Synergic prooxidant,apoptotic and TRPV1 channel activator effects of alpha-lipoic acid and cisplatin in MCF-7 breast cancer cells

Background: Resistance to cisplatin (Cisp) in the treatment of breast cancer is a major obstacle. Alpha-lipoic acid (ALA) has both antioxidant and oxidant properties. ALA has been used on stimulation mechanisms of apoptosis and oxidative stress in the treatment of cancer with a combination of chemotherapeutic agents, although its role on molecular mechanisms in the cancer cells has not been clarified yet. The aim of this study was to evaluate if a combination therapy of ALA with Cisp can alter the effect of this chemotherapy drug in the MCF-7 breast cancer cells.

Materials: The MCF-7 cells were divided into four treatment groups as control, Cisp (0.025?mM), ALA (0.05?mM), and Cisp?+?ALA.

Results: Apoptosis, mitochondrial membrane depolarization, reactive oxygen species (ROS) production, lipid peroxidation, PARP1, caspase 3 and 9 expression levels are increased through activating TRPV1 in the cells by the Cisp and ALA treatments, although cell viability, reduced glutathione and glutathione peroxidase (GPx) values were decreased by the treatments. The Cisp and ALA-induced increase of intracellular free Ca²⁺ concentration was decreased with the TRPV1 blocker, capsazepine.

Conclusions: Apoptosis and oxidant effects of Cisp were increased by activation of TRPV1 channels, but its action on the values was further increased by the ALA treatment. Combination therapy of ALA and Cisp could be used as an effective strategy in the treatment of breast cancer.     

Effect of recombinant erythropoietin on ischemia–reperfusion-induced apoptosis in rat liver

Ischemia–reperfusion (I/R) cannot be avoided in liver transplantation procedures, and apoptosis is a central mechanism of cell death after liver reperfusion. Protective effect of recombinant erythropoietin (rhEPO) on liver apoptosis has not been clearly investigated. This work investigated intraportal (IP) rhEPO-protective effect in a rat model of hepatic I/R-induced apoptosis and its appropriated time and dose of administration. Eight groups were included (n = 10/group): sham-operated, I/R (45 min ischemia and 2 h reperfusion), preconditioned rhEPO I/R (24 h or 30 min before ischemia), and postconditioned rhEPO I/R (before reperfusion) using two different rhEPO doses (1,000 and 5,000 IU/kg). When compared with the sham-operated group, the I/R group showed significant increase of serum levels of aspartate and alanine aminotransferases (AST, ALT), hepatic caspase-9 activity(894.99 ± 176.90 relative fluorescence units (RFU)/mg/min versus 458.48 ± 82.96 RFU/mg/min), and Fas ligand (FasL) expression, histopathological damages, and significant decrease in the antiapoptotic Bcl-xL/apoptotic Bax ratio(0.38 ± 0.21 versus 3.35 ± 0.77) rhEPO-improved ALT and AST but failed to reduce FasL expression in all groups compared with the I/R group. Thirty minutes and 24 h preconditioning with rhEPO (1,000 IU/kg) increased Bcl-xL/Bax ratio and reduced caspase-9 activity, and the same effect was observed when higher dose was given 24 h before ischemia. Preconditioning was more effective than postconditioning in improving caspase-9 activity, and no dose-dependent effect was observed. In conclusion, single IP rhEPO injection 30 min before ischemia has an advantage over rhEPO postconditioning in improving post-hepatic I/R-induced apoptosis with no additional time- and dose-dependent effects which may provide potentially useful guide in liver transplantation procedures.     

