N-acetylcysteine increases the biosynthesis of recombinant EPO in apoptotic Chinese hamster ovary cells

Sodium butyrate (NaBu) is known to enhance the rate of biosynthesis of recombinant proteins in Chinese hamster ovary cells (CHO). Here we demonstrate that supplementation with NaBu during rapid growth brings about abrupt death of the cells. The death of the cells is due to apoptosis, as assessed by intranucleosomal DNA fragmentation. The promotion of apoptotic death of the cells could be partially blocked by treatment with the well-known antioxidant, N-acetylcysteine (NAC). Strikingly, the NAC treatment enhanced the production of recombinant EPO two-fold compared with that of the culture without NAC supplementation. These results showed that NaBu treatment supplemented with NAC not only inhibits apoptosis, but also exerts a synergistic effect on the biosynthesis of recombinant EPO.     

Effect of N-Acetylcystein on butyrate-treated Chinese hamster ovary cells to improve the production of recombinant human interferon-beta-1a

Sodium butyrate (NaBu) is used as a productivity enhancer for the production of therapeutic recombinant proteins in Chinese hamster ovary (CHO) cells. However, NaBu is well-known for having a cytotoxic effect, thereby inducing apoptosis. As an endeavor to reduce this defect, we studied 11 antioxidants known for inhibiting apoptosis, according to a Plackett-Burman statistical design on CHO cells producing recombinant interferon-beta-1a (IFN-beta). None of the antioxidants that we tested were as effective as N-acetylcystein (NAC) from the point of view of maintaining long-term survival of CHO cells and increasing the production of IFN-beta. In 7.5-L perfusion bioreactor cultures, the addition of NaBu and NAC elongated the culture period to almost 200 h throughout production phase and increased the production yield by 2-fold compared to control cultures containing only NaBu. Glycosylation patterns of produced IFN-beta at each run were also compared in IEF analysis. IEF profiles of where NaBu and NAC were added showed to be more isoforms with a lower pI than those of the control run. The sialic acid content was also increased by 17.7% according to HPLC analysis. Taken together, the data obtained demonstrate that the addition of NAC has positive effects on the elongation of the culture period, improving the production and increasing the sialylation of IFN-beta in NaBu-treated CHO cells.     

Effect of sodium butyrate on autophagy and apoptosis in Chinese hamster ovary cells

Sodium butyrate (NaBu), which is widely used in recombinant Chinese hamster ovary cell (rCHO) cultures for high-level expression of therapeutic proteins, is known to induce apoptosis in a dose-dependent manner. Lately, the significance of autophagy has increased in the field of CHO cell culture due to the fact that autophagy is related to the programmed cell death mechanism. To determine the effect of NaBu on autophagy as well as apoptosis of rCHO cells, rCHO cells producing erythropoietin were subjected to NaBu treatment. NaBu treatment up to 5 mM increased cleaved forms of PARP, caspase-3, and Annexin V positive population, confirming the previous results that NaBu induces apoptosis. Concurrently, NaBu treatment increased the level of accumulation of the autophagic marker, LC3-II, independently of nutrient depletion, suggesting that NaBu induces autophagy. To elucidate the potential role of autophagy induced by NaBu, a representative autophagy inducer (rapamycin) or an inhibitor (bafilomycin A1) was added to cultures together with NaBu. It was found that autophagy had the potential role of a positive cell survival mechanism under NaBu treatment. Furthermore, gradual reduction in mitochondrial membrane potential/mass and recruitment of a mitophagy protein, Parkin, to the mitochondria were observed under NaBu treatment, suggesting that this positive function of autophagy might be mediated by the autophagic removal of damaged mitochondria. Taken together, autophagy was observed in rCHO cell culture under NaBu treatments and the results obtained here support the positive effects of autophagy induced by NaBu treatments.     

