Ubiquitin ligase Cbl-b sensitizes leukemia and gastric cancer cells to anthracyclines by activating the mitochondrial pathway and modulating Akt and ERK survival signals

The present study reported that the ubiquitin ligase Cbl-b was up-regulated during anthracycline-induced apoptosis in two cell lines, RBL-2H3 leukemia cells and MGC803 gastric cancer cells. Overexpression of Cbl-b strongly promoted the cytotoxic and apoptosis-inducing effects of anthracyclines, while a dominant negative (DN) Cbl-b mutation abolished these effects in both cell lines. Further investigation revealed that mitochondrial depolarization was enhanced by Cbl-b and decreased by Cbl-b (DN) in RBL-2H3 cells. Moreover, overexpression of Cbl-b significantly suppressed ERK activation, and Cbl-b (DN) strongly enhanced both ERK and Akt activation. Altogether, these results indicate that Cbl-b sensitized both leukemia and gastric cancer cells to anthracyclines by activating the mitochondrial apoptotic pathway and modulating the ERK and Akt survival pathways.     

Freeze-fracture study of plasma membranes in wild type and daunorubicin-resistant Ehrlich ascites tumor and P388 leukemia cells

The plasma membrane is considered to play a major role in the development and maintenance of the multidrug resistance (MDR) phenotype, a role which may in part be mediated by an inducible 170 kD transmembrane protein (P-170). The present freeze-fracture study of plasma membranes of daunorubicin-resistant Ehrlich ascites and P388 leukemia cells demonstrated a significant increase in the density of intramembrane particles (IMP) in the P-face, but not the E-face, of resistant sublines compared with wild type cells. Furthermore, a three-dimensional histogram plot of the diameters of P-face IMPs in Ehrlich ascites tumor cells showed the emergence of a subpopulation of 9 × 11 nm IMPs not found in wild type cells. The size of these IMPs would be consistent with a MW of approximately 340 kD, thus indicating that P-170, shown to be present in both resistant cell lines by Western blot analysis and immunohistochemical staining, exists as a dimer in the plasma membrane. Incubation with the calcium channel blocker verapamil, in concentrations known to inhibit daunorubicin efflux in resistant cells, showed evidence of membrane disturbance in the form of IMP clustering in both wild type and resistant Ehrlich ascites tumor cells. However, incubation with daunorubicin itself did not alter the freeze-fracture morphology of the plasma membranes.     

Reduced creatine-stimulated respiration in doxorubicin challenged mitochondria: Particular sensitivity of the heart

Doxorubicin (DXR) belongs to the most efficient anticancer drugs. However, its use is limited by a risk of cardiotoxicity, which is not completely understood. Recently, we have shown that DXR impairs essential properties of purified mitochondrial creatine kinase (MtCK), with cardiac isoenzyme (sMtCK) being particularly sensitive. In this study we assessed the effects of DXR on respiration of isolated structurally and functionally intact heart mitochondria, containing sMtCK, in the presence and absence of externally added creatine (Cr), and compared these effects with the response of brain mitochondria expressing uMtCK, the ubiquitous, non-muscle MtCK isoenzyme. DXR impaired respiration of isolated heart mitochondria already after short-term exposure (minutes), affecting both ADP- and Cr-stimulated respiration. During a first short time span (minutes to 1 h), detachment of MtCK from membranes occurred, while a decrease of MtCK activity related to oxidative damage was only observed after longer exposure (several hours). The early inhibition of Cr-stimulated respiration, in addition to impairment of components of the respiratory chain involves a partial disturbance of functional coupling between MtCK and ANT, likely due to interaction of DXR with cardiolipin leading to competitive inhibition of MtCK/membrane binding. The relevance of these findings for the regulation of mitochondrial energy production in the heart, as well as the obvious differences of DXR action in the heart as compared to brain tissue, is discussed.     

Screening of terrestrial <Emphasis Type="Italic">Streptomyces</Emphasis> leading to identification of new stereoisomeric anthracyclines

Thirty different Streptomyces strains were isolated from soil samples collected from different places in Egypt. The strains were isolated using Oatmeal agar and ISP medium 2 agar at pH 7.5. The isolate Streptomyces sp. Eg23 was selected for further working up to yield three new streoisomers of anthracycline metabolites. The structures were elucidated as (7R, 9R, 10S)-e-Rhodomycinone (1), (7R, 9R, 10S) Aklavinone (2), and (9R, 10S) 7-Desoxy-z-Rhodomycinone (3) by interpretation of their 1D and 2D NMR spectra. The absolute stereochemistry of 1 was confirmed by modified Mosher’s method. The (7R, 9R, 10S)-e-Rhodomycinone (1) showed activity against human lung cancer cell H460, murine Lewis lung cancer cell LL/2 and human breast cancer cell MCF-7. Compounds 1–3 were known synthetically, but until now have not been isolated as natural products from a microorganism. The results showed that the Streptomyces sp. Eg23 seems to be an interesting target for studying the regio-and stereochemistry of the cyclization step catalysed by polyketide cyclases to produce new anthracyclines through combinatorial biosynthesis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

