Pilot Study on the Molecular Pathogenesis of Pyeloureteral Junction Obstruction: Underdevelopment or Fibrosis?

Background and Objectives: Congenital ureteral stenosis is one of the leading causes of impaired urinary drainage and subsequent dilatation of the urinary collecting system, known as hydronephrosis or ureterohydronephrosis. The mechanism that leads to obstruction is not clearly known. Multiple studies in rat models have shown increased angiotensin II and TGFß levels in obstructed ureteral tissue. The aim of the study is to investigate the expression of fibrosis-related genes in obstructive and normal ureteral tissue. Material and Methods: It is a monocentric pilot study in which nineteen patients were selected prospectively. 17 patients underwent Hynes-Anderson pyeloplasty due to the PUJO; two patients underwent ureteroneocystostomy due to ureterovesical junction obstruction (UVJO); and six patients were chosen for the control group: five underwent nephrectomies due to the kidney tumor and one underwent upper pole heminephrectomy due to the duplex kidney with normal pyeloureteric junctions in all. Tissue RNA was chemically extracted after freezing the biopsy samples in liquid nitrogen, with cDNA synthesis performed immediately after nucleic acid isolation. qPCR was performed to evaluate the relative expression of Tgfb1, Mmp1, Timp1, Pai1, Ctgf, and Vegfa. Expression levels of the Gapdh and Gpi genes (geometric average) were used to calculate the relative expression of the investigated genes. Outliers were removed prior to calculating confidence intervals for the experimental groups, and a Wilcoxon rank-sum test was performed to determine the statistical significance of the differences. Results: Significant differences between healthy and stenotic tissue samples in Ctgf gene expression levels were observed, with the samples from afflicted tissue showing lower expression. No statistical difference in expression levels of Tgfb1, Timp1, Vegfa, Mmp1, and Pai1 was found. Conclusions: These findings suggest that tissue fibrosis, similar to other tissues and organs, is not the leading cause of stenosis, at least at the moment of surgery. Decreased CTGF expression is indicative of the developmental origin of obstruction.

Crosstalk between protein kinases AKT and ERK1/2 in human lung tumor-derived cell models.

There is no doubt that cell signaling manipulation is a key strategy for anticancer therapy. Furthermore, cell state determines drug response. Thus, establishing the relationship between cell state and therapeutic sensitivity is essential for the development of cancer therapies. In the era of personalized medicine, the use of patient-derived ex vivo cell models is a promising approach in the translation of key research findings into clinics. Here, we were focused on the non-oncogene dependencies of cell resistance to anticancer treatments. Signaling-related mechanisms of response to inhibitors of MEK/ERK and PI3K/AKT pathways (regulators of key cellular functions) were investigated using a panel of patients' lung tumor-derived cell lines with various stemness- and EMT-related markers, varying degrees of ERK1/2 and AKT phosphorylation, and response to anticancer treatment. The study of interactions between kinases was the goal of our research. Although MEK/ERK and PI3K/AKT interactions are thought to be cell line-specific, where oncogenic mutations have a decisive role, we demonstrated negative feedback loops between MEK/ERK and PI3K/AKT signaling pathways in all cell lines studied, regardless of genotype and phenotype differences. Our work showed that various and distinct inhibitors of ERK signaling - selumetinib, trametinib, and SCH772984 - increased AKT phosphorylation, and conversely, inhibitors of AKT - capivasertib, idelalisib, and AKT inhibitor VIII - increased ERK phosphorylation in both control and cisplatin-treated cells. Interaction between kinases, however, was dependent on cellular state. The feedback between ERK and AKT was attenuated by the focal adhesion kinase inhibitor PF573228, and in cells grown in suspension, showing the possible role of extracellular contacts in the regulation of crosstalk between kinases. Moreover, studies have shown that the interplay between MEK/ERK and PI3K/AKT signaling pathways may be dependent on the strength of the chemotherapeutic stimulus. The study highlights the importance of spatial location of the cells and the strength of the treatment during anticancer therapy.

Cell-cell and cell-substratum contacts in the regulation of MAPK and Akt signalling: Importance in therapy, biopharmacy and bioproduction.

The use of cultured cells as a tool for research, precision medicine, biopharmacy, and biomanufacturing is constantly increasing. In parallel, the role of cell-cell and cell-substratum contacts in cell functioning is increasingly validated. Adhesion signalling plays a key role here. The activity of cell fate-regulating signalling molecules is an important factor in determining cell behaviour, as well as their response to treatment, depending on cell phenotypic status and location in the body. Three cellular state models (adherent, single cells in suspension, and aggregated cells) were compared for cell signalling, including focal adhesion (FAK), mitogen-activated (MAPK), as well as Akt protein kinases, and transcription factor cJun, by using lung adenocarcinoma A549, muscle-derived stem Myo, as well as primary lung cancer cell lines. Survival of both A549 and Myo cells was dependent on kinases Akt and ERK in detached conditions. Intercellular contacts in aggregates promoted activation of Akt and ERK, and cell survival. Loss of contacts with the substrate increased phosphorylation of MAP kinases JNK and p38, while decreased Akt phosphorylation by processes involving FAK. Unexpectedly, detachment increased phosphorylation of antiapoptotic kinase ERK in A549, while in Myo stem cells ERK phosphorylation was downregulated. JNK target transcription factor cJun protein level was markedly diminished by contacts between cells possibly involving mechanism of proteasomal degradation. Furthermore, studies revealed the opposite dependence of molecules of the same signalling pathway - phospho-cJun and phospho-JNK - on cell culture density. Differences in ERK activation under detachment conditions indicate that targeting of prosurvival kinases during anoikis should be different in different cells. Moreover, the outcome of JNK activation in cells may depend on the amount of cJun, which is determined by cell-cell contacts.

