Endothelin axis and apoptosis

Endothelin axis (endothelin-1, -2 and -3, and endothelin receptors ETAR and ETBR) plays the key role in the various functions of the organism: acts as a modulator of vascular tone, tissue differentiation, growth and development, cell proliferation, and hormone synthesis. In addition to its physiological role, endothelin axis or individual components of the system, can have a significant effect on the tissue remodelling process and tumorigenesis. For example, endothelin-1 modulates mitosis, apoptosis and angiogenesis and can stimulate tumour invasion and metastasis. Increased endothelin-1 expression has been demonstrated in breast, ovarian, prostate and colorectal cancers. In tumour cells, the binding of endothelin-1 to ETA receptor induces signalling pathway for survival. Endothelin-1 triggers the anti-apoptotic signal through phosphatidylinositol 3-kinase (PI3-K)-dependent Akt phosphorylation. Endothelin receptors are coupled with G-proteins and they differ in sensitivity to antagonists. G-proteins are involved in cell signalling via adenylate cyclase, ion channels, PLC, PLA2, PKC, intracellular Ca2+, calcineurin and MAP kinase. It is believed that this activity is mediated by ETAR, since the application of their specific antagonist BQ-123 causes a reverse effect. Endothelin receptor antagonists such as A-127722, BQ-123, BQ-788, etc. act specifically on either proliferation or apoptosis depending on the cell type. Today, certain endothelin receptor antagonists, such as atrasentan, are applied in the treatment of lung and prostate cancer or are at different phases of clinical trials

Toksični učinci Ustilago maydis i fumonizina B1 u štakora

Ustilago maydis and FB1 showed neurotoxicity in Fischer rats, which was indicated by hyperactivity, brain hyperaemia and especially by decreased AChE activity. These pathological changes could be related to the alkaloids of U. maydis and disruption of sphingolipid metabolism by FB1 activity. Pathological changes in liver, lungs, uterus and ovaries were more pronounced in the US+FB1 than in the US group, indicating possible synergism. Increased activity of LDH in the serum was observed only in the US+FB1 group, which led to the conclusion that FB1 is responsible for cytotoxic effects. Such activity of FB1 could increase the toxicity of U. maydis and other co-contaminants of fungal origin that can be found in foods and feeds.Toksično djelovanje kukuruzne snijeti Ustilago maydis i mogući sinergizam s fumonizinom B1 (FB1) ispitivani su na štakorima soja Fischer. Toksični učinci su procijenjeni na temelju organoleptičkog pregleda organa žrtvovanih životinja i mjerenja biokemijskih parametara u serumu (LDH, ALT, GGT, ChE), te homogenatu tkiva jetre i mozga (AChE, ChE, GGT, ALP). U jednoj pokusnoj skupini (US) životinje su hranjene smjesom biomase i snijeti masenog udjela 70%, dok je druga pokusna skupina (US+FB1) primala smjesu biomase i snijeti masenog udjela 70%, te fumonizina B1 (1 mg po kilogramu hrane) tijekom 17 dana. Kontrolna skupina je hranjena standardnom laboratorijskom hranom. Tijekom pokusa životinje su bile ekscitirane. Masa pokusnih životinja bila je na kraju pokusa nešto veća u odnosu na kontrolnu skupinu. Uočene patološke promjene na jetri, plućima, uterusu i ovarijima bile su više izražene u skupini US+FB1. Specifična katalitička aktivnost AChE u homogenatu jetre i mozga bila je značajno smanjena u US skupini (61, odnosno 63%) u odnosu na kontrolu (p < 0,05), što ukazuje na neurotoksično djelovanje snijeti. Osim toga, zabilježena je značajno smanjena aktivnost ovog enzima i ALP u homogenatu jetre US+FB1 skupine (p < 0,05). Specifična katalitička aktivnost LDH u serumu bila je značajno povećana u US+FB1 skupini (165%, odnosno 166%) u odnosu na US i kontrolnu skupinu, što ukazuje da su oštećenja stanične membrane rezultat djelovanja FB1. Neurotoksični učinci u štakora soja Fischer vjerojatno su posljedica djelovanja alkaloida U. maydis i poremećaja metabolizma sfingolipida uslijed djelovanja FB1

Utjecaj različitih koncentracija peroksovanadijevog spoja bpV(phen) na preživljavanje PC12 stanica

