ALBERT

Description: Death-associated protein kinase 1 (DAPK1) expression induced by diverse death stimuli mediates apoptotic activity in various cancers, including ovarian cancer. In addition, mutual interaction between the tumor suppressor p53 and DAPK1 influences survival and death in several cancer cell lines. However, the exact role and connection of DAPK1 and p53 family proteins (p53, p63, and p73) in drug-resistant ovarian cancer cells have not been studied previously. In this study, we investigated whether DAPK1 induction by gliotoxin derived from marine fungus regulates the level of transcriptionally active p63 (TAp63) to promote apoptosis in an autophagy-dependent manner. Pre-exposure of paclitaxel-resistant ovarian cancer cells to gliotoxin inhibited the expression of multidrug resistant-associated proteins (MDR1 and MRP1-3), disrupted the mitochondrial membrane potential, and induced caspase-dependent apoptosis through autophagy induction after subsequent treatment with paclitaxel. Gene silencing of DAPK1 prevented TAp63-mediated downregulation of MDR1 and MRP1-3 and autophagic cell death after sequential treatment with gliotoxin and then paclitaxel. However, pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, had no effect on the levels of DAPK1 and TAp63 or on the inhibition of MDR1 and MRP1-3. These results suggest that DAPK1-mediated TAp63 upregulation is one of the critical pathways that induce apoptosis in chemoresistant cancer cells.

Description: Erythropoietin (Epo) is the primary regulator of erythroid cell proliferation and differentiation mediated through its specific binding to the Epo receptor (EpoR). Epo also is known to have non-hematopoietic actions, including the promotion of wound healing, tissue protection and others. Accumulating evidence demonstrating both Epo and EpoR expression in many types of human cancers has raised concern about the clinical use of Epo and other erythropoiesis stimulating agents (ESAs) in cancer-related anemia, since the presence of functional EpoR on the tumor cells suggests the potential for an adverse effect of Epo treatment by enhancing cancer growth and progression. Previously, we demonstrated that A2780 ovarian cancer cells express the EpoR gene and that long-term Epo treatment of these cells renders them resistant to paclitaxel. We now report the characterization and biological significance of Epo and EpoR expression by four ovarian cancer cell lines. Using semi-quantitative RT-PCR, restriction digestion of the PCR products and DNA sequence analysis, we demonstrated that A2780, CaOV, SKOV, and OVCAR-3 ovarian cancer cell lines examined express Epo and EpoR at the mRNA level. We demonstrated EpoR protein expression both by western blotting and by immunofluorescence, and demonstrated biologically active Epo protein expression by quantitative in vitro bioassay of the cell culture supernatants. The EpoR on A2780 cells are functional, since Epo stimulation of the cells resulted in a time-dependent 5-fold increase in phosphorylation of Erk1/2 that reached a peak in 10 min and then returned to the basal level. None of the cell lines exhibited a short-term (3-day) growth response in culture to exogenous Epo. However, addition of a neutralizing anti-Epo antibody to the cell culture resulted in partial growth inhibition of A2780 cells that was reversed by addition of excess Epo, consistent with an autocrine/paracrine mechanism of Epo growth enhancement of these cells. We also found that long-term Epo treatment of A2780 cells resulted in the development of a phenotype exhibiting enhanced Epo signaling, evidenced by a 20-fold increase in phosphorylation levels of Erk1/2. This phenotype was sustained even after the removal of Epo, suggesting a mechanism underlying the paclitaxel resistance developed after long-term Epo treatment. Our findings have implications for the clinical use of recombinant human Epo and other ESAs to correct and/or prevent anemia in ovarian and, potentially, other cancer patients. They also suggest that endogenous Epo and the EpoR may serve as novel therapeutic targets in the treatment of cancer patients.

