Jiuan-Jiuan Hwang

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.

Matrix metalloproteinase-9 cooperates with transcription factor Snail to induce epithelial-mesenchymal transition.

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)

Functional significance of MMP-9 in tumor necrosis factor-induced proliferation and branching morphogenesis of mammary epithelial cells.

Tissue remodeling is a key process involved in normal mammary gland development, with matrix metalloproteinases (MMPs) playing an important role in this process. Our laboratory has demonstrated that tumor necrosis factor (TNF) stimulates branching morphogenesis of mammary epithelial cells (MEC) within a reconstituted basement membrane. Studies were therefore undertaken to determine whether MMPs might mediate the effects of TNF. Using a primary culture model in which rat MEC grow three-dimensionally within a reconstituted basement membrane, we found that TNF stimulated secretion of MMP-9 but not MMP-2. To determine whether MMP-9 was involved in TNF-induced proliferation and branching morphogenesis, we used a peptide containing the prodomain sequence of MMPs and two MMP inhibitors. Both the prodomain peptide (5 x 10(-4)-10(-3) M), as well as BB-94 (10(-8)-10(-5) M) and CGS 27023A (10(-6)-10(-5) M), inhibited TNF-induced proliferation and branching morphogenesis in a concentration-dependent manner. Finally, to verify the specific requirement for MMP-9, we demonstrated that an MMP-9 neutralizing antibody blocked TNF-induced proliferation and branching morphogenesis. Together, these data suggest that TNF-regulated MMP-9 may play a role in the controlled invasion of the fad pad that occurs during normal mammary gland development and that misregulation of MMP-9 may contribute to the invasiveness of breast cancer.

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.

Functional role of matrix metalloproteinases (MMPs) in mammary epithelial cell development

The extracellular matrix (ECM) is an important regulator of mammary epithelial cell (MEC) function and is remodeled by matrix metalloproteinases (MMPs). To investigate the significance and regulation of MMP activity in normal MEC, we utilized a primary culture model in which rat MEC were grown three dimensionally within a reconstituted basement membrane (RBM) in defined serum‐free medium. Zymograms of culture medium demonstrated that five major gelatinases of 97, 80, 74, 69, and 65 kDa were secreted by MEC and were distinct from gelatinases of RBM origin. Based on molecular weight, p‐aminophenylmercuric acid activation, immunoblotting with MMP‐specific antibodies, inhibition by EDTA, a peptide containing the prodomain sequence of MMP (TMRKPRCGNPDVAN) and two synthetic MMP inhibitors (BB‐94 and CGS 27023A), these were classified as inactive and active forms of MMP‐9 and MMP‐2. The maximal MMP activities occurred when MEC were in a rapid proliferation and branching phase and declined after they underwent functional differentiation. Known regulators of MEC growth and differentiation were evaluated for their ability to modulate gelatinase activity in primary culture. Secretion of one or both MMPs was inhibited by EGF, TGFα, prolactin, and hydrocortisone and stimulated by progesterone. Furthermore, the functional significance of MMPs was demonstrated since three MMP inhibitors blocked branching morphogenesis elicited by the absence of hydrocortisone. Additionally, two synthetic MMP inhibitors not only inhibited epithelial cell growth but also inhibited normal alveolar development of the MEC. Finally, these drugs were found to enhance MMP secretion from MEC, although the activity of the secreted MMPs was inhibited as long as the drug was present.

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.

Role of tissue transglutaminase 2 in the acquisition of a mesenchymal-like phenotype in highly invasive A431 tumor cells

BackgroundCancer progression is closely linked to the epithelial-mesenchymal transition (EMT) process. Studies have shown that there is increased expression of tissue tranglutaminase (TG2) in advanced invasive cancer cells. TG2 catalyzes the covalent cross-linking of proteins, exhibits G protein activity, and has been implicated in the modulation of cell adhesion, migration, invasion and cancer metastasis. This study explores the molecular mechanisms associated with TG2s involvement in the acquisition of the mesenchymal phenotype using the highly invasive A431-III subline and its parental A431-P cells.ResultsThe A431-III tumor subline displays increased expression of TG2. This is accompanied by enhanced expression of the mesenchymal phenotype, and this expression is reversed by knockdown of endogenous TG2. Consistent with this, overexpression of TG2 in A431-P cells advanced the EMT process. Furthermore, TG2 induced the PI3K/Akt activation and GSK3β inactivation in A431 tumor cells and this increased Snail and MMP-9 expression resulting in higher cell motility. TG2 also upregulated NF-κB activity, which also enhanced Snail and MMP-9 expression resulting in greater cell motility; interestingly, this was associated with the formation of a TG2/NF-κB complex. TG2 facilitated acquisition of a mesenchymal phenotype, which was reversed by inhibitors of PI3K, GSK3 and NF-κB.ConclusionsThis study reveals that TG2 acts, at least in part, through activation of the PI3K/Akt and NF-κB signaling systems, which then induce the key mediators Snail and MMP-9 that facilitate the attainment of a mesenchymal phenotype. These findings support the possibility that TG2 is a promising target for cancer therapy.

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)

Regulation of thyroid hormones on the production of testosterone in rats

The effects of a thyroidectomy and thyroxine (T4) replacement on the spontaneous and human chorionic gonadotropin (hCG)‐stimulated secretion of testosterone and the production of adenosine 3′,5′‐cyclic monophosphate (cAMP) in rat testes were studied. Thyroidectomy decreased the basal levels of plasma luteinizing hormone (LH) and testosterone, which delayed the maximal response of testosterone to gonadotropin‐releasing hormone (GnRH) and hCG in male rats. T4 replacement in thyroparathyroidectomized (Tx) rats restored the concentrations of plasma LH and testosterone to euthyroid levels. Thyroidectomy decreased the basal release of hypothalamic GnRH, pituitary LH, and testicular testosterone as well as the LH response to GnRH and testosterone response to hCG in vitro. T4 replacement in Tx rats restored the in vitro release of GnRH, GnRH‐stimulated LH release as well as hCG‐stimulated testosterone release. Administration of T4 in vitro restored the release of testosterone by rat testicular interstitial cells (TICs). The increase of testosterone release in response to forskolin and androstenedione was less in TICs from Tx rats than in that from sham Tx rats. Administration of nifedipine in vitro resulted in a decrease of testosterone release by TICs from sham Tx but not from Tx rats. The basal level of cAMP in TICs was decreased by thyroidectomy. The increased accumulation of cAMP in TICs following administration of forskolin was eliminated in Tx rats. T4 replacement in Tx restored the testosterone response to forskolin. But the testosterone response to androstenedione and the cAMP response to forskolin in TICs was not restored by T4 in Tx rats. These results suggest that the inhibitory effect of a thyroidectomy on the production of testosterone in rat TICs is in part due to: 1) the decreased basal secretion of pituitary LH and its response to GnRH; 2) the decreased response of TICs to gonadotropin; and 3) the diminished production of cAMP, influx of calcium, and activity of 17β‐HSD. T4 may enhance testosterone production by acting directly at the testicular interstitial cells of Tx rats. J. Cell. Biochem. 73:554–562, 1999.