Reactive oxygen metabolites (ROMs), including superoxide anion (O2*-), hydrogen peroxide (H2O2) and hydroxyl radical (*OH), play an important role in carcinogenesis. There are some primary antioxidants such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) which protect against cellular and molecular damage caused by the ROMs. We conducted the present study to determine the rate of O2*- and H2O2 production, and concentration of malondialdehyde (MDA), as an index of lipid peroxidation, along with the SOD, GPx and CAT activities in 54 breast cancer (BC) patients. Forty-two age- and sex-matched patients with minor surgical problems, who had no history of any neoplastic or breast disorders, were taken as controls. The rate of O2*- production was significantly higher (p < 0.001) in BC patients than controls, irrespective of clinical stages and menopausal status. Similarly, H2O2 production was significantly higher in BC patients, especially in stage III and postmenopausal groups, as compared to the respective controls. MDA concentration was also observed significantly elevated in stage II (p < 0.001), stage III (p < 0.01), postmenopausal (p < 0.005), and premenopausal (p < 0.02) group as compared to their corresponding controls. SOD and GPx activities were found significantly raised in all the groups (p < 0.001), except the GPx activity was found a smaller alteration in stage IV (p < 0.02). On the contrary, CAT activity was found significantly depressed in all the study groups. The maximum depression was observed in stage II (-61.8%). Lower CAT activity in our study may be the effect of higher production of ROMs, particularly O2*- and *OH. SOD and GPx, however, were less effected by these higher ROMs production. The results of our study have shown a higher ROMs production and decreased CAT activity, which support the oxidative stress hypothesis in carcinogenesis. The relatively higher SOD and GPx may be due to the response of increased ROMs production in the blood. However, the higher SOD and GPx activities may be inadequate to detoxify high levels of H2O2 into H2O leading to the formation of the most dangerous *OH radical followed by MDA. Therefore, administration of CAT may be helpful in the management of BC patients. However, further elaborate clinical studies are required to evaluate the role of such antioxidant enzymes in BC management.

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Key points Natriuretic peptides (NPs) elicit their effects via multiple NP receptors (including NPR‐A, NPR‐B and NPR‐C, with NPR‐C being relatively poorly understood). We have studied the effects of NPR‐C ablation on cardiac structure, function and arrhythmogenesis using NPR‐C knockout (NPR‐C−/−) mice. NPR‐C−/− mice are characterized by sinoatrial node (SAN) dysfunction and a profound increase in susceptibility to atrial fibrillation. Increased susceptibility to arrhythmias in NPR‐C−/− mice was associated with slowed electrical conduction in the SAN as well as the right and left atria due to enhanced collagen expression and deposition in the atria (structural remodelling), but without changes in action potential morphology (electrical remodelling) in isolated SAN or atrial myocytes. This study demonstrates a critical protective role for NPR‐C in the heart. Abstract Natriuretic peptides (NPs) are critical regulators of the cardiovascular system that are currently viewed as possible therapeutic targets for the treatment of heart disease. Recent work demonstrates potent NP effects on cardiac electrophysiology, including in the sinoatrial node (SAN) and atria. NPs elicit their effects via three NP receptors (NPR‐A, NPR‐B and NPR‐C). Among these receptors, NPR‐C is poorly understood. Accordingly, the goal of this study was to determine the effects of NPR‐C ablation on cardiac structure and arrhythmogenesis. Cardiac structure and function were assessed in wild‐type (NPR‐C+/+) and NPR‐C knockout (NPR‐C−/−) mice using echocardiography, intracardiac programmed stimulation, patch clamping, high‐resolution optical mapping, quantitative polymerase chain reaction and histology. These studies demonstrate that NPR‐C−/− mice display SAN dysfunction, as indicated by a prolongation (30%) of corrected SAN recovery time, as well as an increased susceptibility to atrial fibrillation (6% in NPR‐C+/+ vs. 47% in NPR‐C−/−). There were no differences in SAN or atrial action potential morphology in NPR‐C−/− mice; however, increased atrial arrhythmogenesis in NPR‐C−/− mice was associated with reductions in SAN (20%) and atrial (15%) conduction velocity, as well as increases in expression and deposition of collagen in the atrial myocardium. No differences were seen in ventricular arrhythmogenesis or fibrosis in NPR‐C−/− mice. This study demonstrates that loss of NPR‐C results in SAN dysfunction and increased susceptibility to atrial arrhythmias in association with structural remodelling and fibrosis in the atrial myocardium. These findings indicate a critical protective role for NPR‐C in the heart.