Recombinant human erythropoietin attenuates hepatic injury induced by ischemia/reperfusion in an isolated mouse liver model

Introduction Apoptosis is a central mechanism of cell death following reperfusion of the ischemic liver. Recombinant human erythropoietin (rhEPO) have an important role in the treatment of myocardial ischemia/reperfusion (I/R) injury, by preventing apoptosis. The aim of the study was to investigate the effect of different regimens of rhEPO in preventing apoptosis following I/R-induced hepatic injury. Material and methods Isolated mouse livers were randomly divided into five groups: (1) control group, perfused for the whole study period (105 min); (2) 30-min perfusion followed by 90 min of ischemia and 15 min of reperfusion; (3), (4) and (5) like group 2, but with administration of rhEPO 5,000 units/kg i.p. at 30 min, 24 h, or both 30 min and 24 h respectively, before induction of ischemia. Perfusate liver enzyme levels and intrahepatic caspase-3 activity were measured, and apoptotic cells were identified by morphological criteria, TUNEL assay, and immunohistochemistry for caspase-3. Using immunoblot the expression of the proapoptotic JNK and inhibitor of NFκB (IκBα) were also evaluated. von Willebrand factor (vWF) immunohistochemistry was used as a marker of endothelial cells. Results Compared to the I/R livers, all 3 rhEPO pretreated groups showed: a significant reduction in liver enzyme levels (P < 0.05) and intrahepatic caspase-3 activity (P < 0.05), fewer apoptotic hepatocytes (P < 0.05) and positive vWF staining in numerous endothelial cells lining the sinusoids. EPO decreased JNK phosphorylation and the degradation of the inhibitor of NFκB (IκBα) during I/R. There was no added benefit of the multiple- over the single-dose rhEPO regimen. Conclusion Pretreatment with one dose of rhEPO can attenuate post-I/R hepatocyte apoptotic liver damage. NFκB and JNK activation is likely to play a pivotal role in the pathophysiology of I/R hepatic injury and might have a key role in EPO-mediated protective effects. This effect is associated with the increase in sinusoidal vWF immunostaining suggests an additional effect of rhEPO in liver angiogenesis recovery. These findings have important implications for the potential use of rhEPO in I/R injury during liver transplantation. Edith Hochhauser and Orit Pappo are first two coauthors.     

Knockout of sialidase and pro-apoptotic genes in Chinese hamster ovary cells enables the production of recombinant human erythropoietin in fed-batch cultures

Sialic acid, a terminal monosaccharide present in N-glycans, plays an important role in determining both the in vivo half-life and the therapeutic efficacy of recombinant glycoproteins. Low sialylation levels of recombinant human erythropoietin (rhEPO) in recombinant Chinese hamster ovary (rCHO) cell cultures are considered a major obstacle to the production of rhEPO in fed-batch mode. This is mainly due to the accumulation of extracellular sialidases released from the cells. To overcome this hurdle, three sialidase genes (Neu1, 2, and 3) were initially knocked-out using the CRISPR/Cas9-mediated large deletion method in the rhEPO-producing rCHO cell line. Unlike wild type cells, sialidase knockout (KO) clones maintained the sialic acid content and proportion of tetra-sialylated rhEPO throughout fed-batch cultures without exhibiting a detrimental effect with respect to cell growth and rhEPO production. Additional KO of two pro-apoptotic genes, BAK and BAX, in sialidase KO clones (5X KO clones) further improved rhEPO production without any detrimental effect on sialylation. On day 10 in fed-batch cultures, the 5X KO clones had 1.4-times higher rhEPO concentration and 3.0-times higher sialic acid content than wild type cells. Furthermore, the proportion of tetra-sialylated rhEPO on day 10 in fed-batch cultures was 42.2–44.3% for 5X KO clones while it was only 2.2% for wild type cells. Taken together, KO of sialidase and pro-apoptotic genes in rCHO cells is a useful tool for producing heavily sialylated glycoproteins such as rhEPO in fed-batch mode.     

