The inhibitory effect of salinomycin on the proliferation, migration and invasion of human endometrial cancer stem-like cells.

GOALS: We previously demonstrated that side-population (SP) cells in human endometrial cancer cells (Hec1 cells) and in rat endometrial cells expressing oncogenic human K-Ras protein (RK12V cells) have features of cancer stem cells (CSCs). Hec1-SP cells showed enhanced migration and the potential to differentiate into the mesenchymal cell lineage. In this study, we analyzed the association of the epithelial-mesenchymal transition (EMT) with the properties of these endometrial CSCs. We also assessed the effects of salinomycin (a compound with EMT-specific toxicity) on the proliferative capacity, migration and invasiveness of these endometrial CSCs using Hec1-SP cells. METHOD: We performed microarray expression analysis to screen for up-regulated genes in CSCs using a set of RK12V-SP cells and -non-SP(NSP) cells and used the Metacore package to identify the Gene GO pathway MAPs involved in the up-regulated genes. To analyze their association with EMT, the expression of several EMT associated genes in Hec1-SP cells was investigated by real time PCR and compared with that in Hec1-NSP cells. We assessed the expression of BAX, BCL2, LEF1, cyclinD and fibronectin by real time PCR. We also evaluated the viabilities, migration and invasive activities, and tumorigenicities of these SP cells and NSP cells in the presence or absence of salinomycin. RESULTS: We demonstrated that i) EMT processes were observed in both RK12V-SP cells and Hec1-SP cells, ii) the level of fibronectin was enhanced in Hec1-SP cells and salinomycin reduced the level of fibronectin expression, iii) salinomycin induced apoptosis and inhibited Wnt signaling, and iv) salinomycin inhibited the proliferation, migration, invasiveness and tumorigenicity of these SP cells. CONCLUSION: This is the first report of an inhibitory effect of salinomycin on the properties of endometrial CSCs.

Hypokalemic nephropathy is associated with impaired angiogenesis.

Hypokalemic nephropathy is associated with alterations in intrarenal vasoactive substances, leading to vasoconstriction, salt-sensitivity, and progression of interstitial fibrosis. In this study, we investigated whether hypokalemic nephropathy might also involve impaired renal angiogenesis. Sprague-Dawley rats that were fed low-potassium diets developed peritubular capillary loss that began in the inner stripe of the outer medulla (week 2) and progressed to the outer stripe of the outer medulla (week 4) and cortex (week 12). These changes were associated with increased macrophage infiltration, increased expression of both monocyte chemoattractant protein-1 and TNF-alpha, and a loss of vascular endothelial growth factor and endothelial nitric oxide synthase. Renal thiobarbituric acid-reactive substances, markers of oxidative stress, were increased late in disease. In conclusion, hypokalemic nephropathy is associated with impaired renal angiogenesis, evidenced by progressive capillary loss, reduced endothelial cell proliferation, and loss of VEGF expression.

Specific apoptosis induction in human papillomavirus-positive cervical carcinoma cells by photodynamic antisense regulation.

Human papillomavirus type 18 (HPV18) is frequently detected in cervical cancer cells. The viral proteins E6 and E7 are expressed consistently and have oncogenic activities. The E7 protein binds to a tumor suppressor, the retinoblastoma gene product (pRB), however, leading to the stabilization of tumor suppressor, p53 protein. On the other hand, another viral product, E6, forms complexes with p53 and abrogates its function, resulting in tumor progression. These facts imply that the E6 oncogene is one of the ideal targets for directed gene therapy in HPV-positive cervical cancer. In this study, we tried photodynamic antisense regulation of the antiapoptotic E6 expression using a photocross-linking reagent, 4,5',8-trimethylpsoralen, conjugated oligo(nucleoside phosphorothioate) (Ps-S-Oligo). This photodynamic antisense strategy effectively elicited the apoptotic death of HPV18-positive cervical cancer cells through the selective repression of E6 mRNA and consequent stabilization of p53 protein. E7-mediated signals potentially activated the p53 function and mobilized the p53 pathway to deliver pro-apoptotic signals to the cancer cells, leading to the suppression of in vivo tumorigenesis. An extremely low concentration of cisplatin in addition to Ps-S-Oligos further up-regulated p53 activity, provoking massive apoptotic induction. These results suggest that the photodynamic antisense strategy has the great therapeutic potential in HPV-positive cervical cancers.