Previous studies have shown that ventricular myocytes from female rats have smaller contractions and Ca(2+) transients than males. As cardiac contraction is regulated by the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway, we hypothesized that sex differences in cAMP contribute to differences in Ca(2+) handling. Ca(2+) transients (fura-2) and ionic currents were measured simultaneously (37°C, 2Hz) in ventricular myocytes from adult male and female C57BL/6 mice. Under basal conditions, diastolic Ca(2+), sarcoplasmic reticulum (SR) Ca(2+) stores, and L-type Ca(2+) current did not differ between the sexes. However, female myocytes had smaller Ca(2+) transients (26% smaller), Ca(2+) sparks (6% smaller), and excitation-contraction coupling gain in comparison to males (23% smaller). Interestingly, basal levels of intracellular cAMP were lower in female myocytes (0.7±0.1 vs. 1.7±0.2fmol/μg protein; p<0.001). Importantly, PKA inhibition (2μM H-89) eliminated male-female differences in Ca(2+) transients and gain, as well as Ca(2+) spark amplitude. Western blots showed that PKA inhibition also reduced the ratio of phospho:total RyR2 in male hearts, but not in female hearts. Stimulation of cAMP production with 10μM forskolin abolished sex differences in cAMP levels, as well as differences in Ca(2+) transients, sparks, and gain. To determine if the breakdown of cAMP differed between the sexes, phosphodiesterase (PDE) mRNA levels were measured. PDE3 expression was similar in males and females, but PDE4B expression was higher in female ventricles. The inhibition of cAMP breakdown by PDE4 (10μM rolipram) abolished differences in Ca(2+) transients and gain. These findings suggest that female myocytes have lower levels of basal cAMP due, in part, to higher expression of PDE4B. Lower cAMP levels in females may attenuate PKA phosphorylation of Ca(2+) handling proteins in females, and may limit positive inotropic responses to stimulation of the cAMP/PKA pathway in female hearts.

Phosphodiesterases (PDEs) are critical regulators of cyclic nucleotides in the heart. In ventricular myocytes, the L-type Ca2+ current (ICa,L) is a major target of regulation by PDEs, particularly members of the PDE2, PDE3 and PDE4 families. Conversely, much less is known about the roles of PDE2, PDE3 and PDE4 in the regulation of action potential (AP) properties and ICa,L in the sinoatrial node (SAN) and the atrial myocardium, especially in mice. Thus, the purpose of our study was to measure the effects of global PDE inhibition with Isobutyl-1-methylxanthine (IBMX) and selective inhibitors of PDE2, PDE3 and PDE4 on AP properties in isolated mouse SAN and right atrial myocytes. We also measured the effects of these inhibitors on ICa,L in SAN and atrial myocytes in comparison to ventricular myocytes. Our data demonstrate that IBMX markedly increases spontaneous AP frequency in SAN myocytes and AP duration in atrial myocytes. Spontaneous AP firing in SAN myocytes was also increased by the PDE2 inhibitor erythro-9-[2-hydroxy-3-nonyl] adenine (EHNA), the PDE3 inhibitor milrinone (Mil) and the PDE4 inhibitor rolipram (Rol). In contrast, atrial AP duration was increased by EHNA and Rol, but not by Mil. IBMX also potently, and similarly, increased ICa,L in SAN, atrial and ventricular myocytes; however, important differences emerged in terms of which inhibitors could modulate ICa,L in each myocyte type. Consistent with our AP measurements, EHNA, Mil and Rol each increased ICa,L in SAN myocytes. Also, EHNA and Rol, but not Mil, increased atrial ICa,L. In complete contrast, no selective PDE inhibitors increased ICa,L in ventricular myocytes when given alone. Thus, our data show that the effects of selective PDE2, PDE3 and PDE4 inhibitors are distinct in the different regions of the myocardium indicating important differences in how each PDE family constitutively regulates ion channel function in the SAN, atrial and ventricular myocardium.

Septicaemia is a major threat to survival during the early stages of life. There are several reports that suggest that reactive oxygen species (ROs) play a role in a wide variety of diseases. We estimated the activity of xanthine oxidase (XO), malondialdehyde (MDA) content, creatine phosphokinase (CPK) activity, activities of key enzymatic antioxidants, such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and peroxidase (PO), and non-enzymatic antioxidants, viz. uric acid (UA) and albumin (ALB), in 30 neonates with sepsis and 20 age-matched controls. The babies were categorized as preterm/term, early onset/late onset, and shock/without shock, as per clinical and laboratory investigations. The study was carried out to evaluate the status of antioxidant enzymes and non-enzymatic antioxidants with a view to suggesting the introduction of antioxidant therapy in neonatal sepsis. The activities of serum XO, CPK, SOD and GPx, and the content of MDA were found to be significantly elevated in the neonates with sepsis when compared with controls. Conversely, the activity of PO and the levels of UA and ALB were decreased. The septic, full-term neonates registered significantly higher CPK activity (70%) than the preterm septic neonates. However, infants with late-onset and shock sepsis had a significant decrease in CPK activity (p < 0.05) compared with their corresponding sub-groups. Likewise, UA levels were found to be 28% depressed (p < 0.05) in the babies with late-onset sepsis and 51% increased (p < 0.001) in babies with shock compared with their respective sub-groups. Neonates with septic shock also registered a significant elevation in GPx activity (28%) compared with those without shock. This study suggests increased production of ROs in neonates with sepsis, as evidenced by the positive regulation of XO, SOD and GPx activity. The elevation of antioxidant enzymes, however, was not so effective as to protect from cellular damage and thereby result in higher MDA production. It is evident that antioxidant therapy might be useful in the management of neonates with sepsis but further detailed clinico-biochemical investigations are required to define effective antioxidant therapy.