Systemically delivered Erythropoietin transiently enhances adult hippocampal neurogenesis

Erythropoietin is a primary regulator of erythropoiesis in the hematopoietic system. More recently erythropoietin has been shown to play a role in neurogenesis and provide neurotrophic support to injured CNS tissue. Here the effects of large systemic doses of erythropoietin on basal levels of adult hippocampal neurogenesis in mice were examined. A 7-day period of recombinant human erythropoietin (rhEPO) administration increased the number of bromodeoxyuridine [BrdU(+)] cells in the sub-granular zone (SGZ) by 30%. Analysis of cell phenotype revealed an increase in mitotically active doublecortin(+) neuronal progenitor cells and glial fibrillary acidic protein(+) SGZ radial astrocytes/stem cells but not mature S100beta(+) astrocytes. These effects appeared to be mediated, in part, by mitogen-activated protein kinase signaling and potentially regulated by suppressor of cytokine signaling-3. Hippocampal levels of phosphorylated extracellular signal-related kinase 42/44 and suppressor of cytokine signaling-3 were increased 2-6 h after a single systemic rhEPO injection. However, rhEPO had no observed effect on the long-term survival of new born cells in the SGZ, with similar numbers of BrdU(+) cells and BrdU(+)/NeuN(+) co-labeled cells after 4 weeks. Therefore, systemically delivered rhEPO transiently increased adult hippocampal neurogenesis without any apparent long-term effects.     

Cytogenetic evaluation of arsenic trioxide toxicity in Sprague-Dawley rats

Acute exposure to arsenic trioxide has been reported to induce death and/or multiple organ damage with symptoms including nausea, vomiting, diarrhea, gastrointestinal hemorrhage, cerebral edema, tachycardia, dysrhythmias and hypovolemic shock. Its toxic effects are due to its ability to bind to sulfhydryl groups of proteins and to inhibit energy production. Although the chronic exposure to arsenic trioxide has been linked to various types of cancer, such as skin, liver, lung, bladder and kidney neoplasms, studies of its carcinogenic potential in animals have not been conclusive. In this study, we investigated the genotoxic potential of arsenic trioxide in bone-marrow cells obtained from Sprague-Dawley rats; using chromosomal aberrations (CA), mitotic index (MI) and micronuclei (MN) formation as the toxicological endpoints. Four groups of six male rats each, weighing approximately 60+/-2 g per rat, were injected intraperitoneally, once a day for 5 days with doses of 5, 10, 15 and 20 mg/kg body weight (BW) of arsenic trioxide dissolved in distilled water. A control group was also made of six animals injected with distilled water without chemical. All the animals were sacrificed at the end of the treatment period. Chromosome and micronuclei preparation was obtained from bone-marrow cells following standard protocols. Arsenic trioxide exposure significantly increased the number of structural chromosomal aberrations, the frequency of micronucleated cells and decreased the mitotic index in treated groups when compared with the control group. Our results demonstrate that arsenic trioxide has a clastogenic/genotoxic potential as measured by the bone-marrow CA and MN tests in Sprague-Dawley rats.     

髓芯减压联合自体骨髓间充质干细胞治疗45例股骨头无菌性坏死的临床疗效分析

目的：观察髓芯减压联合自体骨髓间充质干细胞治疗45例股骨头无菌性坏死的的临床效果及安全性。方法：采取随机原则对2011年11月至2012年3月期间在我院骨科住院治疗的54例患者分成两组,对照组接受髓芯减压治疗,治疗组接受髓芯减压联合自体骨髓间充质干细胞移植治疗治疗。结果：术后随访一年,治疗组的Harris评分为（86．78±9．48）分,对照组为（71．18±8．36）分,两组相互比较差异有统计学意义（P〈0．05）;治疗组的优良率为84．38％,对照组的优良率为55．17％,治疗组的优良率明显高于对照组,差异有统计学意义（P〈0．01）。结论：髓芯减压联合自体骨髓间充质干细胞移植治疗股骨头无菌性坏死效果显著,优于单纯髓芯减压治疗。     

Erythropoietin pretreatment protects against acute chemotherapy toxicity in isolated rat hearts