An inhibitory effect on cell proliferation by blockage of the MAPK/estrogen receptor/MDM2 signal pathway in gynecologic cancer.

OBJECTIVE: We previously demonstrated that that the Ras/ER/MDM2 pathway was critical for NIH3T3 cell transformation. In this study, we examined the effect of blocking this pathway on cell growth in gynecologic cancer cells. METHODS: (1) The levels of MDM2, ER, p53 and p21 in endometrial or ovarian cancer cell lines were investigated and compared with that in normal cells by Western blots. (2) The effects of MEK-inhibitor and/or anti-estrogen, and siRNA of MDM2 on cell growth, tumorigenicity in nude mice were examined. RESULTS: The MDM2 level was enhanced in cancer cells compared with normal cells. Treatment with MEK inhibitor(U0126) resulted in a reduced MDM2 level, enhanced p53 and p21 levels and inhibited cell growth by the induction of premature senescence. The effect of MEK inhibitor on cell growth was affected by ER levels and functions. Treatment with low-dose MEK inhibitor in combination with anti-estrogen (ICI182,780) had a more inhibitory effect on cell growth compared to treatment with MEK inhibitor or anti-estrogen alone in cancer cells. Down-regulation of the MDM2 level by siRNA resulted in the inhibition of growth in cancer cells. CONCLUSION: The blockage of the MAPK/ER/MDM2 pathway suppress cell proliferation and it is supposed as a new molecular target therapy in estrogen-dependent gynecologic cancers, such as endometrial or ovarian cancer.

Alteration of glomerulogenesis- and podocyte structure-related gene expression in early diabetic nephropathy.

Diabetic nephropathy is a leading cause of end-stage renal disease. Several pathways, including the renin-angiotensin system, have been postulated as potential mechanisms of diabetic nephropathy. In addition, glomerulogenesis-related molecules are involved in the pathogenesis of diabetic nephropathy, especially at the early stage. They can be divided into three groups by function, that is, fibrosis-related, podocyte differentiation-related and angiogenesis-related molecules. Most of the molecules are expressed in the podocyte and upregulated, even during the normoalbuminuric stage. Expression of several podocyte structure-related molecules are also altered at the normoalbuminuric stage. They can contribute to the structural alteration of the podocyte in diabetic nephropathy. Thus, normalization of the expression of glomerulogenesis-related molecules could be a new target for preventing the initiation and progression of diabetic nephropathy.

Undernutrition in utero augments systolic blood pressure and cardiac remodeling in adult mouse offspring: possible involvement of local cardiac angiotensin system in developmental origins of cardiovascular disease.

Evidence has emerged that undernutrition in utero is a risk factor for cardiovascular disorders in adulthood, along with genetic and environmental factors. Recently, the local expression of angiotensinogen and related bioactive substances has been demonstrated to play a pivotal role in cardiac remodeling, i.e. fibrosis and hypertrophy. The aim of the present study was to clarify the possible involvement of the local cardiac angiotensin system in fetal undernutrition-induced cardiovascular disorders. We developed a mouse model of undernutrition in utero by maternal food restriction, in which offspring (UN offspring) showed an increase in systolic blood pressure (8 wk of age, P < 0.05; and 16 wk, P < 0.01), perivascular fibrosis of the coronary artery (16 wk, P < 0.05) and cardiac cardiomegaly (16 wk, P < 0.01), and cardiomyocyte enlargement, concomitant with a significant augmentation of angiotensinogen (P < 0.05) and endothelin-1 (P < 0.01) mRNA expression and a tendency to increase in immunostaining for both angiotensin II and endothelin-1 in the left ventricles (16 wk). These findings suggest that fetal undernutrition activated the local cardiac angiotensin system-associated bioactive substances, which contributed, at least partly, to the development of cardiac remodeling in later life, in concert with the effects of increase in blood pressure.

Altered gene expression related to glomerulogenesis and podocyte structure in early diabetic nephropathy of db/db mice and its restoration by pioglitazone.