Peroxovanadium compounds are potent insulinomimetic agents and protein tyrosine phosphatase inhibitors. In this study, the potential toxicity of peroxovanadium complex bpV(phen) on rat pheochromocytoma PC12 cells was examined, and the mechanism by which this compound influences cell survival and/or death was explored. BpV(phen) exerted a bimodal effect on PC12 cells: survival enhancing effect at lower and death-inducing effect at higher micromolar concentrations. 1 and 3 μmol dm–3 bpV(phen) intensely induced ERK activation. In contrast, 10 and 100 μmol dm–3 bp (phen) stimulated strong and sustained JNK and p38 MAPK activation as well as caspase-3 activation that preceded bpV(phen)-induced apoptotic cell death. It is suggested that bpV(phen) might exert its action on PC12 cell survival by modulation of MAPKs and caspase-3 activation.Spojevi peroksovanadija snažni su inzulinomimetički agensi i inhibitori tirozinskih fosfataza. U ovom istraživanju ispitivana je moguća toksičnost peroksovanadijevoga kompleksa bpV(phen) na PC12 stanicama feokromocitoma štakora te način na koji ovaj spoj utječe na preživljavanje i/ili umiranje stanica. BpV(phen) dvojako je djelovao na PC12 stanice: primijenjen u nižim mikromolarnim koncentracijama poticao je preživljavanje stanica, dok su više mikromolarne koncentracije poticale umiranje stanica. 1 i 3 μmol dm–3 bpV(phen) snažno je inducirao aktivaciju ERK kinaza. Suprotno tome, 10 i 100 μmol dm–3 bpV(phen) stimulirao je snažnu i dugotrajnu aktivaciju JNK i p38 MAPK kinaza te aktivaciju kaspaze-3, što je prethodilo umiranju stanica procesom apoptoze. Pretpostavlja se da bpV(phen) djeluje na preživljavanje PC12 stanica na način da modulira aktivaciju MAPK kinaza i kaspaze-3

TNF-alpha, CXCL8, big ET-1 and hsCRP in Patients with Chronic Obstructive Pulmonary Disease

COPD (chronic obstructive pulmonary disease) is a chronic progressive inflammatory disease characterised by limitations in lung airflow that is not fully reversible. The concentrations and correlations of TNF-á, CXCL8, big ET-1 and hsCRP were investigated in healthy non-smokers, healthy smokers and patients with COPD in order to study their possible role in the pathophysiology of COPD. The concentrations of TNF-á, CXCL8 and big ET-1 were not statistically different between the experimental groups. No significant differences for the measured analytes were found between smokers and the non-smokers in the control group. The Spearman coefficient of correlation between the concentrations of TNF-á and CXCL8 was r = 0.638 (p<0.0001). However, the concentration of hsCRP was significantly higher in patients with COPD than in the control group (p = 0.0004). hsCRP proved to be a more sensitive diagnostic parameter than TNF-á, CXCL8 and big ET-1 in the systemic circulation in patients with COPD

Okratoksin A izaziva apoptozu u LLC-PK1 stanicama aktivirajući JNK i p38 MAPK

Ochratoxin A (OTA) is a potential inducer of a tubular-interstitial nephropathy in humans and animals. In our study we addressed the question of involvement of apoptosis in the development of OTA-provoked nephrotoxicity. LLC-PK1 kidney cells were treated with nanomolar and micromolar concentrations of OTA for different lengths of time. The apoptotic process was estimated by morphological (haematoxylin/eosin staining, fluorescent staining of DNA free ends – TUNEL assay) and biochemical (MAPKs and Hsps) changes of cells. Forty-eight hours of treatment with 5 10–6 M OTA significantly decreased cell viability and induced apoptosis in 30.7 % of cells. In addition, a transient activation of ERK was observed as well as a strong and prolonged activation of stress kinases, JNK and p38 MAPK, after 12 and 48 hours of treatment. Expression of Hsp72 and Hsp27 was not affected by OTA. The results suggest that apoptosis mediated by activation of JNK and p38 MAPK might play an important role in OTA-induced nephrotoxicity in LLC-PK1 cells.Okratoksin A (OTA) može izazvati tubularno-intersticijsku nefropatiju u ljudi i životinja. Cilj našeg istra- živanja bio je ispitati ulogu apoptoze u nastanku nefrotoksičnosti koju uzrokuje OTA. Naš eksperimentalni model bile su LLC-PK1 bubrežne stanice izložene djelovanju OTA, od nanomolarnih do mikromolarnih koncentracija kroz različita vremenska razdoblja. Apoptoza je u stanicama utvrđena na temelju morfoloških (bojanje stanica hematoksilin/eozinom, fluorescentno bojanje slobodnih krajeva DNA tzv. TUNEL) i biokemijskih (MAP kinaze, Hsps) promjena. 48-satno izlaganje stanica 5 x 10–6 M OTA značajno je smanjilo staničnu vijabilnost i potaknulo apoptozu u 30,7 % stanica. Osim toga, utvrđena je kratkotrajna aktivacija ERK te snažna i dugotrajna aktivacija JNK i p38 MAPK nakon 6-, 12- i 48-satnoga tretiranja. OTA nije utjecao na razinu Hsp72 i Hsp27. Rezultati istraživanja ukazuju da je apoptoza posredovana aktivacijom JNK i p38 MAPK važan događaj u razvoju nefrotoksičnosti izazvanoj djelovanjem OTA na LLC-PK1 stanice