The use of chemotherapeutic agents, such as anthracycline or trastuzumab, in oncology is limited by their cardiac toxicity. Recent experimental studies suggest that recombinant human erythropoietin (rhEPO) can be considered as a protective agent because its administration protects against cardiac ischemic injury, improving functional recovery, and reducing cell death. The aim of this study was to investigate whether pretreatment by rhEPO protects against acute cardiotoxicity induced by doxorubicin and trastuzumab, using the isolated rat heart model. Rats were treated with rhEPO (5000 IU/kg, intraperitoneally [i.p.]) or vehicle. One hour later, hearts were isolated and retrogradely perfused at constant flow. Following 20 mins of stabilization, hearts were perfused for 60 mins with modified-Krebs solution containing 6 mg/l doxorubicin or 10 mg/l trastuzumab. Hearts receiving doxorubicin were paced; those receiving trastuzumab were unpaced. Control hearts were perfused with modified-Krebs solution only. Doxorubicin exposure decreased left ventricular developed pressure (LVDP; approximately -40% of baseline) and increased end diastolic pressure (EDP; approximately +390% of baseline) and coronary perfusion pressure (CPP; approximately +70% of baseline). Incidence of ventricular tachycardia or fibrillation (VT/VF) was also significantly enhanced (86% vs. 0% in control group). Trastuzumab exposure increased CPP and EDP (approximately +70% of baseline for the both) without affecting LVDP. Prior rhEPO treatment significantly prevented doxorubicin-induced deleterious effects on LVDP, EDP, and VT/VF incidence. rhEPO administration also prevented trastuzumab-induced deleterious effects on CPP and EDP. This study shows that pretreatment by rhEPO protects myocardium against functional damage and electrophysiologic injury induced by acute doxorubicin or trastuzumab exposure. Further investigations are required to elucidate the precise mechanisms involved.     

Effects of beta-adrenergic stimulation on bone-marrow function in normal and sublethally irradiated mice. I. The effect of isoproterenol on cAMP content in bone-marrow cells in vivo and in vitro.

The effect of isoproterenol (IPR) on bone-marrow cAMP content was investigated in vivo and in vitro. In unirradiated CFW mice, the bone-marrow cAMP content was found to be elevated by the administration of noradrenaline, adrenaline and isoproterenol. After IPR administration, the increase in cAMP was biphasic with maxima at 1 and 15 min. An increase in cAMP content was also noted in bone-marrow of sublethally-irradiated mice, but no further increase was observed 15 min after the administration of IPR. Elevation of cAMP by either IPR or radiation was prevented by pretreatment with the beta-adrenergic blocking agent--propranolol. IPR was also effective in increasing the cAMP content when added to suspension of bone-marrow cells. This effect was abolished by propranolol. IPR did not increase cAMP levels in bone-marrow cells isolated from irradiated animals. The results suggest that the differentiated bone-marrow cells have beta-adrenergic receptors.     

Erythropoietin Amplifies Stroke-Induced Oligodendrogenesis in the Rat

Background

Erythropoietin (EPO), a hematopoietic cytokine, enhances neurogenesis and angiogenesis during stroke recovery. In the present study, we examined the effect of EPO on oligodendrogenesis in a rat model of embolic focal cerebral ischemia.

Methodology and Principal Findings

Recombinant human EPO (rhEPO) at a dose of 5,000 U/kg (n = 18) or saline (n = 18) was intraperitoneally administered daily for 7 days starting 24 h after stroke onset. Treatment with rhEPO augmented actively proliferating oligodendrocyte progenitor cells (OPCs) measured by NG2 immunoreactive cells within the peri-infarct white matter and the subventricular zone (SVZ), but did not protect against loss of myelinating oligodendrocytes measured by cyclic nucleotide phosphodiesterase (CNPase) positive cells 7 days after stroke. However, 28 and 42 days after stroke, treatment with rhEPO significantly increased myelinating oligodendrocytes and myelinated axons within the peri-infarct white matter. Using lentivirus to label subventricular zone (SVZ) neural progenitor cells, we found that in addition to the OPCs generated in the peri-infarct white matter, SVZ neural progenitor cells contributed to rhEPO-increased OPCs in the peri-infarct area. Using bromodeoxyuridine (BrdU) for birth-dating cells, we demonstrated that myelinating oligodendrocytes observed 28 days after stroke were derived from OPCs. Furthermore, rhEPO significantly improved neurological outcome 6 weeks after stroke. In vitro, rhEPO increased differentiation of adult SVZ neural progenitor cells into oligodendrocytes and enhanced immature oligodendrocyte cell proliferation.

