Biomarkers of epithelial-mesenchymal transition in localized, surgically treated clear-cell renal cell carcinoma

Introduction. It has been suggested that the metastatic potential of neoplastic cells can be predicted on the basis of their biological features, including expression of proteins involved in the epithelial to mesenchymal transition (EMT). Therefore, the purpose of this work was to (1) evaluate the expression of EMT markers: ZEB2, vimentin, N-cadherin, TWIST, PTEN, survivin, E-cadherin, Ki-67 and GLUT-1, (2) assess mutation status of two genes: PIK3CA and KRAS, and (3) investigate the potential relationships between the studied biomarkers and clinicopathological factors in clear-cell renal cell carcinoma (ccRCC).Material and methods. Tumor tissue samples (embedded in paraffin blocks) from 159 patients undergoing radical nephrectomy were analyzed. Proteins expression was evaluated immunohistochemically. DNA mutations were analyzed on DNA isolated from tumor tissue and amplified by real-time PCR detection using suitable fluorescent labeled TaqMan assays.Results. One hundred and seven men and 52 women of mean age of 63.1years were enrolled. Fifty four cancers at pTNM stage I–II and 98 at pTNM III–IV stage were diagnosed. There were 30 Fuhrman grade G1, 61 Fuhrman G2, 49 Fuhrman G3 and 19 Fuhrman G4 tumors. A negative correlation between ZEB2 (p = 0.047, r = –0.172) or E-cadherin expression (p = 0.027, r = –0.191) and TNM was observed. Positive association between grade and Ki-67 (p < 0.001), survivin (p < 0.001), vimentin (p < 0.001) immunoreactivity and negative association between TWIST expression (p = 0.029) or PTEN expression (p = 0.013) were found. Ki-67 expression was positively correlated with survivin (p < 0.001, r = 0.617), vimentin (p = 0.001, r = 0.251) and N-cadherin (p = 0,009, r = 0.207) immunoreactivity which can suggest tumor aggressiveness. TWIST was negatively correlated with E-cadherin (p < 0.001, r = –0.284), vimentin (p < 0.001, r = –0.297) and N-cadherin (p < 0.002, r = –0.241). ZEB2 was not associated with ccRCC grade but was negatively correlated with E-cadherin (p = 0.055, r= –0.153) and PTEN (p = 0.006). GLUT-1 expression was inversely linked to E-cadherin expression (p = 0.022, r= –0.182). Mutations in PIK3CA and KRAS genes were not found in any of the studied ccRCC tumors.Conclusions. Low-grade tumors showed higher expression of ZEB2 and TWIST proteins than high-grade tumors, which can suggest that EMT in ccRCC begins at early stages of tumor development and, therefore, evaluation of these proteins, together with other biomarkers, may be useful for assessment of the tumor metastatic potential

Kinetic studies of novel inhibitors of endomorphin degrading enzymes

Endomorphins (EMs), two endogenous μ-opioid receptor selective ligands, are attractive lead compounds for opioid-based pain management studies. However, these peptides are quickly degraded by peptidases, in particular by dipeptidylpeptidase IV (DPP IV) and aminopeptidase M (APM). Targeting enzymatic degradation is one approach to prolong endomorphin activity. In this study we characterized the action of two new inhibitors of similar to endomorphins structure, Tyr-Pro-Ala-NH2 (EMDB-2) and Tyr-Pro-Ala-OH (EMDB-3), which were designed earlier in our laboratory. The presented data give evidence that EMDB-2 and EMDB-3 are potent inhibitors of enzymes responsible for endomorphin cleavage. These compounds are stable and easily synthesized. EMDB-2 and EMDB-3 are competitive inhibitors of both, DPP IV and APM, with Ki values in micromolar range. They are less potent than diprotin A in protecting EMs against DPP IV but more potent than actinonin in protecting these peptides against APM

Synthesis and Antiproliferative Activity against Cancer Cells of Indole-Aryl-Amide Derivatives