Glomerular injury plays a pivotal role in the development of diabetic nephropathy. To elucidate molecular mechanisms underlying diabetic glomerulopathy, we compared glomerular gene expression profiles of db/db mice with those of db/m control mice at a normoalbuminuric stage characterized by hyperglycemia and at an early stage of diabetic nephropathy with elevated albuminuria, using cDNA microarray. In db/db mice at the normoalbuminuric stage, hypoxia-inducible factor-1alpha (HIF-1alpha), ephrin B2, glomerular epithelial protein 1, and Pod-1, which play key roles in glomerulogenesis, were already upregulated in parallel with an alteration of genes related to glucose metabolism, lipid metabolism, and oxidative stress. Podocyte structure-related genes, actinin 4alpha and dystroglycan 1 (DG1), were also significantly upregulated at an early stage. The alteration in the expression of these genes was confirmed by quantitative RT-PCR. Through pioglitazone treatment, gene expression of ephrin B2, Pod-1, actinin 4alpha, and DG1, as well as that of oxidative stress and lipid metabolism, was restored concomitant with attenuation of albuminuria. In addition, HIF-1alpha protein expression was partially attenuated by pioglitazone. These results suggest that not only metabolic alteration and oxidative stress, but also the alteration of gene expression related to glomerulogenesis and podocyte structure, may be involved in the pathogenesis of early diabetic glomerulopathy in type 2 diabetes.

Expression of progesterone receptor B is associated with G0/G1 arrest of the cell cycle and growth inhibition in NIH3T3 cells.

Previously, we found a significant reduction of progesterone receptor B (PR-B) expression levels in the Ras-mediated NIH3T3 cell transformation, and re-expression of exogenous PR-B eliminated the tumorigenic potential. We hypothesized that this reduction is of biological significance in cell transformation. In the present study, we determined the correlation between PR-B expression and cell cycle progression. In synchronized NIH3T3 cells, we found an increase in PR-B protein and p27 CDK inhibitor levels in the G0/G1 phase and a reduction due to redistribution in the S and G2/M phases. The MEK inhibitor or cAMP stimulation arrested NIH3T3 cells in the G0/G1 phase of the cell cycle. The expression of PR-B and p27 CDK inhibitors was up-regulated by treatment with both the MEK inhibitor and cAMP. Treatment of synchronized cells with a PKA inhibitor in the presence of 1% calf serum resulted in a significant reduction in both PR-B and p27 levels. The decrease in the PR-B levels caused by anti-sense oligomers or siRNA corresponded to the reduction in p27 levels. PR-B overexpression by adenovirus infection induced p27 and suppressed cell growth. Finally, we showed that PR-B modulation involved in the regulation of NIH3T3 cell proliferation was independent of nuclear estrogen receptor (ER) activity but dependent on non-genomic ER activity.

Chronic administration of adrenomedullin attenuates the hypertension and increases renal nitric oxide synthase in Dahl salt-sensitive rats.

Adrenomedullin reduces systemic blood pressure and increases urinary sodium excretion partly through the release of nitric oxide. We hypothesized that chronic adrenomedullin infusion ameliorates salt-sensitive hypertension and increases the expression of renal nitric oxide synthase (NOS) in Dahl salt-sensitive (DS) rats, because the reduced renal NOS expression promotes salt sensitivity. DS rats and Dahl salt-resistant (DR) rats were fed a high sodium diet (8.0% NaCl) for 3 weeks. The high sodium diet resulted in an increase in blood pressure and a reduction of urinary sodium excretion in association with increased renal adrenomedullin concentrations and decreased expression of renal neuronal NOS (nNOS) and renal medullary endothelial NOS (eNOS) in DS rats compared with DR rats. Chronic adrenomedullin infusion partly inhibited the increase of blood pressure and proteinuria in association with a restoration of renal nNOS and medullary eNOS expression in DS rats under the high sodium diet. The immunohistochemical analysis revealed that the restored renal nNOS expression induced by chronic adrenomedullin infusion may reflect the restoration of nNOS expression in the macula densa and inner medullary collecting duct. These results suggest that adrenomedullin infusion has beneficial effects on this hypertension probably in part through restored renal NOS expression in DS rats.

Upregulation of intracardiac adrenomedullin and its receptor system in rats with volume overload-induced cardiac hypertrophy.