Conclusions

Our in vivo and in vitro data indicate that EPO amplifies stroke-induced oligodendrogenesis that could facilitate axonal re-myelination and lead to functional recovery after stroke.     

骨髓刺激后骨髓干细胞移植治疗下肢缺血的研究进展

自体骨髓干细胞移植治疗下肢缺血的研究是近年来令人关注的领域,取得一定的进展,但疗效有待进一步提高。骨髓刺激后自体骨髓干细胞移植治疗肢体缺血取得了较好的疗效;同时它具有抽取骨髓血少、获得细胞量多且安全性高的优点。但其具体作用机理尚不十分明确。内皮祖细胞数量的增加与质量的提高、局部微环境的改变可能是骨髓刺激后自体骨髓干细胞移植促进肢体缺血改善的机制。     

Dynamic Control of Oligosaccharide Modification in the Mammary Gland: Linking Recombinant Human Erythropoietin

We analyzed two transgenic mouse lines that secrete rhEPO in their milk to assess the dynamic control of N-linked oligosaccharides. Since pharmaceutically available epoetin α and β are produced in CHO cells, we compared transgenic mammary gland-derived rhEPO to its CHO cell-derived counterpart. The major glycosyltransferases that determine the N-oligosaccharides patterns of rhEPO include N-acetylglycosaminyltransferase (GnT) and α1,3/4 fucosyltransferase (Fuc-TIV), GnT-III, -V and Fuc-TIV expression in the mouse mammary gland is significantly higher than that in Chinese hamster ovary (CHO)-derived cells, where the protein is not detectable. The data suggest that N-linked sugar chain patterns of recombinant glycoproteins, produced by the mammary gland differ, since GnT-III alters the sugar pattern extensively. In our experiments, rhEPO produced by the transgenic mice contains more tetra-acidic oligosaccharide structures than epoetin α derived from CHO cells, a rhEPO that is widely used therapeutically. Accordingly, we examined milk-derived rhEPO activity, both in vitro and in vivo. The rhEPO protein purified from the milk of mammary glands upregulates the EPO receptor-mediated expression of the STAT5 gene in MCF-7 cells in a dose-dependent manner, similar to the effects of epoetin α. Furthermore, direct injection of rhEPO into the mouse tail vein leads to an increase in the levels of blood components, such as red blood cells and platelets. In light of these findings, we suggest that the mammary glands of transgenic animals provide a sufficient environment to generate rhEPO with post-translational modifications for biopharmaceutical use. These authors are equal contributors to this work.     

EPO/EPOR 在非小性细胞肺癌发展中的作用研究

目的：研究促红细胞生成素（erythropoietin, EPO）及其受体（EPOR）在非小性细胞肺癌中的生物学作用。方法：收集27 例非小 性细胞肺癌（NSCLC）,免疫组织化学方法检测肺癌组织中EPO 和EPOR的表达;观察人源重组EPO（rhEPO）对HCC15 和 HCC1819 细胞活力和细胞周期的影响;分析缺氧对NSCLC细胞EPO 及EPOR 表达的影响。结果：27例非小细胞肺癌的组织标 本中13 例表达EPO,表达率为48 %,25 例表达EPOR,表达率为92 %。rhEPO明显增加了高表达EPOR 的HCC1819 细胞克隆 数,而对低表达EPOR 的HCC15 细胞的克隆形成没有影响。rhEPO增强了HCC1819 的细胞活力,但以siRNA干涉HCC1819 EPOR后,EPO对HCC1819 细胞活力增强作用消失。rhEPO 明显增加了HCC1819 细胞的细胞周期。缺氧促进了HCC1819 细胞的 EPO 的表达,增强了细胞活力。结论：EPO 和EPOR在非小性细胞肺癌中表达增高,EPO 通过EPOR 促进了NSCLC 细胞的增殖, 缺氧诱导了NSCLC 细胞EPO的表达。     

