic PCa. Oligometastatic disease should be considered an individual, heterogeneous entity with distinct metastatic phenotypes and, consequently, wide prognostic variability. In general, patients with oligometastatic disease typically present less biologically aggressive tumors whose metastatic potential is more limited and which are slow-growing. These patients are good candidates for more aggressive treatment approaches. The main aim of the presented review was to evaluate the utility of liquid biopsy for diagnostic purposes in PCa and for use in monitoring disease progression and treatment response, particularly in patients with oligometastatic PCa. Liquid biopsies offer a rapid, non-invasive approach whose use t is expected to play an important role in routine clinical practice to benefit patients. However, more research is needed to resolve the many existing discrepancies with regard to the definition and isolation method for specific biomarkers, as well as the need to determine the most appropriate markers. Consequently, the current priority in this field is to standardize liquid biopsy-based techniques. This review will help to improve understanding of the biology of PCa, particularly the recently defined condition known as "oligometastatic PCa". The presented review of the body of evidence suggests that additional research in molecular biology may help to establish novel treatments for oligometastatic PCa. In the near future, the treatment of PCa will require an interdisciplinary approach involving active cooperation among clinicians, physicians, and biologists.

The Role of MicroRNAs in Early Chondrogenesis of Human Induced Pluripotent Stem Cells (hiPSCs).

Human induced pluripotent stem cells (hiPSCs) play an important role in research regarding regenerative medicine. Particularly, chondrocytes differentiated from hiPSCs seems to be a promising solution for patients suffering from osteoarthritis. We decided to perform chondrogenesis in a three-week monolayer culture. Based on transcriptome analysis, hiPSC-derived chondrocytes (ChiPS) demonstrate the gene expression profile of cells from early chondrogenesis. Chondrogenic progenitors obtained by our group are characterized by significantly high expression of Hox genes, strongly upregulated during limb formation and morphogenesis. There are scanty literature data concerning the role of microRNAs in early chondrogenesis, especially in chondrogenic differentiation of hiPSCs. The main aim of this study was to investigate the microRNA expression profile and to select microRNAs (miRNAs) taking part in early chondrogenesis. Our findings allowed for selection crucial miRNAs engaged in both diminishing pluripotency state and chondrogenic process (inter alia hsa-miR-525-5p, hsa-miR-520c-3p, hsa-miR-628-3p, hsa-miR-196b-star, hsa-miR-629-star, hsa-miR-517b, has-miR-187). These miRNAs regulate early chondrogenic genes such as: HOXD10, HOXA11, RARB, SEMA3C. These results were confirmed by RT-qPCR analysis. This work contributes to a better understanding of the role of miRNAs directly involved in chondrogenic differentiation of hiPSCs. These data may result in the establishment of a more efficient protocol of obtaining chondrocyte-like cells from hiPSCs.

Chondrocytes differentiated from human induced pluripotent stem cells: Response to ionizing radiation.

PURPOSE: Data on the response of chondrocytes differentiated from hiPSCs (hiPSC-DCHs) to ionizing radiation (IR) are lacking. The aim of present study was to assess DNA damage response (DDR) mechanisms of IR-treated hiPSC-DCHs. METHODS AND MATERIALS: The following IR-response characteristics in irradiated hiPSC-DCHs were assessed: 1) the kinetics of DNA DSB formation; 2) activation of major DNA repair mechanisms; 3) cell cycle changes and 4) reactive oxygen species (ROS), level of key markers of apoptosis and senescence. RESULTS: DNA DSBs were observed in 30% of the hiPSC-DCHs overall, and in 60% after high-dose (> 2 Gy) IR. Nevertheless, these cells displayed efficient DNA repair mechanisms, which reduced the DSBs over time until it reached 30% by activating key genes involved in homologous recombination and non-homologous end joining mechanisms. As similar to mature chondrocytes, irradiated hiPSC-DCH cells revealed accumulation of cells in G2 phase. Overall, the hiPSC-DCH cells were characterized by low levels of ROS, cPARP and high levels of senescence. CONCLUSIONS: The chondrocyte-like cells derived from hiPSC demonstrated features characteristic of both mature chondrocytes and "parental" hiPSCs. The main difference between hiPSC-derived chondrocytes and hiPSCs and mature chondrocytes appears to be the more efficient DDR mechanism of hiPSC-DCHs. The unique properties of these cells suggest that they could potentially be used safely in regenerative medicine if these preliminary findings are confirmed in future studies.

Forced differentiation in vitro leads to stress-induced activation of DNA damage response in hiPSC-derived chondrocyte-like cells.

A human induced pluripotent stem cell line (GPCCi001-A) created by our group was differentiated towards chondrocyte-like cells (ChiPS) via monolayer culturing with growth factors. ChiPS are promising because they have the potential to be used in tissue engineering to regenerate articular cartilage. However, their safety must be confirmed before they can be routinely used in regenerative medicine. Using microarray analysis, we compared the ChiPS to both GPCCi001-A cells and chondrocytes. The analysis showed that, compared to both GPCCi001-A cells and chondrocytes, the expression of genes engaged in DNA damage and in the tumor protein p53 signalling pathways was significantly higher in the ChiPS. The significant amount of DNA double strand breaks and increased DNA damage response may lead to incomplete DNA repair and the accumulation of mutations and, ultimately, to genetic instability. These findings provide evidence indicating that the differentiation process in vitro places stress on human induced pluripotent stem cells (hiPSCs). The results of this study raise doubts about the use of stem cell-derived components given the negative effects of the differentiation process in vitro on hiPSCs.

Expression of Pluripotency Genes in Chondrocyte-Like Cells Differentiated from Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (hiPSCs) constitute an important breakthrough in regenerative medicine, particularly in orthopedics, where more effective treatments are urgently needed. Despite the promise of hiPSCs only limited data on in vitro chondrogenic differentiation of hiPSCs are available. Therefore, we compared the gene expression profile of pluripotent genes in hiPSC-derived chondrocytes (ChiPS) to that of an hiPSC cell line created by our group (GPCCi001-A). The results are shown on heatmaps and plots and confirmed by Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) analysis. Unlike the ChiPS, our GPCCi001-A cells maintained their pluripotency state during long-term culture, thus demonstrating that this cell line was comprised of stable, fully pluripotent hiPSCs. Moreover, these chondrocyte-like cells not only presented features that are characteristic of chondrocytes, but they also lost their pluripotency, which is an important advantage in favor of using this cell line in future clinical studies.