Browsing by Subject "Apoptosis"

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  • Barok, Mark; Le Joncour, Vadim; Martins, Ana; Isola, Jorma; Salmikangas, Marko; Laakkonen, Pirjo; Joensuu, Heikki (2020)
    The majority of HER2-positive breast or gastric cancers treated with T-DM1 eventually show resistance to this agent. We compared the effects of T-DM1 and ARX788, a novel anti-HER2 antibody-drug conjugate, on cell growth and apoptosis in HER2-positive breast cancer and gastric cancer cell lines sensitive to T-DM1, gastric cancer cell lines resistant to T-DM1, HER2-negative breast cancer cell lines, and T-DM1-resistant xenograft models. ARX788 was effective in T-DM1-resistant in vitro and in vivo models of HER2-positive breast cancer and gastric cancer. ARX788 showed a pronounced growth inhibitory effect on all five HER2-positive cell lines tested, of which two gastric cancer cell lines had acquired resistance to T-DM1. ARX788 evoked more apoptotic events compared to T-DM1. While JIMT-1 and RN-87 xenograft tumors progressed on T-DM1 treatment, all such tumors responded to ARX788, and four out of the six JIMT-1 tumors and nine out of the twelve RN-87 tumors disappeared during the ARX788 treatment. Mice treated with ARX788 survived longer than those treated with T-DM1. The data support evaluation of ARX788 in patients with HER2-positive breast cancer or gastric cancer including cancers that progress during T-DM1 therapy.
  • Humisto, Anu; Herfindal, Lars; Jokela, Jouni; Karkman, Antti; Bjørnstad, Ronja; Choudhury, Romi R.; Sivonen, Kaarina (2015)
    Cyanobacteria are an inspiring source of bioactive secondary metabolites. These bioactive agents are a diverse group of compounds which are varying in their bioactive targets, the mechanisms of action, and chemical structures. Cyanobacteria from various environments, especially marine benthic cyanobacteria, are found to be rich sources for the search for novel bioactive compounds. Several compounds with anticancer activities have been discovered from cyanobacteria and some of these have succeeded to enter the clinical trials. Varying anticancer agents are needed to overcome increasing challenges in cancer treatments. Different search methods are used to reveal anticancer compounds from natural products, but cell based methods are the most common. Cyanobacterial bioactive compounds as agents against acute myeloid leukemia are not well studied. Here we examined our new results combined with previous studies of anti-leukemic compounds from cyanobacteria with emphasis to reveal common features in strains producing such activity. We report that cyanobacteria harbor specific anti-leukemic compounds since several studied strains induced apoptosis against AML cells but were inactive against non-malignant cells like hepatocytes. We noted that particularly benthic strains from the Baltic Sea, such as Anabaena sp., were especially potential AML apoptosis inducers. Taken together, this review and re-analysis of data demonstrates the power of maintaining large culture collections for the search for novel bioactivities, and also how anti-AML activity in cyanobacteria can be revealed by relatively simple and low-cost assays.
  • Soini, Tea; Pihlajoki, Marjut; Kyronlahti, Antti; Andersson, Leif C.; Wilson, David B.; Heikinheimo, Markku (2017)
    Hepatoblastoma, the most common type of pediatric liver cancer, is treated with a combination of surgery and chemotherapy. An essential drug in the treatment of hepatoblastoma is doxorubicin, which in high doses is cardiotoxic. This adverse effect is due to downregulation of cardiac expression of transcription factor GATA4, leading in turn to diminished levels of anti-apoptotic BCL2 (B-cell lymphoma 2) protein family members. GATA4 is also expressed in early fetal liver, but absent from normal postnatal hepatocytes. However, GATA4 is highly expressed in hepatoblastoma tissue. In this study, we assessed the role of GATA4 in doxorubicin-induced apoptosis of hepatoblastoma cells. Herein, we demonstrate that doxorubicin decreases GATA4 expression and alters the expression pattern of BCL2 family members, most profoundly that of BCL2 and BAK, in the HUH6 hepatoblastoma cell line. Silencing of GATA4 by siRNA prior to doxorubicin treatment sensitizes HUH6 cells to the apoptotic effect of this drug by further shifting the balance of BCL2 family members to the pro-apoptotic direction. Specifically, expression levels of anti-apoptotic BCL2 were decreased and pro-apoptotic BID were increased after GATA4 silencing. On the whole, our results indicate that since high endogenous levels of transcription factor GATA4 likely protect hepatoblastoma cells from doxorubicin-induced apoptosis, these cells can be rendered more sensitive to the drug by downregulation of GATA4.