Description: Abstract 2097 Anemia of inflammation (AI) has been widely associated with chronic rheumatologic, infectious, and cardiovascular disorders and comprises one-third of cases of anemia in the elderly. Although the antimicrobial peptide hepcidin is felt to be the prime regulator of AI, emerging evidence supports the premise that subsets of AI may be mediated via hepcidin-independent inflammatory pathways, such as those modulated by tumor necrosis factor alpha (TNFα), that directly suppress erythropoiesis. A better understanding of the pathophysiology of anemia subtypes is necessary if we are to develop more effective targeted, oral therapies for patients with inflammatory anemia. Our recent analysis of elderly participants in the Third National Health and Nutrition Examination Survey (NHANES III) revealed that vitamin D deficiency was strongly associated with AI in this population, with this subgroup exhibiting a rate of vitamin D deficiency nearly twice that of similarly aged non-anemic individuals. In the current study we aimed to evaluate whether vitamin D treatment may ameliorate TNFα-mediated erythroid colony suppression in humans. Human bone marrow-derived CD34+ cells from 3 healthy adults were first cultured in methylcellulose medium containing IL-3 (10ng/ml), EPO (1U/ml), SCF (50ng/ml) in the presence or absence of TNFα (25 ng/ml) and increasing doses of 1, 25-dihydroxyvitamin D3 [calcitriol, (0.01-1nM)], and erythroid burst-forming units (BFU-E) measured on day 14. The percentage of BFU-E colonies was reduced by 50% at day 14 in the presence of 25ng/ml TNFα (p 〈 0.01, t-test). TNFα-mediated suppression of erythropoiesis was reversed by the addition of vitamin D3 in a dose-dependent manner, with 1nM vitamin D3 resulting in a 50% recovery of BFU-E colony numbers at day 14 (p 〈 0.05, t-test). Vitamin D3 alone exhibited no significant effect on BFU-E formation at all concentrations tested. Based on studies showing that vitamin D receptor may serve as a negative regulator of NF-κB transcriptional activity, we next investigated whether vitamin D3 may alter TNFα-induced NF-κB activation in both K562 and human CD34+ cells. K562 cells pre-incubated with 1nM vitamin D3 for 3, 24, 48 and 72 hours were treated with 5 ng/ml TNFα for 30 minutes, and NF-κB activity in nuclear fraction was determined by an ELISA-format oligonucleotide binding assay. TNFα treatment alone markedly increased the oligonucleotide binding activity of the NF-κB p65 subunit, which was completely blocked by co-incubation with competitor oligonucleotide, confirming the assay specificity. Pre-incubation with vitamin D3 for 3 – 48 hours showed little effect on TNFα-mediated NF-κB activation, but pre-incubation for 72 hours resulted in suppression of TNFα-induced NF-κB activity by 46% (p 〈 0.02, t-test). Under similar conditions, human CD34+ cells pre-incubated with 1 nM vitamin D3 for 48 hours and 72 hours exhibited 38% and 84% suppression of TNFα-induced NF-κB activity, respectively (p 〈 0.01 for both conditions, t-test). Reduced NF-κB activity was correlated with the decreased nuclear translocation of NF-κB in K562 cells with no significant changes in IκB degradation, suggesting that vitamin D3 regulates the NF-κB nuclear translocation independent of IκB. Western analysis of whole cell lysates from both K562 and human CD34+ cells using antibodies recognizing known TNFα-associated signaling pathways revealed robust increases in phospho-p38, phospho-JNK, and phospho-ERK1/2 in response to TNFα stimulation compared with control, with no inhibition of these signaling pathways noted in response to vitamin D3 treatment at all doses tested, suggesting that vitamin D3 does not inhibit these TNFα-induced signaling cascades. Our study indicates that 1, 25-dihydroxyvitamin D3 significantly ameliorates TNFα-mediated suppression of erythroid colony development in human CD34+ cells via mechanisms that involve modulation of NF-κB signaling pathways and supports the design of future clinical trials examining whether vitamin D3 supplementation may prove to be an effective therapy for subgroups of patients with TNFα-mediated inflammatory anemia. Disclosures: No relevant conflicts of interest to declare.

