Routine End-ischemic Hypothermic Oxygenated Machine Perfusion in Liver Transplantation From Donors After Brain Death: A Randomized Controlled Trial.

OBJECTIVE: To assess whether end-ischemic hypothermic oxygenated machine perfusion (HOPE) is superior to static cold storage (SCS) in preserving livers procured from donors after brain death (DBD). BACKGROUND: There is increasing evidence of the benefits of HOPE in liver transplantation, but predominantly in the setting of high-risk donors. METHODS: In this randomized clinical trial, livers procured from DBDs were randomly assigned to either end-ischemic dual HOPE for at least 2 hours or SCS (1:3 allocation ratio). The Model for Early Allograft Function (MEAF) was the primary outcome measure. The secondary outcome measure was 90-day morbidity (ClinicalTrials. gov, NCT04812054). RESULTS: Of the 104 liver transplantations included in the study, 26 were assigned to HOPE and 78 to SCS. Mean MEAF was 4.94 and 5.49 in the HOPE and SCS groups ( P =0.24), respectively, with the corresponding rates of MEAF >8 of 3.8% (1/26) and 15.4% (12/78; P =0.18). Median Comprehensive Complication Index was 20.9 after transplantations with HOPE and 21.8 after transplantations with SCS ( P =0.19). Transaminase activity, bilirubin concentration, and international normalized ratio were similar in both groups. In the case of donor risk index >1.70, HOPE was associated with significantly lower mean MEAF (4.92 vs 6.31; P =0.037) and lower median Comprehensive Complication Index (4.35 vs 22.6; P =0.050). No significant differences between HOPE and SCS were observed for lower donor risk index values. CONCLUSION: Routine use of HOPE in DBD liver transplantations does not seem justified as the clinical benefits are limited to high-risk donors.

Teriflunomide - The common drug with underestimated oxygen - Dependent anticancer potential.

Leflunomide (LFN) is a well-known immunomodulatory and anti-inflammatory prodrug of teriflunomide (TFN). Due to pyrimidine synthesis inhibition TFN also exhibits potent anticancer effect. Because, there is the strict coupling between the pyrimidine synthesis and the mitochondrial respiratory chain, the oxygen level could modify the cytostatic TNF effect. The aim of the study was to evaluate the cytostatic effect of pharmacologically achievable teriflunomide (TFN) concentrations at physiological oxygen levels, i.e. 1% hypoxia and 10% tissue normoxia compared to 21% oxygen level occurred in routine cell culture environment. The TFN effect was evaluated using TB, MTT and FITC Annexin tests for human primary (SW480) and metastatic (SW620) colon cancer cell lines at various oxygen levels. We demonstrated significant differences between proliferation, survival and apoptosis at 1, 10 and 21% oxygen in primary and metastatic colon cancer cell lines (SW480, SW620) under TFN treatment. The cytostatic TFN effect was more pronounced at hypoxia compared to tissue and atmospheric normoxia in both cancer cell lines, however metastatic cells were more resistant to antiproliferative and proapoptotic TFN action. The early apoptosis was predominant in physiological oxygen tension while in atmospheric normoxia the late apoptosis was induced. Our findings showed that anticancer TFN effect is more strong in physiological oxygen compared to atmospheric normoxia. It suggests that results obtained from in vitro studies could be underestimated. Thus, it gives assumption for future comprehensive studies at real oxygen environment involving TNF use in combination with other antitumor agents affecting oxygen-dependent pyrimidine synthesis.

The Real Cytotoxic Effect of Artemisinins on Colon Cancer Cells in a Physiological Cell Culture Setting. How Composition of the Culture Medium Biases Experimental Findings.