Indoles constitute a large family of heterocyclic compounds widely occurring in nature which are present in a number of bioactive natural and synthetic compounds, including anticancer agents or atypical opioid agonists. As a result, exponential increases in the development of novel methods for the synthesis of indole-containing compounds have been reported in the literature. A series of indole-aryl amide derivatives 1-7 containing tryptamine or an indolylacetic acid nucleus were designed, synthesized, and evaluated as opioid ligands. These new indole derivatives showed negligible to very low affinity for mu- and delta-opioid receptor (OR). On the other hand, compounds 2, 5 and 7 showed Ki values in the low mu M range for kappa-OR. Since indoles are well known for their anticancer potential, their effect against a panel of tumor cell lines was tested. The target compounds were evaluated for their in vitro cytotoxicity in HT29, HeLa, IGROV-1, MCF7, PC-3, and Jurkat J6 cells. Some of the synthesized compounds showed good activity against the selected tumor cell lines, with the exception of IGROV1. In particular, compound 5 showed a noteworthy selectivity towards HT29 cells, a malignant colonic cell line, without affecting healthy human intestinal cells. Further studies revealed that 5 caused the cell cycle arrest in the G1 phase and promoted apoptosis in HT29 cells

How tumour cells respirate?

To, że komórki nowotworowe prowadzą inny metabolizm niż komórki prawidłowe sugerował jako pierwszy niemieckibiochemik Otto Warburg na początku ubiegłego wieku. Wykazał, że komórki nowotworowe preferują proces oddychaniabeztlenowego, a nie tlenowego, charakterystyczny dla komórek prawidłowych. Przekształcają duże ilości glukozydo mleczanu w procesie fermentacji mlekowej, prowadzonej nawet w obecności tlenu. Zjawisko to dziś znane jestpod pojęciem efektu Warburga lub glikolizy tlenowej. Biochemik przyczynę tego zjawiska upatrywał w uszkodzeniumitochondriów. Do niedawna nie było wiadomo, jaki proces chemiczny kryje się za „efektem Warburga”. Wiadomo,że wiele komórek proliferujących, w tym nowotworowych, cechuje wzmożone pobieranie glukozy i ograniczeniefosforylacji oksydacyjnej. Ta ścieżka metaboliczna utrzymuje wysoki poziom produkcji mleczanu, nawet w obecnościtlenu. Obecnie sugeruje się, że powstałe metabolity mogą spełniać rolę podobną do onkogenów poprzez zmianęszlaków sygnalizacyjnych i zablokowanie różnicowania komórek. Zmiany te ułatwiają proces onkogenezy i wzrostkomórek. Dużą rolę w zmianie metabolizmu odgrywa kinaza pirogronianowa (PK) — enzym biorący udział w szlakuglikolitycznym, który w komórkach nowotworowych jest zastępowany przez izoformę PKM2, co jest konieczne doprzekierowania metabolizmu na szlak glikolizy tlenowej i jest warunkiem nowotworzenia. Stwierdzono również, żePKM2 odpowiada za homeostazę reakcji redoks, czego dowodem jest uruchomienie szlaku pentozowego, który ograniczaakumulację reaktywnych form tlenu i chroni komórki nowotworowe przed stresem oksydacyjnym, ułatwiając tymsamym ich wzrost. Ostatnio zaproponowano nowy model metabolizmu nowotworu, potwierdzony eksperymentalnie,który nosi nazwę odwrotnego efektu Warburga. Model ten zakłada ścisłą współpracę metaboliczną pomiędzy aktywowanymifi broblastami podścieliska a komórkami nowotworowymi, i dowodzi, że komórki nowotworowe głównieoddychają tlenowo. Proces glikolizy tlenowej (efekt Warburga) natomiast jest przeprowadzany przede wszystkimprzez fi broblasty zrębu nowotworu. Komórki prawidłowe podścieliska, głównie fi broblasty, dzięki zachodzącymw nich pod wpływem stresu oksydacyjnego licznym procesom katabolicznym (autofagia, mitofagia, fermentacjamlekowa) dostarczają komórkom nowotworowym wysokoenergetycznych związków, takich jak mleczan, ketonyczy glutamina, które wykorzystują je jako biopaliwo do reakcji syntez. Komórki te, dzięki dostarczonym substratom,mogą przeprowadzać liczne procesy anaboliczne oraz wytwarzać duże ilości ATP w procesie oddychania tlenowego.Umożliwia to ich wzrost i rozwój, a zatem — progresję nowotworu.Otto Warburg at the beginning of the 20th century suggested that cancer cells exhibit diff erent metabolism than normalcells. He demonstrated that tumour cells prefer aerobic glycolisis rather than oxidative respiration as for normalcells. They convert large amounts of glucose to lactate in the process of glycolysis, and even in the presence of oxygen.The phenomenon is known as the Warburg eff ect or aerobic glycolysis. The biochemist hypothesized that the cause ofthis is mitochondrial damage in tumour cells. The reason why cells undergo the Warburg eff ect is still poorly understood.However it is known that many proliferating cells, also malignant cells, show increased uptake of glucose andrestriction of oxidative phosphorylation. This metabolic pathway facilitates high levels of lactate production, evenin the presence of oxygen. Recent evidence suggests that metabolites themselves can be oncogenic by altering cell signaling and blocking cellular diff erentiation. These changes facilitate the process of oncogenesis and cell growth.The pyruvate kinase (PK), a glycolitic enzyme is replaced by isoform of PKM2 which facilitates aerobic glycolisisin cancer cells. PKM2 is also a regulator of cellular anti-oxidative metabolism which promotes cancer growth byactivating pentose phosphate pathway, maintaining the balance of redox equivalents and activating antioxidantdefence system. Recently there has been proposed a new model of cancer metabolism, which has been provedexperimentally, termed reverse Warburg eff ect. This model explains the role of aerobic glycolysis and lactate productionin fueling tumour growth. This model assumes metabolic cooperation between stromal fi broblasts andtumour cells, and that cancer cells perform oxidative respiration. In activated fi broblasts, oxidative stress in thetumour microenvironment leads to authophagy, mitophagy and aerobic glycolysis, which delivers high-energeticintermediates such as lactate, ketones and glutamine to tumour cells that fuel the anabolic growth. Tumour cellsdue to delivered nutrients can lead anabolic metabolism and produce high amounts of ATP what facilitates tumourgrowth, development and progression