Description: Background : Prognosis of diffuse large B cell lymphoma (DLBCL) is very varied from cure to death. So the prediction for prognosis of DLBCL is very important to make a decision regarding the treatment. Now, international prognostic index (IPI) risk model is widely known as the powerful prognostic indicator for lymphoma. However, many inflammatory factors have been demonstrated as prognostic factors in patients with DLBCL such as C-reactive protein (CRP), ferritin, ¥â2-microglobulin (B2MG), absolute lymphocyte count (ALC) and albumin etc. But LDH is the only inflammatory factor in those of IPI. So we find a new prognostic risk model (inflammatory factors based modified IPI risk model ; inflammatory-IPI) including other inflammatory prognostic factors for predicting more correct survival outcomes and progression free survival (PFS) in patients with DLBCL treated with rituximab combined cyclophosphamide, adriamycin, vincristine and prednisone (RCHOP). Methods: A total of 278 patients who were newly diagnosed with DLBCL and received RCHOP at hospitals throughout South Korea between January 2007 and December 2014 were enrolled retrospectively in the current study. Baseline serum CRP, ferritin, B2MG, ALC, albumin and factors of IPI were measured within 4 weeks before beginning the first line of chemotherapy. Each inflammatory factor of serum CRP 〉 1.5mg/dL, ferritin 〉 500ng/mL, B2MG 〉 3.5mg/L, ALC level 〈 1.0x109/L, serum albumin 〈 3.5g/dL, LDH level 〉 450 IU/L was defined as an abnormal findings, and if the sum of these abnormal findings are not fewer than 2, it is given 1 point as a factor of inflammatory-IPI instead of LDH. After that, sum of this point and the number of negative prognostic factors present at the time of diagnosis (age 〉 60 years, ECOG performance status ¡Ã 2, extranodal site ¡Ã 2, stage III/IV disease) is named the inflammatory-IPI. The inflammatory-IPI risk model were divided into four groups seems like IPI according to the scores ; low risk were 0 or 1 score, low-intermediate risk were 2 scores, and high-intermediate risk were 3 scores, high risk were 4 scores respectively. Results: In univariate analysis, the following factors showed significant higher 5 years overall survival rates (OS) : age (p=0.001), stage III/IV disease (p=0.008), LDH level (p

Description: To study factors associated with anemia and its effect on survival in HIV-infected persons treated with modern combined antiretroviral therapy (cART), we characterized the prevalence of anemia in the Veterans Aging Cohort Study (VACS) and used a candidate gene approach to identify proinflammatory gene single nucleotide polymorphisms (SNPs) associated with anemia in HIV disease. The study comprised 1597 HIV+ and 865 HIV− VACS subjects with DNA, blood, and annotated clinical data available for analysis. Anemia was defined according to World Health Organization criteria (hemoglobin 〈 13 g/dL and 〈 12 g/dL in men and women, respectively). The prevalence of anemia in HIV+ and HIV− subjects was 23.1% and 12.9%, respectively. Independent of HIV status, anemia was present in 23.4% and 8% in blacks and whites, respectively. Analysis of our candidate genes revealed that the leptin −2548 G/A SNP was associated with anemia in HIV+, but not HIV−, patients, with the AA and AG genotypes significantly predicting anemia (P 〈 .003 and P 〈 .039, respectively, logistic regression). This association was replicated in an independent cohort of HIV+ women. Our study provides novel insight into the association between genetic variability in the leptin gene and anemia in HIV+ individuals.