Induction and persistence of micronuclei, sister-chromatid exchanges and chromosomal aberrations in splenocytes and bone-marrow cells of rats exposed to ethylene oxide

Studies on the induction and persistence of ethylene oxide (EO) induced chromosomal alterations in rat bone-marrow cells and splenocytes following in vivo exposure were carried out. Rats were exposed to ethylene oxide either chronically by inhalation (50-200ppm, 4 weeks, 5 days/week, 6h/day) or acutely by intraperitoneal injection (i.p.) at dose levels of 50-100ppm.Spontaneous- and induced-frequencies of micronuclei (MN), sister-chromatid exchanges (SCEs) and chromosomal aberrations were determined in rat bone-marrow cells, and in splenocytes following in vitro mitogen stimulation. Unstable chromosomal aberrations were studied in whole genome using standard Giemsa staining technique and fluorescence in situ hybridisation using probe for chromosome #2 was employed to detect chromosome translocations.Following chronic exposure, the cytogenetic analyses were carried out at days 5 and 21 in rat splenocytes, to study the induction and persistence of sister-chromatid exchanges. Following chronic exposure, ethylene oxide was effective in inducing SCEs, and markedly cells with high frequency SCEs were observed and they in-part persisted until day 21 post-exposure. However, no significant effect was observed in rat splenocytes for induction of MN and chromosomal aberrations. Following acute exposure, both SCEs and MN were increased significantly in rat bone-marrow cells as well as splenocytes.In conclusion, this study indicates that ethylene oxide at the concentrations employed by intraperitoneal injection or inhalation in adult rats is mutagenic and can induce both SCEs and MN.     

Partial recovery of dopaminergic pathway after graft of adult mesenchymal stem cells in a rat model of Parkinson's disease

Cellular therapy with adult stem cells appears as an opportunity for treatment of Parkinson's disease. To validate this approach, we studied the effects of transplantation of rat adult bone-marrow mesenchymal stem cells in a rat model of Parkinson's disease. Animals were unilaterally lesioned in the striatum with 6-hydroxydopamine. Two weeks later, group I did not undergo grafting, group II underwent sham grafting, group III was intra-striatal grafted with cells cultured in an enriched medium and group IV was intra-striatal grafted with cells cultured in a standard medium. Rotational amphetamine-induced behavior was measured weekly until animals were killed 6 weeks later. One week after graft, the number of rotations/min was stably decreased by 50% in groups III and IV as compared with groups I and II. At 8 weeks post-lesion, the density of dopaminergic markers in the nerve terminals and cell bodies, i.e. immunoreactive tyrosine hydroxylase, membrane dopamine transporter and vesicular monoamine transporter-2 was significantly higher in group III as compared with group I. Moreover, using microdialysis studies, we observed that while the rate of pharmacologically induced release of dopamine was significantly reduced in lesioned versus intact striatum in no grafted rats, it was similar in both sides in animals transplanted with mesemchymal stem cells. These data demonstrate that graft of adult mesemchymal stem cells reduces behavioral effects induced by 6-hydroxydopamine lesion and partially restores the dopaminergic markers and vesicular striatal pool of dopamine. This cellular approach might be a restorative therapy in Parkinson's disease.     

The effect of periodic starvation on bone-marrow cells from animals of various ages

At present, little is known of what happens to bone-marrow cells under caloric restriction or periodic starvation. The aim of the study was to investigate the influence of periodic starvation on the proliferative potential and cell morphology of the heterogeneous bone-marrow pool of young and old rats. Periodic starvation reduced the number of bone-marrow cells independently of the animal age, but did not affect cell viability and morphology. In young rats, periodic starvation increased the proportion of differentiated cells in the bone-marrow population; in old animals, it increased the number of undifferentiated blasts and blood-cell precursors. Bone-marrow cell cultivation was accompanied with reduced cell heterogeneity. Periodic starvation increased proliferative potential of bone-marrow cells from old rats. This suggests that periodic starvation at late stages of ontogenesis has an antiaging effect on bone-marrow cells in vivo and in vitro.     