Specific adrenomedullin receptors have been identified as calcitonin receptor-like receptor (CRLR)/receptor activity-modifying proteins (RAMP2 and RAMP3) complexes. Although we have demonstrated that adrenomedullin is increased in volume overload-induced cardiac hypertrophy, it remains unknown whether the adrenomedullin receptor is altered or not. This study sought to investigate the significance of intracardiac adrenomedullin and its receptor system in volume overload-induced cardiac hypertrophy. Left ventricular adrenomedullin levels were higher in aortocaval shunt (ACS) rats than in controls (+58%). The left ventricular gene expressions of adrenomedullin, CRLR, RAMP2 and RAMP3 were increased (+27%, +76%, +108% and +131%, respectively) and the left ventricular collagen gene expressions were also increased (type I: +138%, type III: +87%). The left ventricular adrenomedullin level correlated with the gene expression of type III collagen (R=0.42). These results suggest that intracardiac adrenomedullin and its receptor system are upregulated and may participate in the regulation of cardiac remodeling in volume overload-induced cardiac hypertrophy.

Production and autocrine/paracrine effects of endogenous insulin-like growth factor-1 in rat cardiac fibroblasts.

Insulin-like growth factor (IGF)-1 appears to play an important role in cardiac hypertrophy or remodeling. However, the role of endogenous IGF-1 in the growth of cardiac myocytes and fibroblasts remains unclear. This study investigated the major site of the production of cardiac IGF-1 and the local effects of endogenous IGF-1 secreted from cardiac cells. A significant expression of IGF-1 mRNA was found in cultured neonatal and adult rat cardiac fibroblasts, but not in myocytes. In addition, an in vivo examination by in situ hybridization histochemical analyses demonstrated the IGF-1 transcripts in the interstitial fibrotic tissue of the ventricle. Time-dependent secretion of IGF-1 protein was also observed in cultured cardiac fibroblasts. An antibody against IGF-1 decreased collagen synthesis in cardiac fibroblasts under basal conditions. Fibroblast-conditioned medium, as well as exogenous IGF-1, increased protein synthesis in cardiac myocytes, and this increase was inhibited by antibodies against IGF-1 and IGF-1 receptor, IGF binding protein-3, and IGF-1 receptor antagonist. These observations suggest that IGF-1 is produced and released mainly from cardiac fibroblasts and that endogenous IGF-1 promotes collagen synthesis by cardiac fibroblasts and hypertrophy of myocytes as an autocrine and a paracrine factor. Cardiac IGF-1 may function as an endogenous modulator of cardiac hypertrophy or remodeling.

Direct effects of high glucose and insulin on protein synthesis in cultured cardiac myocytes and DNA and collagen synthesis in cardiac fibroblasts.

The present study examined the direct effects of high glucose and insulin on protein synthesis in cardiac myocytes and DNA and collagen synthesis in cardiac fibroblasts. Cultured rat cardiac myocytes and fibroblasts were grown in media containing normal glucose, high glucose, or osmotic control, and incubated with or without insulin. In cardiac myocytes, high glucose had no effect, but insulin increased protein synthesis and atrial natriuretic peptide (ANP) secretion and gene expression. The extracellular signal-regulated protein kinase (ERK)/mitogen-activated protein kinase (MAPK) inhibitor and the protein kinase C (PKC) inhibitor blocked insulin-induced protein synthesis. In cardiac fibroblasts, high glucose and osmotic control media increased DNA synthesis. Collagen synthesis and fibronectin and transforming growth factor-beta1 (TGF-beta1) mRNA expression were stimulated by high glucose, but not by osmotic control. Insulin increased DNA and collagen synthesis in fibroblasts, and the insulin-induced increase in DNA synthesis was blocked by the phosphatidylinositol 3 kinase (PI3K) inhibitor. Our findings suggest that cardiomyocyte protein synthesis is mainly regulated by insulin rather than high glucose and both high glucose and insulin contribute to fibroblast DNA and collagen synthesis. High glucose accelerates fibroblast DNA synthesis and collagen synthesis, and fibronectin and TGF-beta1 mRNA expression, dependent or independent of osmotic stress. Insulin regulates myocyte protein synthesis and fibroblast DNA synthesis through different intracellular mechanisms.

Inhibitory effect of C-type natriuretic peptide (CNP) on cultured cardiac myocyte hypertrophy: interference between CNP and endothelin-1 signaling pathways.

