The anticancer prodrug butyroyloxymethyl diethylphosphate (AN-7), upon metabolic hydrolysis, releases the histone deacetylase inhibitor butyric acid and imparts histone hyperacetylation. We have ...shown previously that AN-7 increases doxorubicin-induced cancer cell death and reduces doxorubicin toxicity and hypoxic damage to the heart and cardiomyocytes. The cardiofibroblasts remain unprotected against both insults. Herein we examined the selective effect of AN-7 on hypoxic cardiomyocytes and cardiofibroblasts and investigated mechanisms underlying the cell specific response. Hypoxic cardiomyocytes and cardiofibroblasts or H2O2-treated H9c2 cardiomyoblasts, were treated with AN-7 and cell damage and death were evaluated as well as cell signaling pathways and the expression levels of heme oxygenase-1 (HO-1). AN-7 diminished hypoxia-induced mitochondrial damage and cell death in hypoxic cardiomyocytes and reduced hydrogen peroxide damage in H9c2 cells while increasing cell injury and death in hypoxic cardiofibroblasts. In the cell line, AN-7 induced Akt and ERK survival pathway activation in a kinase-specific manner including phosphorylation of the respective downstream targets, GSK-3β and BAD. Hypoxic cardiomyocytes responded to AN-7 treatment by enhanced phosphorylation of Akt, ERK, GSK-3β and BAD and a significant 6-fold elevation in HO-1 levels. In hypoxic cardiofibroblasts, AN-7 did not activate Akt and ERK beyond the effect of hypoxia alone and induced a limited (~1.5-fold) increase in HO-1. The cell specific differences in kinase activation and in heme oxygenase-1 upregulation may explain, at least in part, the disparate outcome of AN-7 treatment in hypoxic cardiomyocytes and hypoxic cardiofibroblasts.
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•HDAC inhibition by AN-7 protects cardiomyocytes not cardiofibroblasts against hypoxia.•Akt and ERK survival pathway activation mediates the AN-7 protection.•HO-1 upregulation via Akt and ERK activation underlies AN-7 protection.•Differences in kinase activation and HO-1 induction explains the discrepancy in protection.
Popeye domain containing1 (Popdc1), also named Bves, is an evolutionary conserved membrane protein. Despite its high expression level in the heart little is known about its membrane localization and ...cardiac functions. The study examined the hypothesis that Popdc1 might be associated with the caveolae and play a role in myocardial ischemia tolerance. To address these issues, we analyzed hearts and cardiomyocytes of wild type and Popdc1-null mice. Immunoconfocal microscopy revealed co-localization of Popdc1 with caveolin3 in the sarcolemma, intercalated discs and T-tubules and with costameric vinculin. Popdc1 was co-immunoprecipitated with caveolin3 from cardiomyocytes and from transfected COS7 cells and was co-sedimented with caveolin3 in equilibrium density gradients. Caveolae disruption by methyl-β-cyclodextrin or by ischemia/reperfusion (I/R) abolished the cellular co-localization of Popdc1 with caveolin3 and modified their density co-sedimentation. The caveolin3-rich fractions of Popdc1-null hearts redistributed to fractions of lower buoyant density. Electron microscopy showed a statistically significant 70% reduction in caveolae number and a 12% increase in the average diameter of the remaining caveolae in the mutant hearts. In accordance with these changes, Popdc1-null cardiomyocytes displayed impaired Ca(+2)i transients, increased vulnerability to oxidative stress and no pharmacologic preconditioning. In addition, induction of I/R injury to Langendorff-perfused hearts indicated a significantly lower functional recovery in the mutant compared with wild type hearts while their infarct size was larger. No improvement in functional recovery was observed in Popdc1-null hearts following ischemic preconditioning. The results indicate that Popdc1 is a caveolae-associated protein important for the preservation of caveolae structural and functional integrity and for heart protection.
Abstract How chemotherapy affects dormant ovarian primordial follicles is unclear. The ‘burnout’ theory, studied only in mice, suggests cyclophosphamide enhances primordial follicle activation. Using ...4-hydroperoxycyclophosphamide (4hc) and phosphoramide mustard (PM), this study assessed how the active cyclophosphamide metabolites 4-hydroxycyclophosphamide (4-OHC) and PM, affect human primordial follicles. Frozen-thawed human ovarian samples were sliced and cultured with basic culture medium (cultured controls) or with 4hc/PM (3 µmol/l/10 µmol/l) (treated samples) for 24–48 h. Follicular counts and classification, Ki67 and anti-Müllerian hormone (AMH) immunohistochemistry and an apoptosis assay were used for evaluation, and 17β-oestradiol and AMH were measured in spent media samples. Generally, there was primordial follicle decrease and elevated developing follicle rates in treated samples compared with cultured ( P = 0.04 to P < 0.0005) and uncultured controls ( P < 0.05 to P < 0.0001). No traces of apoptosis were found. There were almost twicethe levels of AMH and 17β-oestradiol in treated compared with untreated samples (AMH with 4hc 3 µmol/l; P = 0.04). All follicles stained positively for AMHincluded treated samples. Ki67 positive staining was noted in all samples. Cyclophosphamide metabolites seem to enhance human primordial follicle activation to developing follicles, in vitro . Study findings support the ‘burnout’ theory as the mechanism of chemotherapy-induced ovarian toxicity.