  • Vilander, Laura; Kaunisto, Mari A.; Vaara, Suvi; Pettila, Ville; FINNAKI Study Grp (2017)
    Background: Acute kidney injury (AKI) is a multifactorial syndrome, but knowledge about its pathophysiology and possible genetic background is limited. Recently the first hypothesis-free genetic association studies have been published to explore individual susceptibility to AKI. We aimed to replicate the previously identified associations between five candidate single nucleotide polymorphisms (SNP) in apoptosis-related genes BCL2, SERPINA4, SERPINA5, and SIK3 and the development of AKI, using a prospective cohort of critically ill patients with sepsis/ septic shock, in Finland. Methods: This is a prospective, observational multicenter study. Of 2567 patients without chronic kidney disease and with genetic samples included in the Finnish Acute Kidney Injury (FINNAKI) study, 837 patients had sepsis and 627 patients had septic shock. AKI was defined according to the Kidney Disease: Improving Global Outcomes (KDIGO) criteria, considering stages 2 and 3 affected (severe AKI), stage 0 unaffected, and stage 1 indecisive. Genotyping was done using iPLEX (TM) Assay (Agena Bioscience). The genotyped SNPs were rs8094315 and rs12457893 in the intron of the BCL2 gene, rs2093266 in the SERPINA4 gene, rs1955656 in the SERPINA5 gene and rs625145 in the SIK3 gene. Association analyses were performed using logistic regression with PLINK software. Results: We found no significant associations between the SNPs and severe AKI in patients with sepsis/ septic shock, even after adjustment for confounders. Among patients with septic shock (252 with severe AKI and 226 without AKI (149 with KDIGO stage 1 excluded)), the SNPs rs2093266 and rs1955656 were significantly (odds ratio 0.63, p = 0.04276) associated with stage 2-3 AKI after adjusting for clinical and demographic variables. Conclusions: The SNPs rs2093266 in the SERPINA4 and rs1955656 in the SERPINA5 were associated with the development of severe AKI (KDIGO stage 2-3) in critically ill patients with septic shock. For the other SNPs, we did not confirm the previously reported associations.
  • Vilander, Laura M.; Kaunisto, Mari A.; Vaara, Suvi T.; Pettilä, Ville (BioMed Central, 2017)
    Abstract Background Acute kidney injury (AKI) is a multifactorial syndrome, but knowledge about its pathophysiology and possible genetic background is limited. Recently the first hypothesis-free genetic association studies have been published to explore individual susceptibility to AKI. We aimed to replicate the previously identified associations between five candidate single nucleotide polymorphisms (SNP) in apoptosis-related genes BCL2, SERPINA4, SERPINA5, and SIK3 and the development of AKI, using a prospective cohort of critically ill patients with sepsis/septic shock, in Finland. Methods This is a prospective, observational multicenter study. Of 2567 patients without chronic kidney disease and with genetic samples included in the Finnish Acute Kidney Injury (FINNAKI) study, 837 patients had sepsis and 627 patients had septic shock. AKI was defined according to the Kidney Disease: Improving Global Outcomes (KDIGO) criteria, considering stages 2 and 3 affected (severe AKI), stage 0 unaffected, and stage 1 indecisive. Genotyping was done using iPLEXTM Assay (Agena Bioscience). The genotyped SNPs were rs8094315 and rs12457893 in the intron of the BCL2 gene, rs2093266 in the SERPINA4 gene, rs1955656 in the SERPINA5 gene and rs625145 in the SIK3 gene. Association analyses were performed using logistic regression with PLINK software. Results We found no significant associations between the SNPs and severe AKI in patients with sepsis/septic shock, even after adjustment for confounders. Among patients with septic shock (252 with severe AKI and 226 without AKI (149 with KDIGO stage 1 excluded)), the SNPs rs2093266 and rs1955656 were significantly (odds ratio 0.63, p = 0.04276) associated with stage 2–3 AKI after adjusting for clinical and demographic variables. Conclusions The SNPs rs2093266 in the SERPINA4 and rs1955656 in the SERPINA5 were associated with the development of severe AKI (KDIGO stage 2–3) in critically ill patients with septic shock. For the other SNPs, we did not confirm the previously reported associations.
