Protein Tyrosine Kinase Pathways Research Articles

SHED-Dependent Oncogenic Signaling of the PEAK3 Pseudo-Kinase.

Simple SummaryThe human kinome is composed of about 50 pseudo-kinases with unclear function, because they are predicted to be catalytically inactive; however, they are shown to play an important role in cancer, similar to active kinases. Understanding how these pseudo-kinases promote tumor formation despite their catalytic inactivity is a great challenge, which may lead to innovative anti-cancer therapies. The PEAK1 and 2 pseudo-kinases have emerged as important components of the protein tyrosine kinase pathway implicated in cancer progression. They can signal using a scaffolding mechanism via a conserved split helical dimerization (SHED) module. In this study, we uncovered a similar SHED-dependent oncogenic activity for PEAK3, a recently discovered new member of this family. We also show that this new signaling mechanism may be implicated in acute myeloid leukemia.The PEAK1 and Pragmin/PEAK2 pseudo-kinases have emerged as important components of the protein tyrosine kinase pathway implicated in cancer progression. They can signal using a scaffolding mechanism that involves a conserved split helical dimerization (SHED) module. We recently identified PEAK3 as a novel member of this family based on structural homology; however, its signaling mechanism remains unclear. In this study, we found that, although it can self-associate, PEAK3 shows higher evolutionary divergence than PEAK1/2. Moreover, the PEAK3 protein is strongly expressed in human hematopoietic cells and is upregulated in acute myeloid leukemia. Functionally, PEAK3 overexpression in U2OS sarcoma cells enhanced their growth and migratory properties, while its silencing in THP1 leukemic cells reduced these effects. Importantly, an intact SHED module was required for these PEAK3 oncogenic activities. Mechanistically, through a phosphokinase survey, we identified PEAK3 as a novel inducer of AKT signaling, independent of growth-factor stimulation. Then, proteomic analyses revealed that PEAK3 interacts with the signaling proteins GRB2 and ASAP1/2 and the protein kinase PYK2, and that these interactions require the SHED domain. Moreover, PEAK3 activated PYK2, which promoted PEAK3 tyrosine phosphorylation, its association with GRB2 and ASAP1, and AKT signaling. Thus, the PEAK1-3 pseudo-kinases may use a conserved SHED-dependent mechanism to activate specific signaling proteins to promote oncogenesis.

Key roles for MYC, KIT and RET signaling in secondary angiosarcomas.

Background:Angiosarcomas may develop as primary tumours of unknown cause or as secondary tumours, most commonly following radiotherapy to the involved field. The different causative agents may be linked to alternate tumorigenesis, which led us to investigate the genetic profiles of morphologically indistinguishable primary and secondary angiosarcomas.Methods:Whole-genome (18k) c-DNA-mediated annealing, selection, extension and ligation analysis was used to genetically profile 26 primary and 29 secondary angiosarcomas. Key findings were thereafter validated using RT–qPCR, immunohistochemistry and validation of the gene signature to an external data set.Results:In total, 103 genes were significantly deregulated between primary and secondary angiosarcomas. Secondary angiosarcomas showed upregulation of MYC, KIT and RET and downregulation of CDKN2C. Functional annotation analysis identified multiple target genes in the receptor protein tyrosine kinase pathway. The results were validated using RT–qPCR and immunohistochemistry. Further, the gene signature was applied to an external data set and, herein, distinguished primary from secondary angiosarcomas.Conclusions:Upregulation of MYC, KIT and RET and downregulation of CDKN2C characterise secondary angiosarcoma, which implies possibilities for diagnostic application and a mechanistic basis for therapeutic evaluation of RET-kinase-inhibitors in these highly aggressive tumours.

TRPC Channels and Their Splice Variants are Essential for Promoting Human Ovarian Cancer Cell Proliferation and Tumorigenesis

