Ferng-Chun Ke

Effects of luteolin and quercetin, inhibitors of tyrosine kinase, on cell growth and metastasis‐associated properties in A431 cells overexpressing epidermal growth factor receptor

Flavonoids display a wide range of pharmacological properties including anti‐inflammatory. Anti‐mutagenic, anti‐carcinogenic and anti‐cancer effects. Here, we evaluated the effects of eight flavonoids on the tumour cell proliferation, cellular protein phosphorylation, and matrix metalloproteinase (MMPs) secretion. Of the flavonoids examined, luteolin (Lu) and quercetin (Qu) were the two most potent agents, and significantly inhibited A431 cell proliferation with IC50 values of 19 and 21 μM, respectively. 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 μM, both Lu and Qu markedly decreased the levels of phosphorylation of A431 cellular proteins, including EGFR. 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 effect was abolished by EGF treatment. The addition of EGF only marginally diminished the inhibitory effect 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 effectively to inhibit a wide range of protein kinases, including EGFR tyrosine kinase. 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. Examination of the relationship between the chemical structure and inhibitory effects of eight flavonoids 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. This study demonstrates that the inhibitory effects of Lu and Qu, and the stimulatory effects 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.

Binding of Progesterone to Nerve Cell Membranes of Rat Brain Using Progesterone Conjugated to 125I‐Bovine Serum Albumin as a Ligand

Radioiodinated bovine serum albumin conjugated to progesterone was used as a probe to examine binding parameters of steroids to membrane preparations from rat brain tissue. The binding of 11α‐hydroxyprogesterone‐11‐hemisuccinate‐125I‐bovine serum albumin conjugate reached saturation after 30 min of incubation at 5°C. Several bovine serum albumin‐conjugated steroids were then tested for competition displacement studies. Among these steroid conjugates, the bovine serum albumin conjugate at position 3 of progesterone had the highest affinity, with an estimated inhibition constant of 28.5 ± 2.1 nM (n = 3), whereas bovine serum albumin itself and the 17β‐estradiol 6‐(O‐carboxy‐methyl)oxime‐bovine serum albumin conjugate showed no specific displacement. In addition, the binding sites were localized in an axolemma‐enriched fraction of rat brainstem. Specific binding was obtained in tissues from cerebral cortex, brainstem, cerebellum, corpus striatum, and hypothalamus, but little or no binding occurred in uterus, ovary, liver, and spleen. The present data indicate that progesterone‐125I‐bovine serum albumin conjugate can be used as a ligand to study progesterone‐membrane receptor interactions.

Membrane Mechanism Mediates Progesterone Stimulatory Effect on LHRH Release from Superfused Rat Hypothalami in vitro

To determine whether the plasma membrane is a primary site for progesterone (P4) action on the neural LHRH apparatus of hypothalamic tissues derived from ovariectomized, estradiol-primed (OVX + E2) immature rats, immobilized P4 was infused directly to these tissues using a superfusion technique. Two kinds of immobilized P4 with bovine serum albumin (BSA) conjugated at positions 3 or 11, or 11-deoxycorticosterone (DOC) immobilized at position 21 of the steroid molecule, respectively, were tested for structural specificity. Among the three immobilized steroids, only P4 with BSA conjugated at position 3 (P4-3-BSA) was effective in stimulating LHRH release in vitro. P4-3-BSA at 0.5 micrograms/ml, approximately 1.7 X 10(-7) M of P4, increased LHRH levels in the superfusates to about 2.5-fold those of pretreatment levels. In addition, no conversion of P4-3-BSA to free progesterone was detected. This observation demonstrated that the plasma membrane is a primary site for the stimulating effect of P4 on LHRH release from hypothalamic tissue in vitro.

Interplay of PI3K and cAMP/PKA signaling, and rapamycin-hypersensitivity in TGFbeta1 enhancement of FSH-stimulated steroidogenesis in rat ovarian granulosa cells.

