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.
Intestinal metaplasia (IM) is an intermediate step in the progression from premalignant to malignant stages of gastric cancer (GC). The Popeye domain containing (POPDC) gene family encodes three ...transmembrane proteins, POPDC1, POPDC2, and POPDC3, initially described in muscles and later in epithelial and other cells, where they function in cell–cell interaction, and cell migration. POPDC1 and POPDC3 downregulation was described in several tumors, including colon and gastric cancers. We questioned whether IM-to-GC transition involves POPDC gene dysregulation. Gastric endoscopic biopsies of normal, IM, and GC patients were examined for expression levels of POPDC1-3 and several suggested IM biomarkers, using immunohistochemistry and qPCR. Immunostaining indicated lower POPDC1 and POPDC3 labeling in IM compared with normal tissues. Significantly lower POPDC1 and POPDC3 mRNA levels were measured in IM and GC biopsies and in GC-derived cell lines. The reduction in focal IM was smaller than in extensive IM that resembled GC tissues. POPDC1 and POPDC3 transcript levels were highly correlated with each other and inversely correlated with LGR5, OLFM4, CDX2, and several mucin transcripts. The association of POPDC1 and POPDC3 downregulation with IM-to-GC transition implicates a role in tumor suppression and highlights them as potential biomarkers for GC progression and prospective treatment targets.
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.
We studied the unique inhibitor of the histone deacetylases (HDAC) valproate-valpromide of acyclovir (AN446) that upon metabolic degradation release the HDAC inhibitor (HDACI) valproic acid (VPA). ...Among the HDAC inhibitors that we have tested, only AN446, and to a lesser extent VPA, synergized with doxorubicin (Dox) anti-cancer activity. Romidepsin (Rom) was additive and the other HDACIs tested were antagonistic. These findings led us to test and compare the anticancer activities of AN446, VPA, and Rom with and without Dox in the 4T1 triple-negative breast cancer murine model. A dose of 4 mg/kg once a week of Dox had no significant effect on tumor growth. Rom was toxic, and when added to Dox the toxicity intensified. AN446, AN446 + Dox, and VPA + Dox suppressed tumor growth. AN446 and AN446 + Dox were the best inhibitory treatments for tumor fibrosis, which promotes tumor growth and metastasis. Dox increased fibrosis in the heart and kidneys, disrupting their function. AN446 most effectively suppressed Dox-induced fibrosis in these organs and protected their function. AN446 and AN446 + Dox treatments were the most effective inhibitors of metastasis to the lungs, as measured by the gap area. Genes that control and regulate tumor growth, DNA damage and repair, reactive oxygen production, and generation of inflammation were examined as potential therapeutic targets. AN446 affected their expression in a tissue-dependent manner, resulting in augmenting the anticancer effect of Dox while reducing its toxicity. The specific therapeutic targets that emerged from this study are discussed.
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.
The histone deacetylase inhibitor (HDACI) butyroyloxymethyl diethylphosphate (AN-7) synergizes the cytotoxic effect of doxorubicin (Dox) and anti-HER2 on mammary carcinoma cells while protecting ...normal cells against their insults. This study investigated the concomitant changes occurring in heart tissue and tumors of mice bearing a subcutaneous 4T1 mammary tumor following treatment with AN-7, Dox, or their combination. Dox or AN-7 alone led to inhibition of both tumor growth and lung metastases, whereas their combination significantly increased their anticancer efficacy and attenuated Dox- toxicity. Molecular analysis revealed that treatment with Dox, AN-7, and to a greater degree, AN-7 together with Dox increased tumor levels of γH2AX, the marker for DNA double-strand breaks and decreased the expression of Rad51, a protein needed for DNA repair. These events culminated in increased apoptosis, manifested by the appearance of cytochrome-c in the cytosol. In the myocardium, Dox-induced cardiomyopathy was associated with an increase in γH2AX expression and a reduction in Rad51 and MRE11 expression and increased apoptosis. The addition of AN-7 to the Dox treatment protected the heart from Dox insults as was manifested by a decrease in γH2AX levels, an increase in Rad51 and MRE11 expression, and a diminution of cytochrome-c release. Tumor fibrosis was high in untreated mice but diminished in Dox- and AN-7-treated mice and was almost abrogated in AN-7+Dox-treated mice. By contrast, in the myocardium, Dox alone induced a dramatic increase in fibrosis, and AN7+Dox attenuated it. The high expression levels of c-Kit, Ki-67, c-Myc, lo-FGF, and VEGF in 4T1 tumors were significantly reduced by Dox or AN-7 and further attenuated by AN-7+Dox. In the myocardium, Dox suppressed these markers, whereas AN-7+Dox restored their expression. In conclusion, the combination of AN-7 and Dox results in two beneficial effects, improved anticancer efficacy and cardioprotection.
It is with great sadness that we have learned about the passing of Professor David Yaffe (1929-2020, Israel). Yehi Zichro Baruch - May his memory be a blessing. David was a man of family, science and ...nature. A native of Israel, David grew up in the historic years that preceded the birth of the State of Israel. He was a member of the group that established Kibbutz Revivim in the Negev desert, and in 1948 participated in Israel’s War of Independence. David and Ruth eventually joined Kibbutz Givat Brenner by Rehovot, permitting David to be both a kibbutz member and a life-long researcher at the Weizmann Institute of Science, where David received his PhD in 1959. David returned to the Institute after his postdoc at Stanford. Here, after several years of researching a number of tissues as models for studying the process of differentiation, David entered the myogenesis field and stayed with it to his last day. With his dedication to the field of myogenesis and his commitment to furthering the understanding of the People and the Land of Israel throughout the international scientific community, David organized the first ever myogenesis meeting that took place in Shoresh, Israel in 1975. This was followed by the 1980 myogenesis meeting at the same place and many more outstanding meetings, all of which brought together myogenesis, nature and scenery. Herein, through the preparation and publication of this current manuscript, we are meeting once again at a “David Yaffe myogenesis meeting". Some of us have been members of the Yaffe lab, some of us have known David as his national and international colleagues in the myology field. One of our contributors has also known (and communicates here) about David Yaffe’s earlier years as a kibbutznick in the Negev. Our collective reflections are a tribute to Professor David Yaffe. We are fortunate that the European Journal of Translational Myology has provided us with tremendous input and a platform for holding this 2020 distance meeting "Farwell to Professor David Yaffe - A Pillar of the Myogenesis Field".
To investigate the immunocytochemical expression and presence of mRNA transcripts for basic fibroblast growth factor (bFGF) and its four receptors (FGFR-1, -2, -3, and -4) in ovaries from human ...adults and fetuses.
Immunocytochemical and reverse transcription polymerase chain reaction (RT-PCR) study.
Major tertiary care and referral academic centers.
Nine women and girls undergoing laparoscopic ovarian biopsy and 26 women undergoing second- and third-trimester pregnancy terminations.
None.
Microscopic morphometric analysis, immunocytochemistry for bFGF and its receptors, and RT-PCR analyses.
The proteins for bFGF, FGFR-2, FGFR-3, and FGFR-4 were identified in oocytes of all follicular classes. Immunocytochemical expression of bFGF and its receptors was detected in granulosa cells of follicles from adolescents/women but not from fetuses. There was no immunocytochemical expression of FGFR-1. Transcripts of bFGF and its four receptors were identified by RT-PCR in all samples.
The expression of bFGF and its receptors in human ovaries suggests that bFGF might have a role in early folliculogenesis.
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