A DIGE approach for the assessment of differential expression of the CHO proteome under sodium butyrate addition: Effect of Bcl‐xL overexpression

Bcl‐x_L, a member of the Bcl‐2 family, is known to inhibit apoptosis of recombinant Chinese hamster ovary (rCHO) cells induced by the addition of sodium butyrate (NaBu), which is used for the elevated expression of recombinant protein. In order to understand the intracellular effects of Bcl‐x_L overexpression on CHO cells treated with NaBu, changes to the proteome caused by controlled Bcl‐x_L expression in rCHO cells producing erythropoietin (EPO) in the presence of 3 mM NaBu were evaluated using two‐dimensional differential in‐gel electrophoresis (2D‐DIGE) and MS analysis. The consequences of Bcl‐x_L overexpression were not limited to the apoptotic signaling pathway. Out of eight proteins regulated significantly by Bcl‐x_L overexpression in 3 mM NaBu addition culture, four proteins were related to cell survival (Iq motif‐containing GTPase‐activating protein 1), cell proliferation (dihydrolipoamide‐S‐acetyltransferase, guanine nucleotide binding protein alpha interacting 2), and repair of DNA damage (BRCA and CDKN1A interacting protein). Taken together, a DIGE approach reveals that overexpression of Bcl‐x_L not only inhibits apoptosis in the presence of NaBu but also affects cell proliferation and survival in various aspects. Biotechnol. Bioeng. 2010; 105: 358–367. © 2009 Wiley Periodicals, Inc.     

Influence of down-regulation of caspase-3 by siRNAs on sodium-butyrate-induced apoptotic cell death of Chinese hamster ovary cells producing thrombopoietin

Sodium butyrate (NaBu) can enhance the expression of foreign protein of recombinant Chinese hamster ovary (rCHO) cells, but it can also inhibit cell growth and induce cellular apoptosis. Thus, the beneficial effect of using a higher concentration of NaBu on foreign protein expression in rCHO cells is compromised by its growth inhibitory and cytotoxic effects. To overcome this cytotoxic effect of NaBu, an expression vector of small interfering RNAs (siRNAs) targeting against caspase-3, a key effector component in apoptosis, was constructed and transfected into rCHO cells producing human thrombopoietin (hTPO). Using this siRNA strategy, rCHO cells (F21 cells) expressing a low level of caspase-3 proenzyme determined by RT-PCR and Western blot analysis were established. Under the condition of 1-5 mM NaBu addition at the exponential growth phase, down-regulation of caspase-3 in F21 cells could not effectively inhibit NaBu-induced apoptotic cell death. This NaBu-induced apoptotic cell death occurred because F21 cells appeared to compensate for the lack of caspase-3 by increasing the active caspase-7 level. These results suggest that the intracellular caspase's interconnectivity should be taken into consideration for the successful inhibition of apoptosis of rCHO cells.     

Inhibition of sodium butyrate-induced apoptosis in recombinant Chinese hamster ovary cells by constitutively expressing antisense RNA of caspase-3

Sodium butyrate (NaBu) can enhance the expression of genes controlled by some of the mammalian promoters, but it can also inhibit cell growth and induce cellular apoptosis. Thus, the beneficial effect of using a higher concentration of NaBu on a foreign protein expression is compromised by its cytotoxic effect on cell growth. To overcome this cytotoxic effect of NaBu, the expression vector of antisense RNA of caspase-3 was constructed and transfected to recombinant Chinese hamster ovary (rCHO) cells producing a humanized antibody. Using this antisense RNA strategy, rCHO cells (B3) producing a low level of caspase-3 proenzyme were established. When batch cultures of both B3 cells and control cells transfected with antisense RNA-deficient plasmid were performed in the absence of NaBu, both cells showed similar profiles of cell growth and antibody production. Compared with control cell culture, under the condition of 5 mM NaBu addition at the exponential growth phase, expression of antisense RNA of caspase-3 significantly suppressed the NaBu-induced apoptosis of B3 cells and extended culture longevity by >2 days if the culture was terminated at cell viability of 50%. However, compared with control cell culture, the final antibody concentration of B3 cell culture was not increased in the presence of NaBu, which may be due to the loss of cellular metabolic capability resulted from the depolarization of mitochondrial membrane. Taken together, this study suggests that, although expression of antisense RNA of caspase-3 does not improve antibody productivity of rCHO cells, it can suppress NaBu-induced apoptotic cell death of rCHO cells and thereby may reduce problems associated with cellular disintegration.     