Transforming growth factor (TGF) beta1 facilitates FSH-induced differentiation of rat ovarian granulosa cells. The signaling crosstalk between follicle stimulating hormone (FSH) and TGFbeta 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 TGFbeta1-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 3beta-HSD, and their suppression of the FSH plus TGFbeta1 effect was attributed to the reduction of all the three key players, steroidogenic acute regulatory (StAR) protein, P450scc, and 3beta-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, TGFbeta1 had no effect on FSH-activated CREB and PI3K signaling mediators. We further found that rapamycin reduced the TGFbeta1-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 TGFbeta1 regulation of steroidogenesis in rat granulosa cells. Furthermore, we demonstrate for the first time that TGFbeta1 acts in a rapamycin-hypersensitive and mTORC1-independent manner in augmenting FSH-stimulated steroidogenesis in rat granulosa cells.

Potential role of follicle-stimulating hormone (FSH) and transforming growth factor (TGFβ1) in the regulation of ovarian angiogenesis

Angiogenesis occurs during ovarian follicle development and luteinization. Pituitary secreted FSH was reported to stimulate the expression of endothelial mitogen VEGF in granulosa cells. And, intraovarian cytokine transforming growth factor (TGF)β1 is known to facilitate FSH‐induced differentiation of ovarian granulosa cells. This intrigues us to investigate the potential role of FSH and TGFβ1 regulation of granulosa cell function in relation to ovarian angiogenesis. Granulosa cells were isolated from gonadotropin‐primed immature rats and treated once with FSH and/or TGFβ1 for 48 h, and the angiogenic potential of conditioned media (granulosa cell culture conditioned media; GCCM) was determined using an in vitro assay with aortic ring embedded in collagen gel and immunoblotting. FSH and TGFβ1 increased the secreted angiogenic activity in granulosa cells (FSH + TGFβ1 > FSH ≈ TGFβ1 > control) that was partly attributed to the increased secretion of pro‐angiogenic factors VEGF and PDGF‐B. This is further supported by the evidence that pre‐treatment with inhibitor of VEGF receptor‐2 (Ki8751) or PDGF receptor (AG1296) throughout or only during the first 2‐day aortic ring culture period suppressed microvessel growth in GCCM‐treated groups, and also inhibited the FSH + TGFβ1‐GCCM‐stimulated release of matrix remodeling‐associated gelatinase activities. Interestingly, pre‐treatment of AG1296 at late stage suppressed GCCM‐induced microvessel growth and stability with demise of endothelial and mural cells. Together, we provide original findings that both FSH and TGFβ1 increased the secretion of VEGF and PDGF‐B, and that in turn up‐regulated the angiogenic activity in rat ovarian granulosa cells. This implicates that FSH and TGFβ1 play important roles in regulation of ovarian angiogenesis during follicle development. J. Cell. Physiol. 226: 1608–1619, 2011.

Inhibitory effects of a luteinizing hormone-releasing hormone agonist on basal and epidermal growth factor-induced cell proliferation and metastasis-associated properties in human epidermoid carcinoma a431 cells