Artemisinin (ART) and dihydroartemisinin (DHA) are anti-malaria drugs but also exhibit huge anticancer potential based on ferroptosis driven by iron-dependent lipid peroxidation. This study was conducted on primary (SW480), metastatic (SW620) colon cancer, and noncancerous HaCaT cells at pharmacologically relevant drug concentrations (1-8 µM) and in the presence of holotransferrin (TRFi 50 µM) and linoleic acid (LA 20, 40 µM) at physiological levels. ART and DHA showed the growth inhibitory potency which was significantly increased in the presence of LA or/and TRFi. The IC50 for ART or DHA, LA40 and TRFi combination in both cancer cell lines ranged 0.14-0.69 µM whereas no cytotoxic effect was observed for HaCaT cells (SI = 202-480). Almost all experimental settings revealed late apoptosis in both cancer cell lines, but not in normal cells. The percentage of late apoptotic cells increased with LA concentrations and was intensified after TRFi addition. The strongest pro-apoptic effect was exhibited by ART or DHA, LA40, and TRFi combination. More interestingly, we found a stimulatory effect of TRFi on IL-6 synthesis. The present study using LA and TRFi which are inherent blood components revealed high antitumor artemisinin activity in concentrations achievable after drug administration to cancer patients without toxic effects on normal cells.

Design, Synthesis, and In Vitro Antiproliferative Activity of Hydantoin and Purine Derivatives with the 4-Acetylphenylpiperazinylalkyl Moiety.

Cancer represents one of the most serious health problems and the second leading cause of death around the world. Heterocycles, due to their prevalence in nature as well as their structural and chemical diversity, play an immensely important role in anti-cancer drug discovery. In this paper, a series of hydantoin and purine derivatives containing a 4-acetylphenylpiperazinylalkyl moiety were designed, synthesized, and biologically evaluated for their anticancer activity on selected cancer cell lines (PC3, SW480, SW620). Compound 4, a derivative of 3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalene]-2,5-dione, was the most effective against SW480, SW620, and PC3 cancer cell lines. Moreover, 4 has high tumor-targeting selectivity. Based on docking studies, it was concluded that R isomers of 3',4'-dihydro-2'H-spiro[imidazolidine-4,1'-naphthalene]-2,5-dione could be further studied as promising scaffolds for the development of thymidine phosphorylase inhibitors.

Evaluation of the anticancer activity of singly and doubly modified analogues of C20-epi-salinomycin.

In developed countries, cancer is the second leading cause of death, with colon and prostate cancer belonging to the group of most often diagnosed types of neoplastic diseases. The search for new treatment strategies against these types of cancer is thus of top current interest. In this context, salinomycin (SAL), a naturally occurring polyether ionophore, has been identified recently as a very promising anticancer drug candidate towards several tumour cells. In the present work, a broad library of 24 derivatives of C20-epi-salinomycin (2), including C1 singly, C20 singly and C1/C20 doubly modified analogue structures, was screened to identify compounds with improved activity against colon and prostate cancer cells. Our study demonstrated that the growth inhibitory potency of the parent compound on both primary and metastatic colon cancer cells was similar to that of the semisynthetic products derived from SAL, and simultaneously the SAL analogues showed more potent toxic action on metastatic prostate cancer cells than that of the chemically unmodified ionophore. In contrast to the widely used oncological drug doxorubicin, some of the SAL derivatives demonstrated promising anticancer activity with no toxic effects on non-tumour cells, and with more favourable cytotoxicity than that of a reference agent 5-fluorouracil. Mechanistically, the SAL analogues induced late apoptosis in colon cancer cells and necrosis in prostate cancer cells, as well as reduced secretion of interleukin 6 (IL-6) in these cells.

Molecular mechanisms of ethanol biotransformation: enzymes of oxidative and nonoxidative metabolic pathways in human.