Influence of co-down-regulation of caspase-3 and caspase-7 by siRNAs on sodium butyrate-induced apoptotic cell death of Chinese hamster ovary cells producing thrombopoietin

Previously, the expression of caspase-3 siRNA could not effectively inhibit sodium butyrate (NaBu)-induced apoptotic cell death of recombinant Chinese hamster ovary (rCHO) cells producing human thrombopoietin (hTPO). Caspase-3 siRNA expressing cells appeared to compensate for the lack of caspase-3 by increasing active caspase-7 levels. For the successful inhibition of NaBu-induced apoptosis of rCHO cells, both caspase-3 and caspase-7 were down-regulated using the siRNA expression vector system. Co-down-regulation of caspase-3 and caspase-7 increased cell viability and extended culture longevity in serum-free culture in the presence or absence of 1mM NaBu addition. In the cultures with 1mM NaBu addition, the maximum hTPO concentration in rCHO cells with down-regulation of both caspases was approximately 55% higher than that in rCHO cells without down-regulation of caspases and approximately 16% higher than rCHO cells with down-regulation of only caspase-3. However, in the culture with 3mM NaBu, this strategy could not dramatically enhance the culture longevity and hTPO production, compared to Bcl-2 overexpression. The different result in hTPO production between down-regulation of caspases and Bcl-2 overexpression may be because the down-regulation of caspase-3 and caspase-7, unlike Bcl-2 overexpression, could not maintain mitochondrial membrane potential in the presence of 3mM NaBu. Taken together, co-down-regulation of caspase-3 and caspase-7 is effective in regard to extension of culture longevity and enhancement of hTPO production in a serum-free culture in the presence or absence of 1mM NaBu addition.     

端粒酶RNA的反义cDNA对乳腺癌细胞凋亡的影响(英文)

 为了进一步探讨端粒酶RNA(hTR)的反义cDNA对乳腺癌MCF 7细胞凋亡可诱导性的影响 ,构建了能将外源基因整合至细胞基因组的整合型腺病毒载体vAd AAV ,并将hTR全长cDNA反向连接至此载体上 ,获得反义重组腺病毒vAdT AAV .vAd AAV和vAdT AAV分别感染MCF 7细胞后 ,获得两个细胞株MCF 7 vAd AAV和MCF 7 vAdT AAV ,其中MCF 7 vAdT AAV细胞基因组内整合有hTR反义cDNA并能稳定表达 .利用生存曲线、细胞形态学观察、DNA片段分析和流式细胞分析来测定NaBu和无血清DMEM诱导后细胞凋亡的反应性 .通过生存曲线 ,发现NaBu诱导的MCF 7 vAdT AAV细胞比对照组MCF 7和MCF 7 vAd AAV细胞更早出现凋亡 .电镜下 ,NaBu或去血清诱导的MCF 7 vAdT AAV细胞更早出现凋亡形态学指标 .流式细胞分析和DNA片段凝胶电泳实验均显示MCF 7 vAdT AAV细胞对凋亡的抵抗力下降 .研究结果表明 ,端粒酶RNA的反义cDNA使乳腺癌MCF 7细胞的凋亡可诱导性增强     

Butyrated ManNAc analog improves protein expression in Chinese hamster ovary cells