Description: Intracellular reactive oxygen species (ROS) play an important role in the proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs). HSPCs are difficult to be expanded ex vivo while maintaining their stemness when they are exposed to oxidative damage after being released from the bone marrow. There have been efforts to overcome this limitation by using various cytokine cocktails and antioxidants. In this study, we investigated the effects of echinochrome A (Ech A)-a well-established and non-toxic antioxidant-on the ex vivo expansion of HSPCs by analyzing a CD34+ cell population and their biological functions. We observed that Ech A-induced suppression of ROS generation and p38-MAPK/JNK phosphorylation causes increased expansion of CD34+ cells. Moreover, p38-MAPK/JNK inhibitors SB203580 and SP600125 promoted ex vivo expansion of CD34+ cells. We also demonstrated that the activation of Lyn kinase and p110δ is a novel mechanism for Ech A to enhance ex vivo expansion of CD34+ cells. Ech A upregulated phospho-Src, phospho-Lyn, and p110δ expression. Furthermore, the Ech A-induced ex vivo expansion of CD34+ cells was inhibited by pretreatment with the Src family inhibitor PP1 and p110δ inhibitor CAL-101; PP1 blocked p110δ upregulation and PI3K/Akt activation, whereas CAL-101 and PI3K/Akt pathway inhibitor LY294002 did not block Src/Lyn activation. These results suggest that Ech A initially induces Src/Lyn activation, upregulates p110δ expression, and finally activates the PI3K/Akt pathway. CD34+ cells expanded in the presence of Ech A produced equal or more hematopoietic colony-forming cells than unexpanded CD34+ cells. In conclusion, Ech A promotes the ex vivo expansion of CD34+ cells through Src/Lyn-mediated p110δ expression, suppression of ROS generation, and p38-MAPK/JNK activation. Hence, Ech A is a potential candidate modality for the ex vivo, and possibly in vivo, expansion of CD34+ cells.

Description: Recombinant human erythropoietin (EPO, epoetin) is used widely for treatment of chronic anemia due to renal failure, cancer, and other causes. However, considerably high and frequent doses of EPO are required to maintain therapeutic effectiveness, since it has a relatively short in vivo half-life. Thus, alternatives with higher efficacy and/or longer half-life are being developed. We have shown previously that EPO-dimers, either produced by chemical cross-linking of monomeric EPO or expressed as a recombinant fusion protein from COS cells, exhibit enhanced biological properties in vitro and in vivo (Sytkowski, et.al. Proc. Natl. Acad. Sci. USA 95, 1184; Sytkowski, et.al. J. Biol. Chem. 274, 24773). We now report increased activities of EPO-dimer fusion protein and EPO-trimer fusion protein comprised of identical head-to-tail repeats and a 15 or 20-amino acid linker (for dimer), or 17-amino acid linkers (for trimer) produced from stably transfected CHO cells. EPO-fusion proteins were expressed under a CMV promoter with a signal peptide present on the first monomer coding sequence. The EPO-dimer fusion protein was connected with either three or four repeats of Gly-Gly-Gly-Gly-Ser as a 15 or 20-amino acid linker sequence, respectively. The expression levels of EPO-dimer fusion protein from cloned CHO cells to supernatant of protein-free medium ranged from 4 to 40 mg/L determined by EPO-ELISA, and from 2.0×105 to 4.5×106 IU/L determined by in vitro bioassay. We selected clones producing EPO-dimer fusion protein with the greatest extent of glycosylation, as indicated by SDS-PAGE and isoelectric focusing. Subcutaneous injection of mice with three doses of EPO-dimer fusion protein resulted in percent increases in mean hematocrit of 32.6% (300 IU/kg) or 18.2% (100 IU/kg), while equivalent unit doses of EPO-monomer increased mean hematocrit by 12.5% (300 IU/kg) or 6.4% (100 IU/kg). Moreover, a single dose of EPO-dimer fusion protein (100 IU/kg) increased their mean hematocrit by 4.3% within 7 days, while an equivalent unit dose of EPO-monomer had no effect. Importantly, three doses of EPO-trimer fusion protein increased their mean hematocrit by 8.83% per IU injected, which was much greater than that observed with EPO-monomer (0.69%) or EPO-dimer fusion protein (1.81%). The results show that EPO-fusion proteins exhibit biological activities superior to those of EPO-monomer, suggesting important therapeutic advantages.