  • Bolomsky, Arnold; Vogler, Meike; Kose, Murat Cem; Heckman, Caroline A.; Ehx, Gregory; Ludwig, Heinz; Caers, Jo (2020)
    Cell death escape is one of the most prominent features of tumor cells and closely linked to the dysregulation of members of the Bcl-2 family of proteins. Among those, the anti-apoptotic family member myeloid cell leukemia-1 (MCL-1) acts as a master regulator of apoptosis in various human malignancies. Irrespective of its unfavorable structure profile, independent research efforts recently led to the generation of highly potent MCL-1 inhibitors that are currently evaluated in clinical trials. This offers new perspectives to target a so far undruggable cancer cell dependency. However, a detailed understanding about the tumor and tissue type specific implications of MCL-1 are a prerequisite for the optimal (i.e., precision medicine guided) use of this novel drug class. In this review, we summarize the major functions of MCL-1 with a special focus on cancer, provide insights into its different roles in solid vs. hematological tumors and give an update about the (pre)clinical development program of state-of-the-art MCL-1 targeting compounds. We aim to raise the awareness about the heterogeneous role of MCL-1 as drug target between, but also within tumor entities and to highlight the importance of rationale treatment decisions on a case by case basis.
  • Bolomsky, Arnold; Vogler, Meike; Köse, Murat C; Heckman, Caroline A.; Ehx, Grégory; Ludwig, Heinz; Caers, Jo (BioMed Central, 2020)
    Abstract Cell death escape is one of the most prominent features of tumor cells and closely linked to the dysregulation of members of the Bcl-2 family of proteins. Among those, the anti-apoptotic family member myeloid cell leukemia-1 (MCL-1) acts as a master regulator of apoptosis in various human malignancies. Irrespective of its unfavorable structure profile, independent research efforts recently led to the generation of highly potent MCL-1 inhibitors that are currently evaluated in clinical trials. This offers new perspectives to target a so far undruggable cancer cell dependency. However, a detailed understanding about the tumor and tissue type specific implications of MCL-1 are a prerequisite for the optimal (i.e., precision medicine guided) use of this novel drug class. In this review, we summarize the major functions of MCL-1 with a special focus on cancer, provide insights into its different roles in solid vs. hematological tumors and give an update about the (pre)clinical development program of state-of-the-art MCL-1 targeting compounds. We aim to raise the awareness about the heterogeneous role of MCL-1 as drug target between, but also within tumor entities and to highlight the importance of rationale treatment decisions on a case by case basis.
  • Kuuliala, Krista; Louhimo, Johanna; Meri, Seppo (2020)
    Ohjelmoitunutta solukuolemaa tarvitaan elinten kehittymisessä, kudosten homeostaasin ja uusiutumiskyvyn ylläpidossa sekä puolustussolujen toiminnan säätelyssä. Sen ainoana muotona pidettiin pitkään kaspaasientsyymien säätelemää, tulehdusta lietsomatonta apoptoosia. Nykyään tiedetään, ettei tulehdus- ja soluvauriosignaalien laukaisema nekroosikaan ole vain kontrolloimaton solutuho, vaan siitä on aktivoitumiskykyisiä geneettisesti ohjelmoituneita muotoja, joista tunnetuin on nekroptoosi eli tulehduksellisesti ohjelmoitunut nekroottinen solukuolema. Keskeiset solunsisäisessä nekroptoosisignaloinnissa toimivat proteiinit ovat RIP (receptor-interacting protein kinase) 1, RIP3 ja MLKL (mixed lineage kinase domain-like protein). Niiden aktivoituessa, esimerkiksi virus- (muun muassa koronavirus) tai bakteeri-infektion yhteydessä, nekroptoottisesta solusta vapautuu vaarasignaalimolekyylejä. Nekroptoosi aiheuttaa tulehdusreaktion, jolla voi kudostuhon ohella olla regeneratiivisia vaikutuksia. Nekroptoositutkimus on avannut uusia näkymiä immuunipuolustuksen keinoihin ja tulehduksellisten sairauksien patogeneesiin sekä mahdollisuuksiin vaikuttaa näihin.
  • Vilander, Laura M. (2020)
    Genetic predisposition to acute kidney injury in critically ill adults. Laura Vilander, Helsingin yliopisto.