Abstract Tumor progression is substantially dependent on network of multiple factors, including adhesion and homing molecules, which support the malignant metastatic spread. CD44, one of the ...adhesion/homing molecules, has attracted much attention not only because it is expressed on many types of tumors, but also owing to its numerous functions, such as supporting cell migration and transmitting survival signals, thereby being pro-oncogenic by nature. We have used the mouse malignant LB lymphoma cell line as a model for comprehensive in vitro and in vivo analyses of the interaction between CD44 and hyaluronic acid (HA), and its relevance to tumor dissemination. The in vitro studies revealed that LB cells could not bind HA, either under static or dynamic (i.e., shear flow) conditions, unless their CD44 is activated by phorbol ester, deglycosylated (to increase the CD44 positive net charge) or transfected with CD44 variants. In parallel, in vivo experiments showed that LB cell dissemination could be controlled by injection of anti-CD44 monoclonal antibodies or hyaluronidase. Furthermore, LB cells transfected with CD44v4–v10 variant, rather than standard CD44, displayed enhanced invasion of the peripheral lymph nodes. This effect was completely lost if the HA binding site of CD44 were mutated. LB cell accumulation in the lymph nodes is caused by enhanced migration via the afferent lymphatics rather than by accelerated proliferation within the lymph node. This information can be exploited to tailor a “therapeutic suit” that should be maximally effective in inducing tumor resistance, while minimizing destructive side effects.
Abstract Immune tolerance mechanisms supporting normal human pregnancy are exploited by breast cancer and other malignancies. We cloned from human placenta and breast cancer cells the novel human ...immunomodulator named placenta immunosuppressive ferritin (PLIF). PLIF is composed of a ferritin heavy chain–like domain and a novel cytokine-like domain, named C48. Both intact PLIF and C48 inhibit T cell proliferation. Blocking PLIF by specific antibodies in a tolerant breast cancer model in nude mice resulted in tumor cell apoptosis and rejection. This prompted us to study active immune preventive strategies targeting PLIF activity. Currently, we report on the design and synthesis of the novel C24D polypeptide, which inhibits the binding of PLIF to T cells and therefore inhibits the immune suppressive effect of PLIF. The effect of C24D on the generation of anti–breast cancer cytotoxic T lymphocytes (CTLs) was studied in vitro in cultures of MCF-7 (HLA-A2+ ) or T47D (HLA-A2− ) breast cancer cells incubated with peripheral blood mononuclear cells (PBMCs) from healthy blood donors. We found that C24D treatment exclusively induced development of CTLs. On reactivation by their specific target cells, the CTLs secreted interferon-γ and induced target apoptosis. Anti–MCF-7 CTLs were cross-cytotoxic to MDA-MB-231 (HLA-A2+ ) triple-negative breast cancer but not to T47D. Moreover, C24D treatment in vivo inhibited the growth of MCF-7 tumors engrafted in immune-compromised nude mice transfused with naïve allogeneic human PBMCs. Our results demonstrate that C24D treatment breakdown breast cancer induced tolerance enabling the initiation of effective anti-tumor immune response.
Several inflammatory mediators increase calcitriol production by epidermal keratinocytes. In turn calcitriol attenuates the keratinocyte inflammatory response. Since the effect of the in-situ ...generated calcitriol depends also on the sensitivity to the hormone we studied the effect of inflammatory cytokines on the response of HaCaT human keratinocytes to calcitriol by examining the expression and transcriptional activity of VDR. Treatment with TNF, but not with IL-1β or interferon γ, increased VDR protein level, while decreasing the level of its heterodimerization partner RXRα. This was associated with increased VDR mRNA levels. c-Jun N-terminal kinase, but not P38 MAPK or NFκB, was found to participate in the upregulation of VDR by TNF. The functional significance of the modulation of VDR and RXRα levels by TNF is manifested by increased induction of VDR target gene CYP24A1 by calcitriol. Calcitriol, in turn, inhibited the enhanced expression of VDR by TNF. In conclusion, the inflammatory cytokine TNF increases the response of keratinocytes to calcitriol through upregulation of its receptor VDR, which in turn is subject to negative feedback by the hormone accelerating the return of the keratinocyte vitamin D system to its basal activity. We surmise that the increased generation and sensitivity to calcitriol in keratinocytes play a role in the resolution of epidermal inflammation.