TRPC channels are Ca²⁺-permeable cationic channels controlling Ca²⁺ influx response to the activation of G protein-coupled receptors and protein tyrosine kinase pathways or the depletion of Ca²⁺ stores. Here we aimed to investigate whether TRPC can act as the potential therapeutic targets for ovarian cancer. The mRNAs of TRPC1, TRPC3, TRPC4 and TRPC6 were detected in human ovarian adenocarcinoma. The spliced variants of TRPC1β, TRPC3a, TRPC4β, TRPC4γ, and TRPC6 with exon 3 and 4 deletion were highly expressed in the ovarian cancer cells, and a novel spliced isoform of TRPC1 with exon 9 deletion (TRPC1(E9del)) was identified. TRPC proteins were also detected by Western blotting and immunostaining. The expression of TRPC1, TRPC3, TRPC4 and TRPC6 was significantly lower in the undifferentiated ovarian cancer cells, but all-trans retinoic acid up-regulated the gene expression of TRPCs. The expression level was correlated to the cancer differentiation grade. The non-selective TRPC channel blockers, 2-APB and SKF-96365, significantly inhibited the cell proliferation, whilst the increase of TRPC channel activity by trypsin promoted the cell proliferation. Transfection with siRNA targeting TRPC1, TRPC3, TRPC4 and TRPC6 or application of specific blocking antibodies targeting to TRPC channels inhibited the cell proliferation. On the contrary, overexpression of TRPC1, TRPC1(E9del), TRPC3, TRPC4, and TRPC6 increased the cancer cell colony growth. These results suggest that TRPCs and their spliced variants are important for human ovarian cancer development and alteration of the expression or activity of these channels could be a new strategy for anticancer therapy.

TRPC Channels and Their Splice Variants are Essential for Promoting Human Ovarian Cancer Cell Proliferation and Tumorigenesis

TRPC channels are Ca2+-permeable cationic channels controlling Ca2+ influx response to the activation of G protein-coupled receptors and protein tyrosine kinase pathways or the depletion of Ca2+ stores. Here we aimed to investigate whether TRPC can act as the potential therapeutic targets for ovarian cancer. The mRNAs of TRPC1, TRPC3, TRPC4 and TRPC6 were detected in human ovarian adenocarcinoma. The spliced variants of TRPC1β, TRPC3a, TRPC4β, TRPC4γ, and TRPC6 with exon 3 and 4 deletion were highly expressed in the ovarian cancer cells, and a novel spliced isoform of TRPC1 with exon 9 deletion (TRPC1E9del) was identified. TRPC proteins were also detected by Western blotting and immunostaining. The expression of TRPC1, TRPC3, TRPC4 and TRPC6 was significantly lower in the undifferentiated ovarian cancer cells, but all-trans retinoic acid up-regulated the gene expression of TRPCs. The expression level was correlated to the cancer differentiation grade. The non-selective TRPC channel blockers, 2-APB and SKF-96365, significantly inhibited the cell proliferation, whilst the increase of TRPC channel activity by trypsin promoted the cell proliferation. Transfection with siRNA targeting TRPC1, TRPC3, TRPC4 and TRPC6 or application of specific blocking antibodies targeting to TRPC channels inhibited the cell proliferation. On the contrary, overexpression of TRPC1, TRPC1E9del, TRPC3, TRPC4, and TRPC6 increased the cancer cell colony growth. These results suggest that TRPCs and their spliced variants are important for human ovarian cancer development and alteration of the expression or activity of these channels could be a new strategy for anticancer therapy. Keywords: Calcium channels, ovarian cancer, proliferation, SKOV3 cells, TRPC

TRPC Channels and Their Splice Variants are Essential for Promoting Human Ovarian Cancer Cell Proliferation and Tumorigenesis

TRPC channels are Ca²⁺-permeable cationic channels controlling Ca²⁺ influx response to the activation of G protein-coupled receptors and protein tyrosine kinase pathways or the depletion of Ca²⁺ stores. Here we aimed to investigate whether TRPC can act as the potential therapeutic targets for ovarian cancer. The mRNAs of TRPC1, TRPC3, TRPC4 and TRPC6 were detected in human ovarian adenocarcinoma. The spliced variants of TRPC1β, TRPC3a, TRPC4β, TRPC4γ, and TRPC6 with exon 3 and 4 deletion were highly expressed in the ovarian cancer cells, and a novel spliced isoform of TRPC1 with exon 9 deletion (TRPC1(E9del)) was identified. TRPC proteins were also detected by Western blotting and immunostaining. The expression of TRPC1, TRPC3, TRPC4 and TRPC6 was significantly lower in the undifferentiated ovarian cancer cells, but all-trans retinoic acid up-regulated the gene expression of TRPCs. The expression level was correlated to the cancer differentiation grade. The non-selective TRPC channel blockers, 2-APB and SKF-96365, significantly inhibited the cell proliferation, whilst the increase of TRPC channel activity by trypsin promoted the cell proliferation. Transfection with siRNA targeting TRPC1, TRPC3, TRPC4 and TRPC6 or application of specific blocking antibodies targeting to TRPC channels inhibited the cell proliferation. On the contrary, overexpression of TRPC1, TRPC1(E9del), TRPC3, TRPC4, and TRPC6 increased the cancer cell colony growth. These results suggest that TRPCs and their spliced variants are important for human ovarian cancer development and alteration of the expression or activity of these channels could be a new strategy for anticancer therapy.