  • Singh, François; Zoll, Joffrey; Duthaler, Urs; Charles, Anne‑Laure; Panajatovic, Miljenko V.; Laverny, Gilles; McWilliams, Thomas; Metzger, Daniel; Geny, Bernard; Krähenbühl, Stephan; Bouitbir, Jamal (2019)
    Statins inhibit cholesterol biosynthesis and lower serum LDL-cholesterol levels. Statins are generally well tolerated, but can be associated with potentially life-threatening myopathy of unknown mechanism. We have shown previously that statins impair PGC-1β expression in human and rat skeletal muscle, suggesting that PGC-1β may play a role in statininduced myopathy. PGC-1β is a transcriptional co-regulator controlling the expression of important genes in mitochondrial biogenesis, antioxidative capacity and energy metabolism. The principle aim of the current study was to investigate the interaction between atorvastatin and PGC-1β in more detail. We therefore treated wild-type mice and mice with selective skeletal muscle knockout of PGC-1β (PGC-1β(i)skm−/− mice) with oral atorvastatin (5 mg/kg/day) for 2 weeks. At the end of treatment, we determined body parameters, muscle function, structure, and composition as well as the function of muscle mitochondria, mitochondrial biogenesis and activation of apoptotic pathways. In wild-type mice, atorvastatin selectively impaired mitochondrial function in glycolytic muscle and caused a conversion of oxidative type IIA to glycolytic type IIB myofibers. Conversely, in oxidative muscle of wild-type mice, atorvastatin enhanced mitochondrial function via activation of mitochondrial biogenesis pathways and decreased apoptosis. In PGC-1β(i)skm−/− mice, atorvastatin induced a switch towards glycolytic fibers, caused mitochondrial dysfunction, increased mitochondrial ROS production, impaired mitochondrial proliferation and induced apoptosis in both glycolytic and oxidative skeletal muscle. Our work reveals that atorvastatin mainly affects glycolytic muscle in wild-type mice and demonstrates the importance of PGC-1β for oxidative muscle integrity during long-term exposure to a myotoxic agent.
  • Kasica, Natalia; Podlasz, Piotr; Sundvik, Maria; Tamas, Andrea; Reglodi, Dora; Kaleczyc, Jerzy (2016)
    Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide, with known antiapoptotic functions. Our previous in vitro study has demonstrated the ameliorative role of PACAP-38 in chicken hair cells under oxidative stress conditions, but its effects on living hair cells is now yet known. Therefore, the aim of the present study was to investigate in vivo the protective role of PACAP-38 in hair cells found in zebrafish (Danio rerio) sense organs-neuromasts. To induce oxidative stress the 5-day postfertilization (dpf) zebrafish larvae were exposed to 1.5 mM H2O2 for 15 min or 1 h. This resulted in an increase in caspase-3 and p-38 MAPK level in the hair cells as well as in an impairment of the larvae basic behavior. To investigate the ameliorative role of PACAP-38, the larvae were incubated with a mixture of 1.5 mM H2O2 and 100 nM PACAP-38 following 1 h preincubation with 100 nM PACAP-38 only. PACAP-38 abilities to prevent hair cells from apoptosis were investigated. Whole-mount immunohistochemistry and confocal microscopy analyses revealed that PACAP-38 treatment decreased the cleaved caspase-3 level in the hair cells, but had no influence on p-38 MAPK. The analyses of basic locomotor activity supported the protective role of PACAP-38 by demonstrating the improvement of the fish behavior after PACAP-38 treatment. In summary, our in vivo findings demonstrate that PACAP-38 protects zebrafish hair cells from oxidative stress by attenuating oxidative stress-induced apoptosis.
  • Khirug, Stanislav; Soni, Shetal; Saez Garcia, Marta; Tessier, Marine; Zhou, Liang; Kulesskaya, Natalia; Rauvala, Heikki; Lindholm, Dan; Ludwig, Anastasia; Molinari, Florence; Rivera, Claudio (2021)
    A striking result from epidemiological studies show a correlation between low alcohol intake and lower incidence for ischemic stroke and severity of derived brain injury. Although reduced apoptosis and inflammation has been suggested to be involved, little is known about the mechanism mediating this effect in vivo. Increase in intracellular chloride concentration and derived depolarizing GABAAR-mediated transmission are common consequences following various brain injuries and are caused by the abnormal expression levels of the chloride cotransporters NKCC1 and KCC2. Downstream pro-apoptotic signaling through p75NTR may link GABAA depolarization with post-injury neuronal apoptosis. Here, we show that changes in GABAergic signaling, Cl− homeostasis, and expression of chloride cotransporters in the post-traumatic mouse brain can be significantly reduced by administration of 3% ethanol to the drinking water. Ethanol-induced upregulation of KCC2 has a positive impact on neuronal survival, preserving a large part of the cortical peri-infarct zone, as well as preventing the massive post-ischemic upregulation of the pro-apoptotic protein p75NTR. Importantly, intracortical multisite in vivo recordings showed that ethanol treatment could significantly ameliorate stroke-induced reduction in cortical activity. This surprising finding discloses a pathway triggered by low concentration of ethanol as a novel therapeutically relevant target.