Study of Bioreductive Anticancer Agent RH-1-Induced Signals Leading the Wild-Type p53-Bearing Lung Cancer A549 Cells to Apoptosis.

Aziridinylquinone RH-1 (2,5-diaziridinyl-3-hydroxymethyl-6-methyl-cyclohexa-2,5-diene-1,4-dione) is a potential anticancer agent. RH-1 action is associated with NAD(P)H: quinone oxidoreductase (NQO1) which reduces this diaziridinylbenzoquinone into DNA-alkylating hydroquinone and is overexpressed in many tumors. Another suggested mechanism of RH-1 toxicity is the formation of reactive oxygen species (ROS) arising from its redox cycling. In order to improve anticancer action of this and similar antitumor quinones, we investigated the involvement of different signaling molecules in cytotoxicity induced by RH-1 by using wild-type tumor suppressor p53 bearing nonsmall cell lung carcinoma A549 cells as a model. Gradual and prolonged increase of mitogen-activated protein kinases (MAPK) ERK, P38, and JNK phosphorylation was observed during 24-h RH-1 treatment. In parallel, activation of DNA damage-sensing ATM kinase, upregulation, and phosphorylation of TP53 (human p53) took place. Inhibition studies revealed that RH-1-induced A549 apoptosis involved the NQO1-ATM-p53 signaling pathway and ROS generation. TP53 participated in ROS- and DNA damage-induced cell death differently. Moreover, MAP kinase JNK was another TP53 activator and death inducer in A549 cells. At the same time, rapid and prolonged activation of AKT kinase during RH-1 treatment was found, and it proved to be antiapoptotic kinase in our model system. Therefore, we identified that different and opposite cell death regulating signaling pathways, which may counteract one another, are induced in cancer cells during chemotherapeutic RH-1 treatment.

JNK implication in adipocyte-like cell death induced by chemotherapeutic drug cisplatin.

Recent evidence shows that tumor microenvironment containing heterogeneous cells may be involved in cancer initiation, growth and tumor cell response to anticancer therapy. Chemotherapy was designed to make toxic impact on malicious cells in organisms, however, the means to protect healthy cells against chemical toxicity are still unsuccessful. As known, the majority of tumor surrounding cells are cancer-associated adipocytes which influence cancer development, progression and treatment. Targeting the components of tumor microenvironment in combination with conventional cancer treatment may become an effective cancer therapy strategy. However, little is known about adipocyte death mechanisms during combined chemo- and targeted therapy. The importance of c-Jun-NH2-terminal kinase (JNK) signaling in tumor development and treatment has been demonstrated using various in vitro and in vivo cancer models. The aim of this study was to ascertain adipocyte viability during simultaneous stress kinase JNK inhibition and exposure to one of the most commonly used anticancer drugs cis-diamminedichloroplatinum II (cisplatin). Our model involved adipocyte-like cells (ADC) which were obtained during in vitro differentiation of adult rabbit muscle-derived stem cells. Cisplatin induced apoptotic cell death. During 24-hr cisplatin treatment gradual, strong and prolonged increase of both JNK and its target protein c-Jun phosphorylation was found in ADC. Pre-treatment of cells with SP600125 decreased cisplatin-induced activation of c-Jun and promoted apoptosis. Upregulation of proapoptotic Bax and downregulation of antiapoptotic Bcl-2 proteins were found to be regulated in JNK-dependent manner. Thus, the results prove the antiapoptotic role of activated JNK in adipocyte-like cells treated with cisplatin.

Long-term muscle-derived cell culture: multipotency and susceptibility to cell death stimuli.

Improvement in the yield of adult organism stem cells, and the ability to manage their differentiation and survival potential are the major goals in their application in regenerative medicine and in the adult stem cell research. We have demonstrated that adult rabbit muscle-derived cell lines with an unlimited proliferative potential in vitro can differentiate into myogenic, osteogenic, adipogenic and neurogenic lineages. Studies of cell survival in vitro showed that differentiated cells, except neurogenic ones, are more resistant to apoptosis inducers compared to proliferating cells. Resistance to death signals correlated with the level of protein kinase AKT phosphorylation. Skeletal muscle-derived cell lines can be multipurpose tools in therapy. Enhanced resistance of differentiated cells to certain types of damage shows their potential for long-term survival and maintenance in an organism. This article was published online on 29 January 2013. An error was subsequently identified. This notice is included in the online and print versions to indicate that both have been corrected 6 March 2013.