The chemical additive sodium butyrate (NaBu) has been applied in cell culture media as a direct and convenient method to increase the protein expression in Chinese hamster ovary (CHO) and other mammalian cells. In this study, we examined an alternative chemical additive, 1,3,4‐O‐Bu₃ManNAc, for its effect on recombinant protein production in CHO. Supplementation with 1,3,4‐O‐Bu₃ManNAc for two stable CHO cell lines, expressing human erythropoietin or IgG, enhanced protein expression for both products with negligible impact on cell growth, viability, glucose utilization, and lactate accumulation. In contrast, sodium butyrate treatment resulted in a ～20% decrease in maximal viable cell density and ～30% decrease in cell viability at the end of cell cultures compared to untreated or 1,3,4‐O‐Bu₃ManNAc treated CHO cell lines for both products. While NaBu treatment enhanced product yields more than the 1,3,4‐O‐Bu₃ManNAc treatment, the NaBu treated cells also exhibited higher levels of caspase 3 positive cells using microscopy analysis. Furthermore, the mRNA levels of four cell apoptosis genes (Cul2, BAK, BAX, and BCL2L11) were up‐regulated more in sodium butyrate treated wild‐type, erythropoietin, or IgG expressing CHO‐K1 cell lines while most of the mRNA levels of apoptosis genes in 1,3,4‐O‐Bu₃ManNAc treated cell lines remained equal or increased only slightly compared to the levels in untreated CHO cell lines. Finally, lectin blot analysis revealed that the 1,3,4‐O‐Bu₃ManNAc‐treated cells displayed higher relative sialylation levels on recombinant EPO, consistent with the effect of the ManNAc component of this additive, compared to control while NaBu treatment led to lower sialylation levels than control, or 1,3,4‐O‐Bu₃ManNAc‐treatment. These findings demonstrate that 1,3,4‐O‐Bu₃ManNAc has fewer negative effects on cell cytotoxicity and apoptosis, perhaps as a result of a more deliberate uptake and release of the butyrate compounds, while simultaneously increasing the expression of multiple recombinant proteins, and improving the glycosylation characteristics when applied at comparable molarity levels to NaBu. Thus, 1,3,4‐O‐Bu₃ManNAc represents a highly promising media additive alternative in cell culture for improving protein yields without sacrificing cell mass and product quality in future bioproduction processes.

     

Differential induction of autophagy in caspase-3/7 down-regulating and Bcl-2 overexpressing recombinant CHO cells subjected to sodium butyrate treatment

Previous research showed that co-down-regulation of caspase-3/7 in rCHO cells, unlike Bcl-2 overexpression, did not effectively block apoptotic cell death induced by 3mM sodium butyrate (NaBu). Here, it is found that the control of autophagy is also related to this different response to NaBu treatment. With NaBu treatment, co-down-regulation of caspase-3/7 enhanced autophagy induction, whereas Bcl-2 overexpression delayed onset of autophagy induction in a Beclin-1 independent manner. The blockage of autophagy showed a detrimental effect on cell viability even in the Bcl-2 overexpressing cells, which suggests the importance of autophagy control for successful anti-cell death engineering of rCHO cells.     

Calnexin overexpression sensitizes recombinant CHO cells to apoptosis induced by sodium butyrate treatment

Sodium butyrate (NaBu) can enhance the expression of foreign genes in recombinant Chinese hamster ovary (rCHO) cells, but it can also inhibit cell growth and induce cellular apoptosis. In this study, the potential role of calnexin (Cnx) expression in rCHO cells treated with 5 mM NaBu was investigated for rCHO cells producing tumor necrosis factor receptor FC. To regulate the Cnx expression level, a tetracycline-inducible system was used. Clones with different Cnx expression levels were selected and investigated. With regard to productivity per cell (q_p), NaBu enhanced the q_p by over twofold. Under NaBu treatment, Cnx overexpression further enhanced the q_p by about 1.7-fold. However, under NaBu stress, the cells overexpressing Cnx showed a poorer viability profile with a consistent difference of over 25% in the viability when compared to the Cnx-repressed condition. This drop in the viability was attributed to increased apoptosis seen in these cells as evidenced by enhanced poly (ADP-ribose) polymerase cleavage and cytochrome C release. Ca²⁺ localization staining and subsequent confocal imaging revealed elevated cytosolic Ca²⁺ ([Ca²⁺]_c) in the Cnx-overexpressing cells when compared to the Cnx-repressed condition, thus endorsing the increased apoptosis observed in these cells. Taken together, Cnx overexpression not only improved the q_p of cells treated with NaBu, but it also sensitized cells to apoptosis.     