C-type natriuretic peptide (CNP) is known to play a role in the local regulation of vascular tone. We recently found that CNP is also produced by cardiac ventricular cells. However, its local effect on myocyte hypertrophy remains to be elucidated. The present study investigated the effects of CNP on cultured cardiac myocyte hypertrophy and the interaction between CNP and endothelin-1 (ET-1) signaling pathways. CNP attenuated basal and ET-1-augumented protein synthesis, atrial natriuretic peptide secretion, hypertrophy-related gene expression, GATA-4 and MEF-2 DNA binding activities, Ca(2+)/calmodulin-dependent kinase II activity, and ERK phosphorylation. CNP also inhibited ET-1-induced increase in intracellular Ca(2+) concentration. These effects of CNP were mimicked by a cGMP analog, 8-bromo cGMP. However, the inhibitory effects of CNP on the hypertrophic response of myocytes were significantly diminished at high concentrations of ET-1. Although CNP increased intracellular cGMP levels in myocytes, ET-1 suppressed CNP-induced cellular cGMP accumulation. A protein kinase C activator and Ca(2+) ionophore mimicked this suppressive effect of ET-1. We further examined the effect of CNP on the paracrine action of ET-1 secreted from cardiac nonmyocytes. CNP and 8-bromo cGMP significantly inhibited ET-1 secretion from nonmyocytes. Although nonmyocyte-conditioned medium increased the protein synthesis in myocytes through endogenous ET-1 action, this increase was significantly attenuated by pretreatment of nonmyocytes with CNP and 8-bromo cGMP. These findings demonstrate that CNP inhibits ET-1-induced cardiac myocyte hypertrophy via a cGMP-dependent mechanism, and conversely, ET-1 inhibits CNP signaling by a protein kinase C- and Ca(2+)-dependent mechanism, suggesting mutual interference between CNP and ET-1 signaling pathways.

Ventricular nonmyocytes inhibit doxorubicin-induced myocyte apoptosis: involvement of endogenous endothelin-1 as a paracrine factor.

A cross-talk between cardiac myocytes and nonmyocytes via humoral factors plays an important role in the development of cardiac growth. However, it remains to be elucidated whether humoral factors produced from nonmyocytes have a protective effect on acute myocardial injury. The present in vitro study investigated the antiapoptotic effect of nonmyocytes on doxorubicin (DOX)-induced myocyte apoptosis and its molecular mechanism. Myocyte-nonmyocyte coculture and treatment with nonmyocyte-conditioned media significantly attenuated DOX-induced myocyte apoptosis. Treatment with nonmyocyte-conditioned media stimulated the phosphorylation of ERK, Akt, and cAMP response element-binding protein (CREB) in myocytes. Nonmyocyte-conditioned media also increased protein levels of Bcl-2 but not Bcl-xL and decreased caspase-3 activation induced by DOX. MAPK kinase-specific inhibitor PD98059, phosphatidylinositol-3 kinase-Akt inhibitor LY294002, and CREB antisense oligonucleotide significantly blocked the antiapoptotic effect of nonmyocyte-conditioned media. A considerable amount of endothelin (ET)-1 production was detected in nonmyocytes but not in myocytes. Exogenous ET-1 mimicked nonmyocyte-conditioned media-mediated ERK and CREB phosphorylation and Bcl-2 protein increase but not Akt phosphorylation. In addition, ET-A receptor antagonists BQ123 and BQ485 partially blocked nonmyocyte-conditioned media-mediated antiapoptotic effect, ERK and CREB phosphorylation, and Bcl-2 protein increase. Nonmyocyte-conditioned media and exogenous ET-1 unchanged protein levels of manganese superoxide dismutase and oxidative stress-related product levels augmented by DOX. The present findings demonstrate that cardiac nonmyocytes inhibit DOX-induced myocyte apoptosis, at least in part, via ET-1 secretion-mediated CREB activation independent of the decrease in oxidative stress.

Gene expression, secretion, and autocrine action of C-type natriuretic peptide in cultured adult rat cardiac fibroblasts.

