Despite similar clinical endpoints, heart failure resulting from dilated cardiomyopathy (DCM) or hypertrophic cardiomyopathy (HCM) appears to develop through different remodeling and molecular pathways. Current understanding of heart failure has been facilitated by microarray technology. We constructed an in-house spotted cDNA microarray using 10,272 unique clones from various cardiovascular cDNA libraries sequenced and annotated in our laboratory. RNA samples were obtained from left ventricular tissues of precardiac transplantation DCM and HCM patients and were hybridized against normal adult heart reference RNA. After filtering, differentially expressed genes were determined using novel analyzing software. We demonstrated that normalization for cDNA microarray data is slide-dependent and nonlinear. The feasibility of this model was validated by quantitative real-time reverse transcription-PCR, and the accuracy rate depended on the fold change and statistical significance level. Our results showed that 192 genes were highly expressed in both DCM and HCM (e.g., atrial natriuretic peptide, CD59, decorin, elongation factor 2, and heat shock protein 90), and 51 genes were downregulated in both conditions (e.g., elastin, sarcoplasmic/endoplasmic reticulum Ca2+-ATPase). We also identified several genes differentially expressed between DCM and HCM (e.g., alphaB-crystallin, antagonizer of myc transcriptional activity, beta-dystrobrevin, calsequestrin, lipocortin, and lumican). Microarray technology provides us with a genomic approach to explore the genetic markers and molecular mechanisms leading to heart failure.

1 Flavonoids display a wide range of pharmacological properties including anti-in¯ammatory. Anti-mutagenic, anti-carcinogenic and anti-cancer eects. Here, we evaluated the eects of eight avonoids on the tumour cell proliferation, cellular protein phosphorylation, and matrix metalloproteinase (MMPs) secretion. 2 Of the¯avonoids examined, luteolin (Lu) and quercetin (Qu) were the two most potent agents, and signi®cantly inhibited A431 cell proliferation with IC 50 values of 19 and 21 mM, respectively. 3 The epidermal growth factor (EGF) (10 nM) promoted growth of A431 cells (+25+4.6%) and mediated epidermal growth factor receptor (EGFR) tyrosine kinase activity and autophosphorylation of EGFR were inhibited by Lu and Qu. At concentration of 20 mM, both Lu and Qu markedly decreased the levels of phosphorylation of A431 cellular proteins, including EGFR. 4 A431 cells treated with Lu or Qu exhibited protuberant cytoplasmic blebs and progressive shrinkage morphology. Lu and Qu also time-dependently induced the appearance of a ladder pattern of DNA fragmentation, and this eect was abolished by EGF treatment. 5 The addition of EGF only marginally diminished the inhibitory eect of luteolin and quercetin on the growth rate of A431 cells, treatment of cellular proteins with EGF and luteolin or quercetin greatly reduced protein phosphorylation, indicating Lu and Qu may act eectively to inhibit a wide range of protein kinases, including EGFR tyrosine kinase. 6 EGF increased the levels of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9), while Lu and Qu appeared to suppress the secretion of these two MMPs in A431 cells. 7 Examination of the relationship between the chemical structure and inhibitory eects of eight avonoids reveal that the double bond between C2 and C3 in ring C and the OH groups on C3' and C4' in ring B are critical for the biological activities. 8 This study demonstrates that the inhibitory eects of Lu and Qu, and the stimulatory eects of EGF, on tumour cell proliferation, cellular protein phosphorylation, and MMP secretion may be mediated at least partly through EGFR. This study supports the idea that Lu and Qu may have potential as anti-cancer and anti-metastasis agents.