1H NMR-visible mobile lipid domains correlate with cytoplasmic lipid bodies in apoptotic T-lymphoblastoid cells

The presence of nuclear magnetic resonance (NMR)-visible mobile lipid (ML) domains in apoptotic lymphoblasts suggests alterations in neutral lipid metabolism and compartmentation during programmed cell death. The detection of similar ML signals in activated lymphocytes raises questions about common mechanisms of ML formation during apoptosis and upon lymphoblast stimulation. Structure and subcellular localization of ML domains were therefore investigated by NMR, fluorescence and electron microscopy in Jurkat T-lymphoblasts either induced to apoptosis (by anthracyclines or dexamethasone or by serum deprivation) or activated by phorbol myristate acetate (PMA) plus ionomycin. ML contents in drug-treated cells correlated linearly with apoptosis, irrespective of the specific inducer and cell cycle arrest phase (r=0.993, P<0.001). Similar ML levels were measured in drug-induced apoptotic cells (A≈30–40%) and in non-apoptotic PMA/ionomycin-treated lymphoblasts (72 h). Lower ML contents were instead formed in serum-deprived apoptotic cells, with respect to controls. Increases in ML signals were associated, in either apoptotic or activated cells, with the accumulation of cytoplasmic, osmophilic lipid bodies (diameter≤1.0 μm), surrounded by own membrane, possessing intramembrane particles. The results support the hypothesis that ML are formed in the cytoplasm of drug-induced apoptotic cells during an early, ‘biochemically active’ phase of programmed cell death.     

Thiol Oxidation Induced by Oxidative Action of Adriamycin

To clarify the mechanism of the cardiotoxic action of adriamycin (ADM), the participation of free radicals from ADM in cardiotoxicity was investigated through the protective action of glutathione (GSH) or by using electron spin resonance (ESR). Oxidation of ADM by horseradish peroxidase and H₂O₂ (HRP-H₂O₂) was blocked by GSH concentration dependently. Inactivation of creatine kinase (CK) induced during interaction of ADM with HRP-H₂O₂ was also protected by GSH. Other anthracycline antitumor drugs that have a p-hydroquinone structure in the B ring also inactivated CK, and GSH inhibited the inactivation of CK. These results suggest that ADM was activated through oxidation of the p-hydroquinone in the B ring by HRP-H₂O₂. Although ESR signals of the oxidative ADM B ring semiquinone were not detected, glutathionyl radicals were formed during the interaction of ADM with HRP-H₂O₂ in the presence of GSH. ADM may be oxidized to the ADM B ring semiquinone and then reacts with the SH group. However, ESR signals of ADM C ring semiquinone, which was reductively formed by xanthine oxidase (XO) and hypoxanthine (HX) under anaerobic conditions, were not diminished by GSH, but they completely disappeared with ferric ion. These results indicate that oxidative ADM B ring semiquinones oxidized the SH group in CK, but reductive ADM C ring semiquinone radicals may participate in the oxidation of lipids or DNA and not of the SH group.     

Leukocyte 8-oxo-7,8-dihydro-2′-deoxyguanosine and comet assay in epirubicin-treated patients

Epirubicin fights cancer through topoisomerase II inhibition, hence producing DNA strand breaks that finally lead to cell apoptosis. But anthracyclines produce free radicals that may explain their adverse effects. Dexrazoxane—an iron chelator—was proven to decrease free radical production and anthracycline cardiotoxicity.

In this article, we report the concentrations of cellular 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo) relative to 2′-deoxyguanosine (dGuo), and comet assay results from a study including 20 cancer patients treated with epirubicin. Plasma concentrations of vitamins A, E, C and carotenoids are also reported. All data were obtained before and immediately after epirubicin infusion. The ratios of 8-Oxo-dGuo to dGuo were measured in leukocyte DNA by HPLC-coulometry after NaI extraction of nucleic acids. Vitamins A and E and carotenoids were measured by HPLC-spectrophotometry. Vitamin C was measured by HPLC-spectrofluorimetry.