Ethanol, as a small-molecule organic compound exhibiting both hydrophilic and lipophilic properties, quickly pass through the biological barriers. Over 95% of absorbed ethanol undergoes biotransformation, the remaining amount is excreted unchanged, mainly with urine and exhaled air.The main route of ethyl alcohol metabolism is its oxidation to acetaldehyde, which is converted into acetic acid with the participation of cytosolic NAD+ - dependent alcohol (ADH) and aldehyde (ALDH) dehydrogenases. Oxidative biotransformation pathways of ethanol also include reactions catalyzed by the microsomal ethanol oxidizing system (MEOS), peroxisomal catalase and aldehyde (AOX) and xanthine (XOR) oxidases. The resulting acetic acid can be activated to acetyl-CoA by the acetyl-CoA synthetase (ACS).It is also possible, to a much smaller extent, non-oxidative routes of ethanol biotransformation including its esterification with fatty acids by ethyl fatty acid synthase (FAEES), re-esterification of phospholipids, especially phosphatidylcholines, with phospholipase D (PLD), coupling with sulfuric acid by alcohol sulfotransferase (SULT) and with glucuronic acid using UDP-glucuronyl transferase (UGT, syn. UDPGT).The intestinal microbiome plays a significant role in the ethanol biotransformation and in the initiation and progression of liver diseases stimulated by ethanol and its metabolite - acetaldehyde, or by lipopolysaccharide and ROS.

Telmisartan Influences the Antiproliferative Activity of Linoleic Acid in Human Colon Cancer Cells.

Aim: Linoleic acid (LA) and telmisartan as PPARgamma agonists exhibit anticancer activity. The LA effect is observed for high non-achievable in vivo concentrations and in short treatment period, therefore we evaluate the effect of supplemental LA and pharmacological telmisartan plasma concentrations on human primary (SW480) and metastatic (SW620) colon cancer cells and immortal keratinocytes (HaCaT) cells in long-term treatment. Methods: Cell viability and proliferation were determined by TB and MTT and pro-apoptotic effect was measured by Annexin V binding assays, respectively.Results: LA decreased cancer cell viability and proliferation in a concentration-dependent manner, whereas no significant effect was found for HaCaT cells. Telmisartan (0.2 µM) suppresses antiproliferative effect of 60 µM LA on cancer cells in short-term treatment. Long-term administration of 60 µM LA reduced cancer cells viability after one week, while telmisartan delayed this effect by two weeks. Growth of all cell lines with 20 µM LA was unchanged during all treatment time. Telmisartan decreased late apoptosis of cancer and normal cells with 60 and 120 µM LA. Conclusion: The cytotoxic LA action depends not only on its concentration but also duration of treatment. Telmisartan exhibits biphasic but not synergistic effect on LA cytotoxicity in cancer cells.

[Effect of antioxidants on human primary and metastatic colon cancer cells at hypoxia and normoxia].

THE AIM: Evaluation of some antioxidants on human colon cancer cells viability and proliferation at various oxygen levels. MATERIAL AND METHODS: Human primary (SW480) and metastatic (SW620) colon cancer cells were cultured at hypoxia (1% oxygen), tissues (10% oxygen) and atmospheric (21% oxygen) normoxia with quercetin, epigallocatechin gallate, lipoic acid, hydroxycitric acid, their mixture, and without studied compounds (control). Antioxidants were used at physiological concentrations. The cell viability was determined by trypan blue dye exclusion and proliferation by MTT assay. RESULTS: The viability of each line ranged from 80% to 97%, and it was independent on the compound and oxygen availability. At hypoxia the cell count of both lines was lower than for the controls in the presence of each studied compound. At tissue normoxia the cell count of primary cancer cells was decreased only with epigallocatechin gallate, whereas metastatic cells were sensitive for each antioxidant. CONCLUSIONS: Our results indicated, that the studied antioxidants were not cytotoxic at physiological levels for both pirmary and metastatic colon cancer. Their cytostatic effect depend on the type of cell, oxygen availability and antioxidant concentration.

Effect of different concentrations of oxygen on expression of sigma 1 receptor and superoxide dismutases in human colon adenocarcinoma cell lines.