Differences in the prognosis of HPV16-positive patients with squamous cell carcinoma of head and neck according to viral load and expression of P16

Purpose To evaluate the impact of HPV16 load (VL-the number of virus genome copies per cell) and P16 expression on prognosis of patients with squamous cell carcinomas (SCCs) of head and neck (HN). Materials and methods HPV16 presence was assessed in the group of 109 patients with HNSCCs by quantitative polymerase chain reaction (qPCR). VL (assessed by qPCR) and P16 expression (evaluated by immunohistochemistry) were analysed only in the subgroup of HPV16-positive tumours. These features were correlated with 5-year overall survival (OS) and disease-free survival (DFS). Results HPV16 infection was found in 36 tumours (33.0%). Virus-positive patients had better OS and DFS than those without infection (P = 0.041 and 0.005). Among HPV16-positive HNSCCs, 18 (50.0%) had higher VL (median value > 6764.3 copies/cell) and 25 (73.5%) P16 over expression. The significant differences in OS and DFS (P = 0.008 and 0.004) were noticed according to VL, wherein 100% DFS was found for patients with higher VL. According to P16 expression, significant difference was found only for OS (P = 0.020). In multivariate analysis, VL (P = 0.045; HR = 2.795; CI 0.121-1.060) and the level of smoking (P = 0.023, HR = 2.253; CI 1.124-4.514) were independent factors affecting DFS of HPV16-positive patients. Conclusion On the basis of viral load, it is possible to differentiate prognosis of patients with HPV16-positive HNSCCs. In this subgroup, viral load has stronger prognostic potential than P16 expression