Resistance of solid tumor cells to anoikis, apoptosis induced by cell detachment from the extracellular matrix, is thought to be critical for the ability of these cells to grow anchorage independently within thee-dimensional tumor masses and from metastases. ␤-Catenin, a major oncoprotein, can inhibit anoikis of cancer cells via unknown mechanisms. In an effort to identify these mechanisms we found that ␤-catenin blocks anoikis of malignant kidney and intestinal epithelial cells and promotes their anchorage-independent growth by down-regulating death-associated protein kinase-2 (DAPk-2), a pro-apoptotic protein whose cellular functions have so far remained unexplored. We found that ␤-catenin-induced down-regulation of DAPk-2 requires the presence of the transcription factor Tcf-4, a known mediator of ␤-catenin signaling. We also observed that DAPk-2 contributes to the execution of anoikis of the non-malignant epithelial cells. Thus, ␤-catenin-induced down-regulation of DAPk-2 represents a novel signaling mechanism by which ␤-catenin promotes the survival of malignant epithelial cells following their detachment from the ECM and enables these cells to grow in an anchorage-independent manner.Epithelial cells of many organs grow in vivo as monolayers that are attached to a form of the extracellular matrix (ECM) 3 called the basement membrane (BM). Detachment from the ECM triggers apoptosis of these cells (1, 2), a phenomenon called anoikis (3). By contrast, carcinomas, cancers of epithelial origin, represent three-dimensional disorganized multicellular masses in which cell-BM contacts are significantly altered. It is known in this regard that during tumor progression cancer cells often secrete BM-degrading enzymes, and this allows tumors to invade adjacent tissues (4). Furthermore, at advanced stages of the disease clumps of cancer cells detach from the tumor and migrate to other organs where they give rise to metastases (5, 6). Despite the fact that carcinoma cells tend to be deprived of normal contacts with the BM during tumor progression, a significant fraction of these cells remains viable (5, 6). Numerous studies indicate that this viability is a critical prerequisite for carcinoma progression. First, cancer cells can typically survive and grow without adhesion to the ECM as colonies in soft agar, and this ability represents one of the most stringent criteria for malignant transformation that are presently used (7,8). Second, we and others found that activation of major oncoproteins, such as Ras (9, 10) or ␤-catenin (11), can block anoikis of various types of cancer cells. Third, several studies, including ours, indicate that treatments reversing anoikis resistance of cancer cells suppress their ability to form primary tumors (12-14) and metastases (6,14). Finally, we found that spontaneous acquisition of anoikis resistance by the non-malignant epithelial cells is sufficient for their in vivo tumorigenicity (15). Thus, anoikis resistance of tumor cells represents a potential therapeutic target. However, molecul...

2-Methoxyestradiol (2-ME(2)), a promising anticancer drug, induces growth arrest and apoptosis in various androgen-dependent (LNCaP) and -independent (DU145 and PC-3) prostate cancer cell lines. Moreover, flow cytometric analysis indicated a novel dual impact of 2-ME(2) on the cell division cycle of prostate cancer cells. Chronic exposure of high doses of 2-ME(2) enhance the accumulation of cells in S and G2/M phases, while cell numbers in the G1 phase were reduced significantly by this treatment. Because cyclin B1 overexpression, induction of cdc2 phosphorylation, and its regulatory proteins wee1 and phospho-cdc25C (interphase and mitotic forms) by 2-ME(2) treatment correlated with the induction of apoptosis, growth arrest at the G2/M phase, and accumulation of the S phase, we reasoned that cyclin B1 and cdc2 phosphorylation and its upstream regulatory molecular networks may be associated with the ultimate impacts of 2-ME(2). Because phosphorylation of cdc2 and upregulation of wee1 by 2-ME(2) can be abolished by both extracellular receptor kinase (ERK) inhibitor (U0126) and c-Jun N-terminal kinase (JNK) inhibitor (SP600125), our studies indicate that the 2-ME(2)-induced upregulation of wee1 and subsequent cdc2 phosphorylation are mediated through mitogen-activated protein kinase (MAPK)-ERK-JNK signaling pathways.