CONTEXT: Tumor cells due to distance from capillary vessels exist in different oxygenation conditions (anoxia, hypoxia, normoxia). Changes in cell oxygenation lead to reactive oxygen species production and oxidative stress. Sigma 1 receptor (Sig1R) is postulated to be stress responding agent and superoxide dismutases (SOD1 and SOD2) are key antioxidant enzymes. It is possible that they participate in tumor cells adaptation to different concentrations of oxygen. OBJECTIVE: Evaluation of Sig1R, SOD1, and SOD2 expression in different concentrations of oxygen (1%, 10%, 21%) in colon adenocarcinoma cell lines. MATERIALS AND METHODS: SW480 (primary adenocarcinoma) and SW620 (metastatic) cell lines were cultured in standard conditions in Dulbecco's modified Eagle's medium for 5 days, and next cultured in Hypoxic Chamber in 1% O2, 10% O2, 21% O2. Number of living cells was determined by trypan blue assay. Level of mRNA for Sig1R, SOD1, and SOD2 was determined by standard PCR method. Statistical analysis was conducted using Statistica 10.1 software. RESULTS: We observed significant changes in expression of Sig1R, SOD1, SOD2 due to different oxygen concentrations. ANOVA analysis revealed significant interactions between studied parameters mainly in hypoxia conditions in SW480 cells and between Sig1R and SOD2 in SW620 cells. It also showed that changes in expression of studied proteins depend significantly on type of the cell line. CONCLUSION: Changes of Sig1R and SOD2 expression point to mitochondria as main organelle responsible for survival of tumor cells exposed to hypoxia or oxidative stress. Studied proteins are involved in intracellular response to stress related with different concentrations of oxygen.

Lactate Formation in Primary and Metastatic Colon Cancer Cells at Hypoxia and Normoxia.

High glucose consumption and lactate synthesis in aerobic glycolysis are a hallmark of cancer cells. They can form lactate also in glutaminolysis, but it is not clear how oxygen availability affects this process. We studied lactate synthesis at various oxygen levels in human primary (SW480) and metastatic (SW620) colon cancer cells cultured with L-Ser and/or L-Asp. Glucose and lactate levels were determined colorimetrically, amino acids by HPLC, expression of AST1-mRNA and AST2-mRNA by RT-PCR. In both lines glucose consumption and lactate synthesis were higher at 10% than at 1% oxygen, and lactate/glucose ratio was increased above 2.0 by L-Asp. AST1-mRNA expression was independent on oxygen and cell line, but AST2-mRNA was lower at hypoxia in SW480. We conclude that, in both cell lines at 1% hypoxia, lactate is formed mainly from glucose but at 10% normoxia also from L-Asp. At 10% normoxia, lactate synthesis is more pronounced in primary than metastatic colon cancer cells.

[Aspartate aminotransferase--key enzyme in the human systemic metabolism]. / Aminotransferaza asparaginianowa--kluczowy enzym w metabolizmie ogólnoustrojowym czlowieka.

Aspartate aminotransferase is an organ-nonspecific enzyme located in many tissues of the human body where it catalyzes reversible reaction of transamination. There are two aspartate aminotransferase isoforms--cytoplasmic (AST1) and mitochondrial (AST2), that usually occur together and interact with each other metabolically. Both isoforms are homodimers containing highly conservative regions responsible for catalytic properties of enzyme. The common feature of all aspartate aminotransfeses is Lys - 259 residue covalent binding with prosthetic group - pyridoxal phosphate. The differences in the primary structure of AST isoforms determine their physico-chemical, kinetic and immunological properties. Because of the low concentration of L-aspartate (L-Asp) in the blood, AST is the only enzyme, which supply of this amino acid as a substrate for many metabolic processes, such as urea cycle or purine and pyrimidine nucleotides in the liver, synthesis of L-arginine in the kidney and purine nucleotide cycle in the brain and the skeletal muscle. AST is also involved in D-aspartate production that regulates the metabolic activity at the auto-, para- and endocrine level. Aspartate aminotransferase is a part of the malate-aspartate shuttle in the myocardium, is involved in gluconeogenesis in the liver and kidney, glyceroneogenesis in the adipose tissue, and synthesis of neurotransmitters and neuro-glial pathway in the brain. Recently, the significant role of AST in glutaminolysis - normal metabolic pathway in tumor cells, was demonstrated. The article is devoted the role of AST, known primarily as a diagnostic liver enzyme, in metabolism of various human tissues and organs.