Median 8-oxo-dGuo/dGuo ratios increased significantly from 0.34 to 0.48 lesions per 100,000 bases while per cent of tail DNA increased from 3.47 to 3.94 after chemotherapy 8-Oxo-dGuo/dGuo and per cent of tail DNA medians remained in the normal range. Only vitamin C decreased significantly from 55.4 to 50.3?μM Decreases in vitamins A, E, lutein and zeaxanthin were not significant, but concentrations were below the lower limit of the normal range both before and after chemotherapy. Only the correlation between comet assay results and vitamin C concentrations was significant ( ).

This study shows that cellular DNA is damaged by epirubicin-generated free radicals which produce the mutagenic modified base 8-oxo-dGuo and are responsible for strand breaks. However, strand breaks are created not only by free radicals but also by topoisomerase II inhibition. In a previous study we did not find any significant change in urinary 8-oxo-dGuo excretion after adriamycin treatment. However, 8-oxo-dGuo may have increased at the end of urine collection as DNA repair and subsequent kidney elimination are relatively slow processes. In another study, authors used GC-MS to detect 8-oxo-dGuo in DNA and did not find any change after prolonged adriamycin infusion. Reasons for these apparent discrepancies are discussed.     

Stable RNA interference of ErbB-2 gene synergistic with epirubicin suppresses breast cancer growth in vitro and in vivo

Overexpression of human epidermal growth factor receptor-2 (Her2, ErbB-2) contributes to the progression and metastasis of breast cancer, implying that Her2 gene is a suitable target of RNA interference (RNAi) for breast cancer therapy. Here, we employed plasmid-mediated expression of 2 different Her2-shRNAs (pU6-Her2shRNAs) efficiently silenced the target gene expression on Her2 expressing SKBR-3 breast cancer cells in both mRNA and protein levels. Consequently, pU6-Her2shRNA increased apoptosis and reduced proliferation of SKBR-3 cells assayed by TUNEL and MTT, respectively. In vivo, intra-tumor injection of pU6-Her2shRNA inhibited the growth of SKBR-3 tumors inoculated subcutaneously in nude mice. Furthermore, pU6-Her2shRNA synergized the tumor suppression effect of epirubicin to SKBR-3 cells in vitro and implanted subcutaneously in nude mice. Therefore, we concluded that stable silencing of Her2 gene expression with plasmid expressing shRNA may hold great promise as a novel therapy for Her2 expressing breast cancers alone or in combination with anthracycline chemotherapy.     

Anthracycline cardiotoxicity in transgenic mice overexpressing SR Ca2+-ATPase

Chronic anthracycline administration results in a time- and dose-dependent cardiomyopathy. The Ca-ATPase of the sarcoplasmic reticulum, SERCA2, has been implicated as a principal target for anthracycline-induced cardiotoxicity. This hypothesis predicts that improved SERCA2 function would provide protection from cardiotoxic effects of anthracycline administration. Doxorubicin was administered (1.7 mg/kg three times weekly; cumulative dose of 20 mg/kg) to 10 transgenic mice that overexpressed SERCA2 and to 10 isogenic littermates. Survival was monitored for 60 days and histologic comparisons were made of cardiac tissue. Survival in the transgenic mice was worse (1/10 60-day survivors) compared to isogenic control mice (7/10 60-day survivors). There was a greater degree of histologic damage exhibited in hearts from transgenic mice compared to isogenic controls when all available hearts were examined. These data do not support a role of SERCA2 in ameliorating anthracycline cardiotoxicity.     

Erythropoietin protects cardiac myocytes against anthracycline-induced apoptosis

The cardiotoxic adverse effects of anthracycline antibiotics limit their therapeutic utility as essential components of chemotherapy regimens for hematologic and solid malignancies. Here we show that the hematopoietic cytokine erythropoietin attenuates doxorubicin-induced apoptosis of primary neonatal rat ventricular cardiomyocytes in a dose-dependent manner. Erythropoietin treatment induced rapid, time-dependent phosphorylation of MAP kinases (MAPK) Erk1/2 and the phosphatidylinositol 3-kinase substrate Akt. Treatment of cardiomyocytes with inhibitors of phosphatidylinositol 3-kinase (LY294002) or Akt (Akti-1/2) abolished the protective effect of erythropoietin, whereas treatment with MAPK kinase (MEK1) inhibitor U0126 did not. Erythropoietin also induced the phosphorylation of GSK-3beta, a downstream target of PI3K-Akt. Because phosphorylation is known to inactivate GSK-3beta, we investigated whether GSK-3beta inhibition is cardioprotective. We found that GSK-3beta inhibitors SB216763 or lithium chloride blocked doxorubicin-induced cardiomyocyte apoptosis in a manner similar to erythropoietin, suggesting that GSK-3beta inhibition is involved in erythropoietin-mediated cardioprotection. Erythropoietin may serve as a novel cardioprotective agent against anthracycline-induced cardiotoxicity.