Sodium butyrate inhibits aerobic glycolysis of hepatocellular carcinoma cells via the c‐myc/hexokinase 2 pathway

Aerobic glycolysis is a well‐known hallmark of hepatocellular carcinoma (HCC). Hence, targeting the key enzymes of this pathway is considered a novel approach to HCC treatment. The effects of sodium butyrate (NaBu), a sodium salt of the short‐chain fatty acid butyrate, on aerobic glycolysis in HCC cells and the underlying mechanism are unknown. In the present study, data obtained from cell lines with mouse xenograft model revealed that NaBu inhibited aerobic glycolysis in the HCC cells in vivo and in vitro. NaBu induced apoptosis while inhibiting the proliferation of the HCC cells in vivo and in vitro. Furthermore, the compound inhibited the release of lactate and glucose consumption in the HCC cells in vitro and inhibited the production of lactate in vivo. The modulatory effects of NaBu on glycolysis, proliferation and apoptosis were related to its modulation of hexokinase 2 (HK2). NaBu downregulated HK2 expression via c‐myc signalling. The upregulation of glycolysis in the HCC cells induced by sorafenib was impeded by NaBu, thereby enhancing the anti‐HCC effect of sorafenib in vitro and in vivo. Thus, NaBu inhibits the expression of HK2 to downregulate aerobic glycolysis and the proliferation of HCC cells and induces their apoptosis via the c‐myc pathway.     

Involvement of N-acetylcysteine-sensitive pathways in ricin-induced apoptotic cell death in U937 cells

We have found that the antioxidant N-acetylcysteine (NAC) strongly inhibited ricin-induced apoptotic cell death in U937 cells (human myeloid leukemia), as judged by cytotoxicity, nuclear morphological change, and DNA fragmentation. Consistent with these observations, a significant depletion of cellular glutathione was observed in ricin-treated cells, and NAC prevented the decrease in cellular glutathione. On the other hand, among the caspase inhibitors tested, Z-Asp-CH2-DCB, which inhibited ricin cytotoxicity, also suppressed ricin-mediated glutathione depletion, while NAC did not affect the generation of caspase-3 like activity in ricin-treated cells. These results suggest that glutathione loss takes place downstream from caspase activation during the ricin-induced apoptotic process. Treatment with a specific inhibitor of glutathione biosynthesis, buthionine sulfoximine (BSO) failed to induce apoptosis, and had no effect on the overall extent of ricin-induced apoptosis, even though the glutathione level was decreased to less than 5% of the control level. However, NAC still protected against ricin-induced apoptosis in the BSO-treated cells. We conclude that glutathione loss is one of several apoptotic changes caused by ricin, but is not a sufficient factor for the progress of apoptosis. NAC may prevent ricin-induced apoptosis through maintaining an intracellular reducing condition by acting as a thiol supplier.     

Effect of sodium butyrate on the production, heterogeneity and biological activity of human thrombopoietin by recombinant Chinese hamster ovary cells