Significance of arginase determination in body fluids of patients with hepatocellular carcinoma and liver cirrhosis before and after surgical treatment.

OBJECTIVE: To assess the utility of arginase activity and expression in diagnosis of liver diseases. DESIGN AND METHODS: Arginase activity, sensitivity and specificity were determined in serum of 140 patients including 50 with HCC, 60 with LC, 30 with choledocholithiasis (CDL) and 90 healthy controls. In HCC and LC arginase activity in serum was studied before and after tumor resection or liver transplantation. Arginase sensitivity in HCC was compared to that of alpha-fetoprotein (AFP) and aminotransferases (AST, ALT). In LC the activity was determined also in bile before and after transplantation. The expression of arginase isoenzymes in serum was studied by Western blotting. RESULTS: In HCC and LC the preoperative arginase activity was significantly higher compared to controls, and it decreased after surgery. The sensitivity of arginase in HCC was much higher than that of AFP, AST and ALT (96, 40, 20 and 18%, respectively). In HCC it was higher than in LC (93%) and CDL (33%). The specificity of arginase was above 80%. In bile of cirrhotic patients the highest activity was immediately after liver transplantation. It decreased with time but increased dramatically at the time of the graft rejection. Arginase AII was present in serum of HCC and LC but not the control cases. CONCLUSIONS: The increase of arginase activity in serum accompanied by the presence of isoenzyme AII can be useful in HCC and LC diagnosis. The determination of arginase activity in bile may be helpful in monitoring liver graft recipients.

[Evaluation of beta2-microglobuline expression level in gastrointestinal tract tumors due to usefulness as a reference gene in PCR method]. / Ocena poziomu ekspresji beta2-mikroglobuliny w nowotworach przewodu pokarmowego pod wzgledem przydatnosci jako genu referencyjnego w metodzie PCR.

UNLABELLED: Quantitative and semi-quantitative determination of gene expression by PCR plays an important role in studying of tumors initiation and progression mechanisms. Selection of appropriate reference gene is a critical factor influencing the results of gene expression analysis. One of the most commonly used reference genes in PCR is beta2-microglobuline (beta2-M). Recent studies showed however that expression of some common reference genes might be unstable, therefore it is necessary to verify again their usefulness. The aim of the study was to determine the level of beta2-M mRNA in normal and tumor tissues of gastrointestinal tract due to adequate selection of reference gene in gene expression studies. MATERIAL AND METHODS: Samples were taken from 253 patients operated on for gastrointestinal tract tumors: 22 with oral cavity cancer, 12 with benign and 50 with malignant liver tumors, 86 with colorectal cancer, and 83 with metachronous metastases to liver. Also 56 patients with liver cirrhosis were studied, which was treated as pre-tumor state. Together 309 patients were studied. RNA was isolated from tissues by Chomczynski method. The expression level of 12-M was determined by reverse transcriptase PCR (RT-PCR) and given in terms of optical density values. RESULTS: Expression of beta2-M was observed in all studied tissues. There were no differences between normal and tumor tissue. The level of expression of beta2-M was different due to type of studied tissue (oral cavity, liver, colon). CONCLUSIONS: The lack of significant differences in beta2-M expression level in normal and tumor tissues indicated that beta2-M can be used as reference gene in studies of gene expression in gastrointestinal tract tumors. On the other hand differences of beta2-M expression level in different types of tissues point to its tissue specificity and suggest application in PCR of more than one reference gene.

L-arginine as a factor increasing arginase significance in diagnosis of primary and metastatic colorectal cancer.