C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, is known to be synthesized in the central nervous system and vascular endothelial cells, in contrast to atrial natriuretic peptide and brain natriuretic peptide. However, there have been no studies concerning CNP production in cultured cardiac cells. Here, we examined the production and the local effect of CNP in cultured ventricular cells. Under serum-free conditions, adult rat cardiac fibroblasts secreted immunoreactive CNP time dependently. TGF-beta1, basic fibroblast growth factor, and endothelin-1 significantly stimulated CNP secretion. Northern blot analysis detected significant expressions of CNP and its specific receptor (guanylyl cyclase-B) mRNA in cardiac fibroblasts. CNP stimulated intracellular cGMP production in fibroblasts more intensely than atrial and brain natriuretic peptides. CNP inhibited both DNA and collagen syntheses of cardiac fibroblasts, and these inhibitory effects by CNP were stronger than by atrial and brain natriuretic peptides. The inhibition by CNP of DNA and collagen syntheses was reproduced by a cGMP analog, 8-bromo cGMP. The present findings demonstrate that CNP is synthesized in and secreted from cardiac fibroblasts and suggest that CNP has a suppressive effect on fibroblast proliferation and extracellular matrix production, probably via the guanylyl cyclase-B-mediated cGMP-dependent process. CNP produced by cardiac fibroblasts may play a role as an autocrine regulator against excessive cardiac fibrosis.

Tubulointerstitial disease: role of ischemia and microvascular disease.

PURPOSE OF REVIEW: Tubulointerstitial injury is characteristic of aging-associated renal injury and progressive renal disease. Salt-sensitive hypertension is also associated with tubulointerstitial inflammation, especially when accompanied by microvascular disease. Here we summarize recent studies on the pathogenesis and consequences of tubulointerstitial disease, emphasizing the role of ischemia and the microvasculature. RECENT FINDINGS: Tubulointerstitial injury occurs via several mechanisms of which one of the most important is chronic ischemia. Recent studies suggest that chronic vasoconstriction may contribute to the renal injury associated with angiotensin II, catecholamines, nitric oxide inhibition, hypokalemia, hyperuricemia, and cyclosporine nephropathy. Salt-sensitivity may result as a consequence of the tubulointerstitial inflammatory response to these conditions, and this appears to be perpetuated by the development of preglomerular vascular disease. With progression of tubulointerstitial disease there is also a loss of peritubular capillaries, and stimulating microvascular growth with angiogenic factors can stabilize renal function in these models. SUMMARY: Ischemia secondary to vasoconstriction or to structural changes of the renal vasculature may have important consequences both in terms of mediating salt-sensitive hypertension and renal progression. Angiogenic factors may have potential benefit in preventing or treating these conditions.

Endothelin a receptor blockade and endothelin B receptor blockade improve hypokalemic nephropathy by different mechanisms.

Hypokalemia causes renal tubulointerstitial injury with an elevation in renal endothelin-1 (ET-1). It was hypothesized that hypokalemic tubulointerstitial injury is ameliorated by the blockade of ET-A receptors (ETA), whereas ET-B receptor (ETB) antagonism may exacerbate the injury, because ETB is thought to mediate vasodilation. Rats were fed a K(+)-deficient diet alone (LC) or with an ETA-selective antagonist ABT-627 (LA) or an ETB-selective antagonist A-192621 (LB) for 8 wk. Control rats were on a normal K(+) diet alone or with the ETA-selective or ETB-selective antagonists. The severity of hypokalemia was not significantly different among LA, LB, and LC. LC developed tubulointerstitial injury with an elevation of renal preproET-1 mRNA level. There was an increase in tubular osteopontin expression, macrophage infiltration, collagen accumulation, and tubular cell hyperplasia. ETA blockade significantly ameliorated all parameters for renal injury in the cortex without suppressing local ET-1 and ETA expression. By contrast, ETB blockade significantly reduced local ET-1 and ETA expression and improved the injury to a similar extent in the cortex. In the medulla, ETA or ETB blockade only partially blocked renal injury. ETA blockade did not affect BP in normokalemic or hypokalemic rats. ETB blockade induced a BP elevation with a decrease in urinary Na(+) excretion in normokalemic but not in hypokalemic rats. These results indicate that ET-1 can mediate hypokalemic renal injury in two different ways: by directly stimulating ETA and by locally promoting endogenous ET-1 production via ETB. Thus, ETA as well as ETB blockade may be renoprotective in hypokalemic nephropathy.