Human thrombopoietin (hTPO) is a heavily glycosylated protein with 6 and 24 potential N- and O-glycosylation sites, respectively. To determine the effect of sodium butyrate (NaBu) on the production and quality of hTPO in recombinant Chinese hamster ovary (rCHO) cells, NaBu (0-10 mM) was added to the cultures of exponentially growing cells. NaBu addition significantly increased both the specific and volumetric hTPO production, although it decreased the cell viability by apoptosis in a dose-dependent manner. The highest hTPO concentration of 82.2 +/- 5.6 microgml-1 was obtained in the culture with 3 mM NaBu addition. Compared with the culture without NaBu addition, the culture with 3 mM NaBu resulted in a 6.4-fold increase in qTPO and a 3.3-fold increase in the final hTPO concentration on day 7. However, NaBu deteriorated the quality of hTPO, resulting from increased heterogeneity, reduced acidic hTPO isoforms, reduced alpha(2 --> 3) sialylation, and decreased in vivo biological activity. We also found that the biological activity of hTPO in the culture with 3 mM NaBu addition collected on day 7 was 72% of that in the culture without NaBu addition. Taken together, the use of NaBu or its optimal concentration for high-level expression of a heavily glycosylated protein like hTPO should be determined by considering its detrimental effect on the quality of glycoprotein.     

Suppression of arsenic trioxide-induced apoptosis in HeLa cells by N-acetylcysteine

Arsenic trioxide (ATO) can affect many biological functions such as apoptosis and differentiation in various cells. We investigated the involvement of ROS and GSH in ATO-induced HeLa cell death using ROS scavengers, especially N-acetylcysteine (NAC). ATO increased intracellular O(2)(*-) levels and reduced intracellular GSH content. The ROS scavengers, Tempol, Tiron and Trimetazidine, did not significantly reduce levels of ROS or GSH depletion in ATO-treated HeLa cells. Nor did they reduce the apoptosis induced by ATO. In contrast, treatment with NAC reduced ROS levels and GSH depletion in the ATO-treated HeLa cells and prevented ATO-induced apoptosis. Treatment with exogenous SOD and catalase reduced the depletion of GSH content in ATO-treated cells. Catalase strongly protected the cells from ATO-induced apoptosis. In addition, treatment with SOD, catalase and NAC slightly inhibited the G1 phase accumulation induced by ATO. In conclusion, NAC protects HeLa cells from apoptosis induced by ATO by up-regulating intracellular GSH content and partially reducing the production of O(2)(*-).     

Enhanced human thrombopoietin production by sodium butyrate addition to serum-free suspension culture of bcl-2-overexpressing CHO cells

When sodium butyrate (NaBu) was added to serum-free suspension culture of recombinant CHO (rCHO) cells for enhanced expression of human thrombopoietin (hTPO), apoptotic cell death of rCHO cells was induced in a dose-dependent manner and hTPO quality was deteriorated in regard to sialic acid and acidic isoform contents. To overcome these problems, we overexpressed Bcl-2 protein, an antiapoptotic protein, in rCHO cells producing hTPO. Compared to serum-free suspension culture of control cells without Bcl-2 overexpression (R-neo cells) and NaBu addition, a more than 10-fold increase in the maximum hTPO concentration was obtained in serum-free suspension culture of cells with Bcl-2 overexpression (R-bc12-14 cells) and 3 mM NaBu addition. Both the enhanced specific productivity endowed by NaBu and the extended culture longevity provided by the antiapoptotic effect of Bcl-2 overexpression contributed to the enhancement of maximum hTPO concentration. The problem of quality reduction of hTPO induced by NaBu was not solved by Bcl-2 overexpression, but it was not that significant. Compared to the culture in the absence of NaBu, the percentage of hTPO isoforms in pI 3-5 with high in vivo biological activity produced by R-bc12-14 cells was decreased by approximately 18% in the presence of 3 mM. As a result, a more than 6-fold increase in the production of hTPO isoforms in pI 3-5 was achieved in R-bcl2-14 cell culture with 3 mM NaBu addition. Taken together, the data obtained suggest that Bcl-2 overexpression in rCHO cells and NaBu addition in serum-free suspension culture can be an effective means to enhance the production of highly glycosylated protein such as hTPO.     