Flavonoid diosmetin increases ATP levels in kidney cells and relieves ATP depleting effect of ochratoxin A

Diosmetin (DIOS) is a flavone aglycone commonly occurring in citrus species and olive leaves, in addition it is one of the active ingredients of some medications. Based on both in vitro and in vivo studies several beneficial effects are attributed to DIOS but the biochemical background of its action seems to be complex and it has not been completely explored yet. Previous investigations suggest that most of the flavonoid aglycones have negative effect on ATP synthesis in a dose dependent manner. In our study 17 flavonoids were tested and interestingly DIOS caused a significant elevation of intracellular ATP levels after 6- and 12-h incubation in MDCK kidney cells. In order to understand the mechanism of action, intracellular ATP and protein levels, ATP/ADP ratio, cell viability and ROS levels were determined after DIOS treatment. In addition, impacts of different enzyme inhibitors and effect of DIOS on isolated rat liver mitochondria were also tested. Finally, the influence of DIOS on the ATP depleting effect of the mycotoxin, ochratoxin A was also investigated. Our major conclusions are the followings: DIOS increases intracellular ATP levels both in kidney and in liver cells. Inhibition of glycolysis or citric acid cycle does not decrease the observed effect. DIOS-induced elevation of ATP levels is completely abolished by the inhibition of ATP synthase. DIOS is able to completely reverse the ATP-depleting effect of the mycotoxin, ochratoxin A. Most probably the DIOS-induced impact on ATP system does not originate from the antioxidant property of DIOS. Based on our findings DIOS may be promising agent to positively influence ATP depletion caused by some metabolic poisons

Apoptosis of Leukemic Cells: A Case Report

Transformation of leukemic cells is associated with delay in maturation and in apoptosis, and to altered responsiveness to growth factors. However, some studies have revealed that Fas (CD95/APO1) which mediates apoptotic signal and decrease of anti-apoptotic Bcl-2 are frequently observed in acute myeloid leukemia (AML) M4/M5 leukemic cells. The aim of the study was to compare cytomorphology and cytochemistry of bone marrow (BM) apoptotic leukemic cells to preserved peripheral blood (PB) leukemic cells in our patient, a 76-year-old man with AML-M5b treated at Zagreb University Hospital Center. BM and PB of the AL patient were analyzed after Pappenheim and cytochemical stainings, and leukemic cells were classified according to FAB and WHO classification. Analysis of PB revealed leukocytosis and 80–90% monocytic cells (46% monoblasts, 29% promonocytes and 11% monocytes). Only a few preserved monoblasts and promonocytes were found in BM, together with numerous morphologically altered cells with characteristic chromatin condensation and pyknosis of nucleus, as well as nuclear fragmentation and formation of apoptotic bodies. Thus, cytomorphology of PB leukemic cells pointed to proliferation of immature monocytic cells, and cytomorphology of BM to cell apoptosis. Cytochemistry of PB monocytic cells and BM apoptotic cells confirmed monocytic cell lineage because esterase was strongly positive in almost all BM apoptotic leukemic cells and PB leukemic cells, and esterase was completely inhibited with sodium fluoride. On the basis of these findings, AML-M5b was diagnosed in our patient. There are many possible explanations for our observation of BM leukemic cell apoptosis in a patient with AML-M5. The most reliable one is that apoptosis was induced ex vivo after BM aspiration in course of the air drying of BM specimen before staining. Mass BM leukemic cell apoptosis that was recorded in contrast to numerous preserved leukemic cells in PK could be probably connected to unfavorable ratio of relatively low concentration of cytokines in relation to high leukemic cell number in BM aspirated cytologic specimen