  • Teirilä, Laura; Heikkinen-Eloranta, Jenni; Kotimaa, Juha; Meri, Seppo; Lokki, A. Inkeri (2019)
    Preeclampsia is a serious vascular complication of the human pregnancy, whose etiology is still poorly understood. In preeclampsia, exacerbated apoptosis and fragmentation of the placental tissue occurs due to developmental qualities of the placental trophoblast cells and/or mechanical and oxidative distress to the syncytiotrophoblast, which lines the placental villi. Dysregulation of the complement system is recognized as one of the mechanisms of the disease pathology. Complement has the ability to promote inflammation and facilitate phagocytosis of placenta-derived particles and apoptotic cells by macrophages. In preeclampsia, an overload of placental cell damage or dysregulated complement system may lead to insufficient clearance of apoptotic particles and placenta-derived debris. Excess placental damage may lead to sequestration of microparticles, such as placental vesicles, to capillaries in the glomeruli of the kidney and other vulnerable tissues. This phenomenon could contribute to the manifestations of typical diagnostic symptoms of preeclampsia: proteinuria and new-onset hypertension. In this review we propose that the complement system may serve as a regulator of the complex tolerance and clearance processes that are fundamental in healthy pregnancy. It is therefore recommended that further research be conducted to elucidate the interactions between components of the complement system and immune responses in the context of complicated and healthy pregnancy.
  • Torregrosa-Munumer, Ruben; Vara, Elena; Fernandez-Tresguerres, Jesus Angel; Gredilla, Ricardo (2021)
    Purpose Aging is known to play a critical role in the etiopathogenesis of several diseases. Among them, cardiovascular disorders are especially relevant since they are becoming the first cause of death in western countries. Resveratrol is a polyphenolic compound that has been shown to exert beneficial effects at different levels, including neuronal and cardiovascular protection. Those effects of resveratrol are related, at least in part, to its antioxidant and anti-inflammatory properties. In the current investigation we were interested in exploring whether the positive effects of resveratrol at cardiac level were taking place even when the supplementation started in already old animals. Methods Old male rats were supplemented with resveratrol during 10 weeks. Using RT-PCR, we analyzed the effects of resveratrol supplementation on the expression of different genes related to inflammation, oxidative stress and apoptosis in rat heart. Results Resveratrol reverted age-related changes in inflammatory, oxidative and apoptotic markers in the rat heart. Among others, the expression of two major inflammatory markers, INF-gamma and TNF-alpha and two oxidative markers, heme oxygenase-1 and nitric oxide synthase, were increased with aging, and resveratrol supplementation reduced the level of some of these to those observed in the heart of young animals. Moreover, age-related changes in apoptotic markers in rat heart tend to be also reverted by resveratrol treatment. Conclusion Our results suggest that resveratrol might exert beneficial effects as an anti-aging compound to revert age-related changes in cardiac function.
  • Adem, Jemal; Eray, Mine; Eeva, Jonna; Nuutinen, Ulla; Pelkonen, Jukka (2017)
    CD40 is a cell surface receptor which belongs to tumor necrosis factor receptor (TNFR) family members. It transmits signals that regulate diverse cellular responses such as proliferation, differentiation, adhesion molecule expression and apoptosis. Unlike other TNFR family members (TRAIL-R, Fas-R and TNFR1), the CD40 cytoplasmic tail lacks death domain. However, CD40 is capable of inducing apoptosis in different types of cancer cells including lymphoma. The apoptotic effect of CD40 is linked to the involvement of Fas, TRAIL or receptor interacting protein 1 (RIP1) kinase. We have previously shown that CD40 activation has anti-apoptotic or apoptotic effect in follicular lymphoma (FL) cell lines. In this study, we investigated the mechanism by which CD40 mediates apoptosis in a follicular lymphoma cell line, HF4.9. We show here that CD40-induced apoptosis was dependent on caspase-8 activation because caspase-8 specific inhibitor, Z-IETD-FMK completely prevented apoptosis. Therefore, the involvement of TRAIL, Fas and RIP1 in caspase-8 activation was examined. The exogenous TRAIL-induced apoptosis was fully prevented by anti-TRAIL neutralizing antibody. However, the antibody had no effect on CD40-induced apoptosis indicating that CD40 did not induce the expression of endogenous TRAIL in HF4.9 cells. Moreover, the cells were not sensitive to Fas-mediated apoptosis. Interestingly, RIP1 specific inhibitor, necrostatin-1 decreased CD40-induced apoptosis, which showed that RIP1 has a role in caspase-8 activation. In conclusion, the survival or apoptotic effects of CD40-mediated signaling might be related to the differentiation stages of FL cells.