The purpose of this study was to investigate the effects of a potent LHRH agonist, [D‐Trp6]LHRH on the basal and EGF‐induced cell proliferation and the metastasis‐associated properties in A431 human epidermoid carcinoma. [D‐Trp6]LHRH time‐dependently inhibited the basal and EGF‐stimulated growth of A431 cancer cells. It is assumed that phosphorylation/dephosphorylation of cellular proteins is highly related to cell growth. This study demonstrates that [D‐Trp6]LHRH decreased the basal and EGF‐induced total cellular kinase activity, particularly the tyrosine phosphorylation of several cellular proteins including the EGFR. In contrast, [D‐Trp6]LHRH did not cause detectable changes in basal and EGF‐stimulated serine/threonine phosphorylation of A431 cellular proteins. The inhibitory effect of [D‐Trp6]LHRH on A431 cell proliferation was associated with apoptosis as evidenced by the cell morphology and DNA integrity (ladder pattern), the expression of interleukin 1β‐converting enzyme (ICE) and activation of caspase. Furthermore, EGF could rescue the remaining attached A431 cells following [D‐Trp6]LHRH treatment for 48 hr, which suggests that limited exposure to [D‐Trp6]LHRH did not channel all cells to irreversible apoptotic process. We also determined the effects of [D‐Trp6]LHRH on metastasis‐associated properties in A431 cells. [D‐Trp6]LHRH reduced both basal and EGF‐stimulated secretion of MMP‐9 and MMP‐2. In addition, [D‐Trp6]LHRH suppressed the basal and EGF‐induced invasive activity of A431 cells based on an in vitro invasion assay. In conclusion, this study indicates that [D‐Trp6]LHRH may act partly through activating tyrosine phosphatase activity to inhibit cell proliferation and the metastasis‐associated properties of A431 cancer cells. Our work suggests that [D‐Trp6]LHRH may be therapeutically useful in limiting the tumor growth and metastasis of some neoplasms.

Effects of dietary flavonoids, luteolin, and quercetin on the reversal of epithelial–mesenchymal transition in A431 epidermal cancer cells

Highly invasive A431‐III cells, which are derived from parental A431‐P cells, were originally isolated by three successive passages through a Boyden chamber using a Matrigel‐coated membrane support. The greater invasion potential shown by A431‐III cells was due to their increased ability to spread/migrate, which was associated with enhanced MMP activity. The tumor progression events evoked by A431‐P cells compared to A431‐III cells may help identify useful strategies for evaluating the epithelial–mesenchymal transition (EMT) and these cell lines could be a reliable model for evaluating tumor metastasis events. Using this approach, we evaluated the effects of luteolin and quercetin using the A431‐P/A431‐III EMT model. These flavonoids reversed cadherin switching, downregulated EMT markers, and nullified the invasion ability of A431‐III cells. Overexpression of MMP‐9 resulted in induction of the EMT in A431‐P cells and this could be reversed by treating with luteolin or quercetin. Cotreatment of A431‐P and A431‐III cells with epidermal growth factor (EGF) plus luteolin or quercetin resulted in a more epithelial‐like morphology, led to reduced levels of EGF‐induced markers of EMT, and caused the restoration of cell–cell junctions. E‐cadherin was decreased by EGF, but increased by luteolin and quercetin. Our results suggest that luteolin and quercetin are potentially beneficial agents that target and prevent the occurrence of EMT in epidermal carcinoma cells. These chemicals also have the ability to attenuate tumor progression in A431‐III cells. Luteolin and quercetin show inherent potential as chemopreventive/antineoplastic agents and do this by abating tumor progression through a reversal of EMT. (Cancer Sci 2011; 102: 1829–1839)

The Modulatory Role of Transforming Growth Factor β1 and Androstenedione on Follicle-Stimulating Hormone-Induced Gelatinase Secretion and Steroidogenesis in Rat Granulosa Cells