One of the most fundamental biological processes in tumor metastasis is the process of epithelial-mesenchymal transition (EMT). During EMT, zinc-finger-family of transcription factors such as Snail, Slug and Twist, and matrix metalloproteinases (MMPs) are upregulated, and this correlates with increased tumor cell invasion and motility. We previously obtained a highly invasive A431-III tumor subline, which is a rich source of MMP-9 and observed a plausible link between MMP levels and the promotion of EMT. To gain further understanding of EMT, we investigated the contribution of distinct MMPs to the induction of EMT. Exposing A431, cervical carcinoma parental cells, to MMP-9 stimulated a phenotypic alteration and cells became spindle-like as shown for A431-III cells. In the present communication, we document changes in gene expression profiles of A431-P and A431-III cells, including those of genes involved in cell adhesion, cytoskeleton reorganization, polarity, migration and transcription. Treatment of both A431-P and A431-III cells with GM6001, a broad spectrum MMP inhibitor, resulted in the diminution of vimentin and fibronectin, indicating a role for MMP-9 in the induction of EMT. Abrogation of MMP-9-mediated cell-cell contact in both A431-P and A431-III cells using MMP-9 siRNA resulted in decreased cell invasion, motility and altered cytoskeleton arrangement together with a reduction in Snail expression. Knockdown of Snail resulted in similar changes along with diminished MMP-9 expression. These data suggest a higher capacity of MMP-9 than that of Snail in eliciting the development of EMT in A431 cells. Based on these findings, we speculate that the overexpression of MMP-9 in A431-III cells might directly induce (or stimulate) EMT and that the transcriptional factor, Snail, could cooperatively engage in this phenomenon. (Cancer Sci 2011; 102: 815-827) T he spread of cancer through metastasis is considered to be responsible for the majority of cancer mortalities.(1) Alteration in matrix remodeling-related proteolysis in cancers is linked to unregulated tumor growth and cancer cell invasion and metastasis. Matrix metalloproteinases (MMPs) are the most noteworthy proteolytic enzymes that are associated with tumorigenesis.(2) The MMPs belong to a rapidly growing family of zinc-dependent endopeptidases that currently includes more than 25 members classified as collagenases, gelatinases, stromelysins, matrilysins and membrane-associated MMP.(3,4) Enhanced levels of certain MMPs are associated with cancer growth and are regarded as prime candidates functioning during tumor invasion, metastasis and angiogenesis, and, in some instances, overexpression correlates with poor clinical outcomes.(4) It is believed that MMPs degrade the extracellular matrix (ECM) and thus enable tumor cells to migrate, invade and spread to various secondary sites, where they form metastases.(5,6) Tumor cells require the action of more than one MMP and more general degradative enzymes to cross the tissue barriers they encounter in the proc...

Transforming growth factor (TGF)b1 facilitates FSH-induced differentiation of rat ovarian granulosa cells. The signaling crosstalk between follicle stimulating hormone (FSH) and TGFb receptors remains unclear. This study was to investigate the interplay of cAMP/protein kinase A (PKA) and phosphatidylinositol-3-kinase (PI3K) signaling including mammalian target of rapamycin (mTOR)C1 dependence in FSH-and TGFb1-stimulated steroidogenesis in rat granulosa cells. To achieve this aim, inhibitors of PKA (PKAI), PI3K (wortmannin), and mTORC1 (rapamycin) were employed. PKAI and wortmannin suppressions of the FSH-increased progesterone production were partly attributed to decreased level of 3b-HSD, and their suppression of the FSH plus TGFb1 effect was attributed to the reduction of all the three key players, steroidogenic acute regulatory (StAR) protein, P450scc, and 3b-HSD. Further, FSH activated the PI3K pathway including increased integrin-linked kinase (ILK) activity and phosphorylation of Akt(S473), mTOR(S2481), S6K(T389), and transcription factors particularly FoxO1(S256) and FoxO3a(S253), which were reduced by wortmannin treatment but not by PKAI. Interestingly, PKAI suppression of FSH-induced phosphorylation of cAMP regulatory element-binding protein (CREB(S133)) disappeared in the presence of wortmannin, suggesting that wortmannin may affect intracellular compartmentalization of signaling molecule(s).In addition, TGFb1 had no effect on FSH-activated CREB and PI3K signaling mediators. We further found that rapamycin reduced the TGFb1-enhancing effect of FSH-stimulated steroidogenesis, yet it exhibited no effect on FSH action. Surprisingly, rapamycin displayed a suppressive effect at concentrations that had no effect on mTORC1 activity. Together, this study demonstrates a delicate interplay between cAMP/PKA and PI3K signaling in FSH and TGFb1 regulation of steroidogenesis in rat granulosa cells. Furthermore, we demonstrate for the first time that TGFb1 acts in a rapamycinhypersensitive and mTORC1-independent manner in augmenting FSH-stimulated steroidogenesis in rat granulosa cells.

Expression of MR increased in AF, thus augmenting the genomic effects of aldosterone. Aldosterone induced atrial ionic remodeling and calcium overload through a genomic pathway, which was attenuated by spironolactone. These results suggest that aldosterone may play a role in AF electrical remodeling and provide insight into the treatment of AF with MR blockade.