  • Mutanen, Annika; Barrett, Meredith; Feng, Yongjia; Lohi, Jouko; Rabah, Raja; Teitelbaum, Daniel H.; Pakarinen, Mikko P. (2019)
    Background: Although serial transverse enteroplasty (STEP) improves function of dilated short bowel, a significant proportion of patients require repeat surgery. To address underlying reasons for unsuccessful STEP, we compared small intestinal mucosal characteristics between initial and repeat STEP procedures in children with short bowel syndrome (SBS). Methods: Fifteen SBS children, who underwent 13 first and 7 repeat STEP procedures with full thickness small bowel samples at median age 1.5 years (IQR 0.7-3.7) were included. The specimens were analyzed histologically for mucosal morphology, inflammation and muscular thickness. Mucosal proliferation and apoptosis was analyzed with MIB1 and Tunel immunohistochemistry. Results: Median small bowel length increased 42% by initial STEP and 13% by repeat STEP (p - 0.05), while enteral caloric intake increased from 6% to 36% (p 0.07) during 14 (12-42) months between the procedures. Abnormal mucosal inflammation was frequently observed both at initial (69%) and additional STEP (86%, p 0.52) surgery. Villus height, crypt depth, enterocyte proliferation and apoptosis as well as muscular thickness were comparable at first and repeat STEP (p>0.05 for all). Patients, who required repeat STEP tended to be younger (p 0.057) with less apoptotic crypt cells (p-0.031) at first STEP. Absence of ileocecal valve associated with increased intraepithelial leukocyte count and reduced crypt cell proliferation index (p Conclusions: No adaptive mucosal hyperplasia or muscular alterations occurred between first and repeat STEP. Persistent inflammation and lacking mucosal growth may contribute to continuing bowel dysfunction in SBS children, who require repeat STEP procedure, especially after removal of the ileocecal valve. (C) 2018 Elsevier Inc. All rights reserved.
  • Tuuminen, Raimo; Holmstrom, Emil; Raissadati, Alireza; Saharinen, Pipsa; Rouvinen, Eeva; Krebs, Rainer; Lemstrom, Karl B. (2016)
    Background: In transplantation-associated ischemia/reperfusion injury (Tx-IRI), tumor necrosis factor alpha and damage-associated molecular patterns promote caspase-8 and -9 apoptotic and receptor-interacting protein kinase-1 and-3 (RIPK1/3) necroptotic pathway activation. The extent of cell death and the counterbalance between apoptosis and regulated necrosis eventually determine the immune response of the allograft. Although simvastatin prevents Tx-IRI, its role in apoptotic and necroptotic activity remains unsolved. Methods: Rat allograft donors and recipients were treated with a single-dose of simvastatin 2 h prior to allograft procurement and reperfusion, respectively. Intragraft caspase-3, -8, and -9 and RIPK1 and-3 mRNA expression was analysed by quantitative RT-PCR and protein activity measured by immunohistochemistry and luminescent assays 6 h after reperfusion. Lactate and lactate dehydrogenase (LDH) levels were analysed from allograft recipient and from hypoxic endothelial cell cultures having treated with activated simvastatin. Results: When compared to without cold ischemia, prolonged 4-hour cold ischemia significantly enhanced intragraft mRNA expression of caspase-3 and-9, and RIPK1 and -3, and elevated protein activity of caspase-9 and RIPK1 in the allografts. Simvastatin pretreatment decreased mRNA expression of caspase-3 and -9, and RIPK1 and-3 and protein activity of caspase-9 and RIPK1 in the allografts. Intragraft caspase-8 mRNA expression remained constant regardless of cold ischemia or simvastatin pretreatment. Simvastatin pretreatment attenuated lactate and LDH levels, both in the allograft recipients and in hypoxic endothelial cell cultures. Conclusions: The beneficial effects of simvastatin pretreatment in cardiac allograft IRI may involve prevention of apoptosis and necroptosis. (C) 2016 Elsevier B.V. All rights reserved.