Abstract To investigate the potential roles of matrix metalloproteinases (MMPs) in ovarian granulosa cell differentiation, we studied the interactive effects of FSH and local ovarian factors, transforming growth factor β1 (TGFβ1) and androstenedione, on gelatinase secretion and progesterone production in rat ovarian granulosa cells. Granulosa cells of eCG-primed immature rats were treated once with various doses of FSH and TGFβ1 and androstenedione alone or in combinations for 2 days. Conditioned media were analyzed for gelatinase activity using gelatin-zymography/densitometry and progesterone levels using enzyme immunoassay. Cell lysates were analyzed for steroidogenic acute regulatory (StAR) and cholesterol side-chain-cleavage (P450scc) enzyme protein levels. This study demonstrates for the first time that FSH dose-dependently increased the secretion of a major 63-kDa gelatinase and minor 92- and 67-kDa gelatinases. TGFβ1 also dose-dependently increased the secretion of 63-kDa gelatinase, while androstenedione alone had no effect. The 92-kDa gelatinase was identified as the pro-MMP9 that could be cleaved by aminophenylmercuric acetate into the 83-kDa active form. Importantly, we show that TGFβ1 and androgen act in an additive manner to enhance FSH stimulatory effects both on the secretion of gelatinases and the production of progesterone. We further show by immunoblotting that the enhancing effect of TGFβ1 and androstenedione on FSH-stimulated steroidogenesis is partly mediated through the increased level of StAR protein and/or P450scc enzyme. In conclusion, this study indicates that, during antral follicle development, TGFβ1 and androgen act to enhance FSH promotion of granulosa cell differentiation and that the process may involve the interplay of modulating cell- to-matrix/cell-to-cell interaction and steroidogenic activity.

High-pressure liquid chromatographic separation of a mixture of corticosteroids, androgens, and progestins

Fifteen steroids including corticosteroids, androgens, progestins, and their derivatives were completely separated by reverse-phase high-pressure liquid chromatography on a ChemcoPak 7 ODS-H column in 50 min. The elution procedures were first with water:methanol:acetonitrile:isopropanol 55:32:6.5:7.5 (v/v) for 15 min and followed with a linear gradient elution for 35 min from 0 to 80% of water:methanol:n-butanol 40:40:20 (v/v). The applicability of this method was successfully demonstrated in the analyses of the biological samples of carp plasma, testis, and head kidney.

CREB coactivator CRTC2/TORC2 and its regulator calcineurin crucially mediate follicle‐stimulating hormone and transforming growth factor β1 upregulation of steroidogenesis

In vitro and in vivo studies implicate that follicle‐stimulating hormone (FSH) and transforming growth factor β1 (TGFβ1) play crucial physiological roles in regulating ovarian granulosa cell function essential to fertility control in females. FSH induces cAMP and calcium signaling, thereby activating transcription factor CREB to upregulate steroidogenic gene expression, and TGFβ1 greatly enhances FSH‐stimulated steroidogenesis. A CREB coactivator CRTC2/TORC2 was identified to function as a cAMP and calcium‐sensitive coincidence sensor. This led us to explore the role of CRTC2 and its regulator calcineurin in FSH and TGFβ1‐stimulated steroidogenesis. Primary culture of granulosa cells from gonadotropin‐primed immature rats was used. Immunoblotting analysis shows that FSH rapidly and transiently induced dephosphorylation/activation of CRTC2, and FSH + TGFβ1 additionally induced late‐phase CRTC2 dephosphorylation. Immunofluorescence analysis further confirms FSH ± TGFβ1 promoted CRTC2 nuclear translocation. Using selective inhibitors, we demonstrate that FSH activated CRTC2 in a PKA‐ and calcineurin‐dependent manner, and TGFβ1 acting through its type I receptor (TGFβRI)‐modulated FSH action in a calcineurin‐mediated and PKA‐independent fashion. Next, we investigated the involvement of calcineurin and CRTC2 in FSH and TGFβ1‐stimulated steroidogenesis. Calcineurin and TGFβRI inhibitor dramatically reduced the FSH ± TGFβ1‐increased progesterone synthesis and protein levels of StAR, P450scc, and 3β‐HSD enzyme. Furthermore, chromatin‐immunoprecipitation and immunoprecipitation analyses demonstrate that FSH ± TGFβ1 differentially increased CRTC2, CREB, and CBP binding to these steroidogenic genes, and CREB nuclear association with CRTC2 and CBP. In all, this study reveals for the first time that CRTC2 and calcineurin are critical signaling mediators in FSH and TGFβ1‐stimulated steroidogenesis in ovarian granulosa cells. J. Cell. Physiol. 227: 2430–2440, 2012.