Overexpression of bcl-2 inhibits sodium butyrate-induced apoptosis in Chinese hamster ovary cells resulting in enhanced humanized antibody production

Sodium butyrate (NaBu) can enhance the expression of genes from some of the mammalian promoters including cytomegalovirus (CMV) and simian virus 40 (SV40), but it can also inhibit cell growth and induce cellular apoptosis. Thus, the beneficial effect of using a higher concentration of NaBu on a foreign protein expression is compromised by its cytotoxic effect on cell growth. To overcome this cytotoxic effect of NaBu, a survival protein, human Bcl-2, was overexpressed in recombinant Chinese hamster ovary (CHO) cells (SH2-0.32), producing a humanized antibody directed against the S surface antigen of hepatitis B virus. When batch cultures of both control cells transfected with bcl-2-deficient plasmid (SH2-0.32-Deltabcl-2) and cells transfected with bcl-2 expression plasmid (14C6-bcl-2) were performed in the absence of NaBu, both cells showed similar profiles of cell viability and antibody production. Compared with the SH2-0.32-Deltabcl-2 culture, under the condition of NaBu addition at the exponential growth phase, overexpression of the bcl-2 gene considerably suppressed the NaBu-induced apoptosis of 14C6-bcl-2 by inhibiting caspase 3 activity and extending culture longevity by >2 days. As a result, the final antibody concentration of 14C6-bcl-2 culture was twofold higher than that of SH2-0.32-Deltabcl-2 culture in the presence of NaBu and threefold higher than that of SH2-0.32-Deltabcl-2 and 14C6-bcl-2 cultures in the absence of NaBu.     

Erythropoietin protects cardiac myocytes from hypoxia-induced apoptosis through an Akt-dependent pathway

Apoptosis is a contributing cause of myocyte loss in ischemic heart disease. Recent work has shown that erythropoietin (EPO) offers protection against apoptosis in a wide variety of tissues. We demonstrate that the erythropoietin receptor (EPOR) is expressed in the neonatal rat ventricular myocyte (NRVM). Exposure of NRVMs to hypoxia, with recombinant human EPO, significantly decreased apoptosis as measured by TUNEL, flow cytometry, and caspase 3/7 like activity when compared to hypoxia treatment alone. EPO administered at the initiation of coronary artery occlusion in the rat significantly decreased apoptosis in the myocardial ischemic region. In the NRVM, EPO increased the activity of Akt. The anti-apoptotic effect of EPO was abrogated by co-treatment with LY294002, a specific blocker of phosphatidylinositol 3-kinase (PI3-K). Our study demonstrates that EPO inhibits apoptosis in the NRVM exposed to hypoxia, through an Akt-dependent pathway. EPO also inhibits apoptosis in the in vivo rat model of myocardial ischemia.     

Prothymosin-alpha plays a defensive role in retinal ischemia through necrosis and apoptosis inhibition

Prothymosin-alpha (ProTalpha) causes a switch in cell death mode from necrosis to neurotrophin-reversible apoptosis in primary cultured cortical neurons. In the present study, post-ischemic administration (3 or 24 h, intravenously) of recombinant mouse ProTalpha without neurotrophins completely prevented ischemia-induced retinal damage accompanying necrosis and apoptosis, as well as dysfunction assessed by electroretinogram. Treatments with anti-erythropoietin (EPO) or brain-derived neurotrophic factor (BDNF) immunoglobulin G (IgG) reversed ProTalpha-induced inhibition of apoptosis. ProTalpha upregulated retinal EPO and BDNF levels in the presence of ischemia. Moreover, intravitreous administration of anti-ProTalpha IgG or an antisense oligodeoxynucleotide for ProTalpha accelerated ischemia-induced retinal damage. We also observed that ischemia treatment caused a depletion of ProTalpha from retinal cells. Altogether, these results suggest that the systemic administration of ProTalpha switches ischemia-induced necrosis to apoptosis, which in turn is inhibited by neurotrophic factors upregulated by ProTalpha and ischemia. ProTalpha released upon ischemic stress was found to have a defensive role in retinal ischemia.