Description: Abstract 2507 Poster Board II-484 Although the etiology of anemia remains unexplained in one third of elderly subjects, increasing evidence implicates chronic inflammation in the pathogenesis of anemia in the elderly population. We hypothesize that in addition to classical anemia of inflammation (AI) mediated by hepcidin, a significant proportion of unexplained anemia involves the overexpression of proinflammatory cytokines like TNFα that promote direct suppression of erythroid progenitors to a degree more prominent than the effects of hepcidin, resulting in the development of unexplained anemia marked by absence of the expected biochemical profile of AI. A better understanding of the pathophysiology of anemia subtypes is necessary if we are to develop effective targeted, oral therapies for patients. In the current study, we aimed to evaluate whether resveratrol, a flavanol found in high concentration in red wine that appears to possess potent anti-inflammatory properties, ameliorates TNFα-mediated erythroid colony suppression in human CD34+ cells. Human peripheral blood CD34+ cells from 3 healthy adults were first cultured in methylcellulose medium in the presence of IL-3 (10ng/ml), EPO (1U/ml), SCF (50ng/ml) and increasing doses of TNFα (0-100ng/ml) and erythroid burst-forming units (BFU-E) measured on day 14. Consistent with prior experiments, the percentage of BFU-E colonies was reduced by 60% at day 14 in the presence of 25 and 100ng/ml TNFα (p 〈 0.01, t-test), which was reversed to baseline with the addition of the neutralizing anti-TNFα antibody infliximab (p 〈 0.01, t-test). In order to determine whether the addition of resveratrol moderated TNFα-mediated erythroid colony suppression, human CD34+ cells were initially pre-incubated with increasing doses of resveratrol (0-50μM) for 72 hours and cultured in methylcellulose medium containing IL-3 (10ng/ml), EPO (1U/ml), and SCF (50ng/ml) in the presence or absence of 25ng/ml TNFα and varying concentrations of resveratrol (0-50μM) and BFU-E measured on day 14. We noted 50% recovery in BFU-E colony numbers at day 14 in CD34+ cells grown in the presence of 10μM resveratrol and TNFα in comparison with TNFα alone (p 〈 0.02, t-test). To better characterize TNFα-mediated pathways influenced by resveratrol, we analyzed in parallel CD34+ cells pre-incubated for 72 hours with 10μM resveratrol and grown in serum-free liquid culture containing similar concentrations of IL-3, SCF, and EPO in the presence or absence of 25ng/ml of TNFα and 10μM resveratrol. Total RNA and protein were isolated at multiple timepoints after the addition of TNFα on culture day 4 and TNFα-mediated gene and protein expression as well as hematopoietic microRNAs miR155 and miR451 expression analyzed by real-time RT-PCR and/or Western blot. We found that TNFα-treated CD34+ cells exhibited a 5-fold increased expression of miR155 without change in miR451 expression at 12 hours post TNFα treatment compared with control cells. However, CD34+ cells cultured in the presence of 10μM resveratrol and TNFα revealed downregulation of miR155 expression to baseline levels without change in miR451 expression (p 〈 0.01, t-test). Resveratrol treatment also resulted in 2-fold increased expression of GATA-1 without an appreciable change in GATA-2 (p 〈 0.01). Western analysis of whole cell lysates using antibodies recognizing known TNFα-associated signaling pathways revealed robust increases in phospho-p38MAPK, phospho-JNK, and phospho-ERK 1/2 in response to TNFα stimulation compared with control, with no inhibition of these signaling pathways noted in response to resveratrol treatment. We next examined TNFα-mediated NF-κB signaling in the presence or absence of resveratrol by Western analysis and found that the addition of resveratrol resulted in a 50% reduction in phospho-NF-κB levels in response to TNFα, with total NF-κB levels unchanged in all conditions. These data indicate that resveratrol significantly ameliorates TNFα-mediated erythroid colony suppression in human CD34+ cells via novel mechanisms that incorporate downregulation of both miR155 and NF-κB-associated signaling pathways, providing important evidence that resveratrol may be a potentially effective agent in the treatment of TNFα-mediated anemia and inflammation. Disclosures: No relevant conflicts of interest to declare.