The possibility of maturing human primordial follicles in vitro would assist fertility restoration without the danger of reseeding malignancies. Leukaemia inhibitory factor (LIF) and certain culture ...matrices may promote human follicular growth. The present study compared human primordial follicular growth on novel culture matrices, namely human recombinant vitronectin (hrVit), small intestine submucosa (SIS), alginate scaffolds and human recombinant virgin collagen bioengineered in tobacco plant lines (CollPlant). The frozen-thawed ovarian samples that were used had been obtained from girls or young women undergoing fertility preservation. In the first part of the study, 20 samples were cultured for 6 days on hrVit or SIS with basic culture medium alone or supplemented with one of two concentrations of LIF (10ngmL
and 100ngmL
), with and without LIF-neutralising antibody. In the second part of the study, 15 samples were cultured for 6 days on alginate scaffolds or CollPlant matrices with basic culture medium. Follicular development was assessed by follicular counts and classification, Ki67 immunohistochemistry and 17β-oestradiol and anti-Müllerian hormone measurements in spent media samples. Primordial follicular growth was not enhanced by LIF. Despite some significant differences among the four matrices, none appeared to have a clear advantage, apart from significantly more Ki67-stained follicles on alginate and CollPlant matrices. Further studies of other culture matrices and medium supplements are needed to obtain an optimal system.
ERK6, a mitogen-activated protein (MAP) kinase-related serine/threonine kinase, is highly expressed in human skeletal muscle and appears to function as a signal transducer during differentiation of ...myoblasts to myotubes. In transfected 293 cells, activation of the 45-kDa enzyme results in tyrosine-phosphorylated 46- and 56-kDa forms, which phosphorylate myelin basic protein. Overexpression of wild-type ERK6 or the inactive mutant Y185F has no effect on fibroblast and myoblast proliferation, but it enhances or inhibits C2C12 cell differentiation to myotubes, respectively. Our findings suggest ERK6 to be a tissue-specific, differentiation signal-transducing factor that is connected to phosphotyrosine-mediated signaling pathways distinct from those activating other members of the MAP kinase family such as ERK1 and ERK2.
The recently cloned human gene named “placental immunoregulatory ferritin” (PLIF) is a pregnancyrelated immunomodulator. Recombinant PLIF and its bioactive domain C48 are immune-suppressive and ...induce pronounced IL-10 production by immune cells. PLIF is expressed in the placenta and breast cancer cells. Blocking PLIF in pregnant mice by anti-C48 antibodies inhibited placental and fetal growth and modulated the cytokine network. It has been revealed that anti-C48 treatment inhibited MCF-7 tumor growth in nude mice. However, this significant effect was observed only in those transfused with human peripheral blood mononuclear cells. Blocking PLIF in tumor-engrafted human immune cell transfused mice resulted in massive infiltration of human CD45+ cells (mainly CD8+ T cells), both intratumorally and in the tumor periphery, and a significant number of caspase-3+ cells. In vitro, antiC48 treatment of MCF-7 tumor cells cocultured with human lymphocytes induced a significant increase in interferon-γ secretion. We conclude that blocking PLIF inhibits breast cancer growth, possibly by an effect on the cytokine network in immune cells and on breakdown of immunosuppression.
Objective Cloned placenta immunoregulatory ferritin (PLIF) contains a novel, nonferritin bioactive domain (C-48) with immunodulatory activity. We documented that treatment of whole human bone marrow ...cells with PLIF and its subcloned C48 proteins resulted in myeloid progenitor cell growth and differentiation and T-cell suppression via an effect on the cytokine network. We tested whether this differential effect supports allogeneic bone marrow transplantation with long-lasting tolerance without any further treatments. Materials and Methods Splenocyte-enriched C3H (H2k ) whole bone marrow was transplanted into C57Bl (H2b ) recipients after total body irradiation. Recipients were injected with recombinant C48 (3mg/kg, intraperitoneal) for 21 days or with glutathione S-transferase. Animals were monitored for survival, chimerism, and clinical signs of graft-vs-host disease (GVHD). Next, chimera whole bone marrow was transplanted to secondary myeloablated C57Bl (H2b ) hosts without treatment. Results Mice that received C48 treatment following allogeneic splenocyte-enriched bone marrow transplantation demonstrated full-donor chimerism without GVHD mortality, and normal blood cell counts in 75% of recipients. Secondary transplants from the full chimera to myeloblated C57Bl hosts showed 100% engraftment, no GVHD mortality, and no impairment in the long-term hematopoietic reconstitution potential. Allogeneic response of spleen cells from secondary chimeras against donor C3H (H2k ) and recipient C57Bl (H2b ) were similar to syngeneic response, whereas reactivity to third party (DBA H2d ) was significantly enhanced. Conclusions Findings of this study provide the proof of concept that C48—a novel, single, bifunctional therapeutic modality enabled successful allogeneic, unmanipulated bone marrow transplantation without GVHD, and with lasting specific tolerance.
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