OBJECTIVE: The usefulness of simultaneous L-arginine and arginase determination in diagnosis of primary and metastatic colorectal cancer. METHODS: L-arginine and arginase were determined before and after surgery in serum from 43 patients with colorectal cancer (CRC), 24 with colorectal cancer liver metastasis (CRCLM), and 39 control subjects (10 patients with non-malignant diseases and 29 healthy blood donors). RESULTS: Preoperative L-arginine concentration in the patient groups was 2-fold higher, whereas arginase activity was over 3- and 6-fold higher in CRC and CRCLM when compared with control. The values of both parameters lowered significantly after surgery. The sensitivity of single parameter in CRC was 79% for L-arginine and 81% for arginase, and in CRCLM it was 83% for each parameter. The combination of L-arginine with arginase improved the sensitivity to 93% and 100% in CRC and CRCLM, respectively. The specificity of L-arginine and arginase calculated for 39 subjects was 87% and 82%. CONCLUSION: Simultaneous determination of L-arginine and arginase increases the value of arginase itself in diagnosis and follow up of patients with CRC and CRCLM.

[New insights into arginase. Part I. Structure and characteristics]. / Nowe oblicza arginazy. Czesc I. Struktura i wlasciwosci.

Arginase (amidinohydrolase, EC 3.5.3.1) is present in all living organisms, i.e. bacteria, yeasts, plants, invertebrates, and vertebrates. In ureolitic organisms, arginase expresses the highest activity in the liver, where it takes part in ammonia detoxifi cation. Arginase activity is much lower in extrahepatic tissues and its physiological function is still poorly understood; however, it seems to be involved in L-arginine metabolism. Arginase is a homotrimer consisting of 20- to 40-kDa subunits acting at a pH of 10 and in the presence of manganese ions. Proline, ornithine, and NG-hydroxy-L-arginine, an intermediate in the biosynthesis of NO, are known as competitive arginase inhibitors. Two arginase isoenzymes, AI (the so-called "hepatic arginase") and AII ("extrahepatic arginase") are present in mammalian tissues. There are signifi cant differences between the isoenzymes regarding their subcellular localization, isoelectric point, substrate affinity, and immunological cross-reactivity. Arginase isoenzymes AI and AII have high substrate specifi city, but the affi nity to L-arginine is higher for isoenzyme AI than AII. Both isoenzymes exist in most tissues and their expressions change depending on the functional state and metabosynlic requirements. Besides differences in the amino-acid content of the arginase isoforms within one or different species, they have highly conserved regions responsible for the structure and catalytic properties of arginase.

[New insights into arginase. Part II. Role in physiology and pathology]. / Nowe oblicza arginazy. Czesc II. Rola w fizjologii i patologii.

Arginase (amidinohydrolase, EC 3.5.3.1) is known as the last enzyme in the urea cycle in the liver, but it is also present in extrahepatic tissues. Arginase hydrolyzes L-arginine to L-ornithine and urea and its biochemical and physiological role varies depending on the organism and tissue. Besides its participation in ammonia detoxification, arginase is involved in the synthesis of polyamines, crucial for the proper course of many metabolic processes, proline, the main connective tissues protein, and glutamates, amino acids which take part in nitric metabolism, important in the nervous system and also a substrate for protein synthesis. The competition of arginase with nitric oxide synthase (NOS) for the common substrate L-arginine indicates its participation in the regulation of nitric oxide (NO) synthesis. The physiological role of arginase and its common occurrence indicate its engagement in many pathologies. Due to its competition with NOS for arginine and its participation in proline synthesis, arginase plays an important role in such diseases as cerebral stroke, trauma, inflammation, and depression, whereas its participation in polyamine synthesis indicates arginase's engagement in the development of neoplastic diseases in the human organism.

[Serum arginase activity in patients with liver cirrhosis and hepatocellular carcinoma]. / Arginaza w surowicy krwi chorych z marskoscia i rakiem watrobowokomórkowym.

Preoperative and postoperative arginase activity was determined in blood serum of 25 patients with liver cirrhosis and 25 patients with hepatocellular carcinoma. The rise of serum arginase activity was observed in the majority of patients before the surgery and the decrease after tumor resection or liver transplantation. The preoperative values of serum arginase activity were similar in both groups of patients. A presence of additional, anionic arginase isoform (All) was demonstrated in serum of studied patients, which was absent in healthy subjects. Thus, our results indicate that the arginase activity cannot differentiate liver cirrhosis and hepatocellular carcinoma. However, arginase isoform All seems to be specific for studied liver diseases.