Expression and characterization of human N-acetylglucosaminyltransferases and alpha2,3-sialyltransferase in insect cells for in vitro glycosylation of recombinant erythropoietin

The glycans linked to the insect cell-derived glycoproteins are known to differ from those expressed mammalian cells, partly because of the low level or lack of glycosyltransferase activities. GnT II, GnT IV, GnT V, and ST3Gal IV, which play important roles in the synthesis of tetraantennary-type complex glycan structures in mammalian cells, were overexpressed in Trichoplusia ni cells by using a baculovirus expression vector. The glycosyltransferases, expressed as a fusion form with the IgG-binding domain, were secreted into the culture media and purified using IgG sepharose resin. The enzyme assay, performed using pyridylaminated-sugar chain as an acceptor, indicated that the purified glycosyltransferases retained their enzyme activities. Human erythropoietin expressed in T. ni cells (rhEPO) was subjected to in vitro glycosylation by using recombinant glycosyltransferases and was converted into complex-type glycan with terminal sialic acid. The presence of N-acetylglucosamine, galactose, and sialic acid on the rhEPO moiety was detected by a lectin blot analysis, and the addition of galactose and sialic acid to rhEPO was confirmed by autoradiography using UDP-14C-Gal and CMP-14C-Sia as donors. The in vitro glycosylated rhEPO was injected into mice, and the number of reticulocytes among the red blood cells was counted using FACS. A significant increase in the number of reticulocytes was not observed in the mice injected with in vitro glycosylated rhEPO as compared with those injected with rhEPO.     

Did Clinical Trials in Which Erythropoietin Failed to Reduce Acute Myocardial Infarct Size Miss a Narrow Therapeutic Window?

Background

To test a hypothesis that in negative clinical trials of erythropoietin in patients with acute myocardial infarction (MI) the erythropoietin (rhEPO) could be administered outside narrow therapeutic window. Despite overwhelming evidence of cardioprotective properties of rhEPO in animal studies, the outcomes of recently concluded phase II clinical trials have failed to demonstrate the efficacy of rhEPO in patients with acute MI. However, the time between symptoms onset and rhEPO administration in negative clinical trials was much longer that in successful animal experiments.

Methodology/Principal Findings

MI was induced in rats either by a permanent ligation of a descending coronary artery or by a 2-hr occlusion followed by a reperfusion. rhEPO, 3000 IU/kg, was administered intraperitoneally at the time of reperfusion, 4 hrs after beginning of reperfusion, or 6 hrs after permanent occlusion. MI size was measured histologically 24 hrs after coronary occlusion. The area of myocardium at risk was similar among groups. The MI size in untreated rats averaged ∼42% of area at risk, or ∼24% of left ventricle, and was reduced by more than 50% (p<0.001) in rats treated with rhEPO at the time of reperfusion. The MI size was not affected by treatment administered 4 hrs after reperfusion or 6 hrs after permanent coronary occlusion. Therefore, our study in a rat experimental model of MI demonstrates that rhEPO administered within 2 hrs of a coronary occlusion effectively reduces MI size, but when rhEPO was administered following a delay similar to that encountered in clinical trials, it had no effect on MI size.

Conclusions/Significance

The clinical trials that failed to demonstrate rhEPO efficacy in patients with MI may have missed a narrow therapeutic window defined in animal experiments.     

Cytogenetic effects of methotrexate on human cells in vivo: comparison between results obtained by chromosome studies on bone-marrow cells and blood lymphocytes and by the micronucleus test

Cytogenetic studies were performed in 22 patients treated with methotrexate (MTX). In some patients, metaphases from both bone-marrow cells and peripheral blood cells were studied. In the bone-marrow preparations an increased number of structural chromosomal aberrations was present, whereas abnormalities were not observed in the peripheral blood cells. An examination of the bone-marrow chromosomes must therefore be included in the study of the possible chromosome-breaking effect of chemical agents. The results obtained with the micronucleus test and chromosome studies were compared in 10 patients treated with MTX. The micronucleus test was more sensitive than the chromosome analysis as regards the clastogenic effect of MTX.