Design, Synthesis and Functional Analysis of Cyclic Opioid Peptides with Dmt-Tic Pharmacophore

The opioid receptors are members of the G-protein-coupled receptor (GPCR) family and are known to modulate a variety of biological functions, including pain perception. Despite considerable advances, the mechanisms by which opioid agonists and antagonists interact with their receptors and exert their effect are still not completely understood. In this report, six new hybrids of the Dmt-Tic pharmacophore and cyclic peptides, which were shown before to have a high affinity for the mu-opioid receptor (MOR) were synthesized and characterized pharmacologically in calcium mobilization functional assays. All obtained ligands turned out to be selective antagonists of the delta-opioid receptor (DOR) and did not activate or block the MOR. The three-dimensional structural determinants responsible for the DOR antagonist properties of these analogs were further investigated by docking studies. The results indicate that these compounds attach to the DOR in a slightly different orientation with respect to the Dmt-Tic pharmacophore than Dmt-Tic psi[CH2-NH]Phe-Phe-NH2 (DIPP-NH2[psi]), a prototypical DOR antagonist peptide. Key pharmacophoric contacts between the DOR and the ligands were maintained through an analogous spatial arrangement of pharmacophores, which could provide an explanation for the predicted high-affinity binding and the experimentally observed functional properties of the novel synthetic ligands

Synthesis, Selected Transformations, and Biological Activity of Alkoxy Analogues of Lepidilines A and C

Condensation of diacetyl monooxime with formaldimines derived from alkoxyamines in glacial acetic acid at room temperature leads to corresponding 2-unsubstituted imidazole N-oxides bearing an alkoxy substituent at the N(1) atom of the imidazole ring. Subsequent O-benzylation afforded, depending on the type of alkylating agent, either symmetric or nonsymmetric alkoxyimidazolium salts considered as structural analogues of naturally occurring imidazole alkaloids, lepidilines A and C. Some of the obtained salts were tested as precursors of nucleophilic heterocyclic carbenes (NHCs), which in situ reacted with elemental sulfur to give the corresponding N-alkoxyimidazole-2-thiones. The cytotoxic activity of selected 4,5-dimethylimidazolium salts bearing either two benzyloxy or benzyloxy and 1-adamantyloxy groups at N(1) and N(3) atoms was evaluated against HL-60 and MCF-7 cell lines using the MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. Notably, in two cases of alkoxyimidazolium salts, no effect of the counterion exchange (Br− → PF6−) on the biological activity was observed

Functional selectivity of EM-2 analogs at the mu-opioid receptor

The mu opioid receptor agonists are the most efficacious pain controlling agents but their use is accompanied by severe side effects. More recent developments indicate that some ligands can differentially activate receptor downstream pathways, possibly allowing for dissociation of analgesia mediated through the G protein from the opioid-related side effects mediated by β-arrestin pathway. In an effort to identify such biased ligands, here we present a series of thirteen endomorphin-2 (EM-2) analogs with modifications in positions 1, 2, and/or 3. All obtained analogs behaved as mu receptor selective agonists in calcium mobilization assay carried out on cells expressing opioid receptors and chimeric G proteins. A Bioluminescence Resonance Energy Transfer (BRET) approach was employed to determine the ability of analogs to promote the interaction of the mu opioid receptor with G protein or β-arrestin 2. Nearly half of the developed analogs showed strong bias towards G protein, in addition four compounds were nearly inactive towards β-arrestin 2 recruitment while blocking the propensity of EM-2 to evoke mu-β-arrestin 2 interaction. The data presented here contribute to our understanding of EM-2 interaction with the mu opioid receptor and of the transductional propagation of the signal. In addition, the generation of potent and selective mu receptor agonists strongly biased towards G protein provides the scientific community with novel tools to investigate the in vivo consequences of biased agonism at this receptor