104 G-PROTEIN COUPLED RECEPTOR (GPCR) SIGNALLING IN SPONTANEOUSLY HYPERTENSIVE RATS (SHR)

Objectives: Nuclear transcription factor, Peroxisome Poliferator Activated Receptorγ (PPARγ) has been implicated in blood pressure regulation and vascular signalling that involves G-protein coupled receptors (GPCR). GPCR function depends on relative activation of multiple signalling pathways which is still not clear. Therefore, we have investigate the expression of genes that are related to GPCR signalling in hypertension and modulation by PPARγ. Design & Methods: Male SHRs and WKY controls were treated for 2 months with / with-out GW1929 (30 mg/kg orally), a PPARγ ligand and expression of genes were determined by PCR array. RNAs from aortic homogenates were used in this array analysis that included 84 genes from 16 different pathways involved in GPCR signalling. Results: In SHR, expression of 5 genes were up-regulated (3-5 fold) compared to their WKY controls. Among the up-regulated genes, expression of Cdkn1b and S1pr2 remains unchanged in GW1929 treated SHR. On the contrary expression of Ccl2, Ctgf and Gnas genes were significantly (p<0.05) reduced in GW1929 treated SHR. Similarly, in SHR, expression of 8 genes were significantly reduced (3-12 fold). Among these genes, Agtr1b, Ucp1, IL2, Drd1a were unchanged in GW1929 treated SHR while improving the expression of Bai1, Grm1, Grm5, Oprk1 gene (p<0.05) expression. Conclusion: This data suggests that GPCR signalling genes are involved in the regulation of blood pressure in SHR and PPARγ ligand-mediated blood pressure reduction may involve transcriptional regulation of these genes. We also suggests that cAMP, PKC, PI-3 kinase, Protein tyrosine kinase and Rho kinase pathways are involved in the regulation of blood pressure in SHR

An Essential Regulatory Role of Downstream of Kinase-1 in the Ovalbumin-Induced Murine Model of Asthma

The downstream of kinase (DOK)-1 is involved in the protein tyrosine kinase (PTK) pathway in mast cells, but the role of DOK-1 in the pathogenesis of asthma has not been defined. In this study, we have demonstrated a novel regulatory role of DOK-1 in airway inflammation and physiologic responses in a murine model of asthma using lentiviral vector containing DOK-1 cDNA or DOK-1-specific ShRNA. The OVA-induced inflammatory cells, airway hyperresponsiveness, Th2 cytokine expression, and mucus response were significantly reduced in DOK-1 overexpressing mice compared to OVA-challenged control mice. The transgenic introduction of DOK-1 significantly stimulated the activation and expression of STAT-4 and T-bet, while impressively inhibiting the activation and expression of STAT-6 and GATA-3 in airway epithelial cells. On the other hand, DOK-1 knockdown mice enhanced STAT-6 expression and its nuclear translocation compared to OVA-challenged control mice. When viewed in combination, our studies demonstrate DOK-1 regulates allergen-induced Th2 immune responses by selective stimulation and inhibition of STAT-4 and STAT-6 signaling pathways, respectively. These studies provide a novel insight on the regulatory role of DOK-1 in allergen-induced Th2 inflammation and airway responses, which has therapeutic potential for asthma and other allergic diseases.

Protein tyrosine kinase and mitogen-activated protein kinase signalling pathways contribute to differences in heterophil-mediated innate immune responsiveness between two lines of broilers

Protein tyrosine phosphorylation mediates signal transduction of cellular processes with protein tyrosine kinases (PTKs) regulating virtually all signalling events. The mitogen-activated protein kinase (MAPK) super-family consists of three conserved pathways that convert receptor activation into cellular functions: extracellular response kinases (ERK), c-Jun N-terminal kinases (JNK) and p38. Previously conducted studies using two chicken lines (A and B) show line A heterophils are functionally more responsive and produce a differential cytokine/chemokine profile compared with line B, which also translates to increased resistance to bacterial challenges. Therefore, we hypothesize the differences between the lines result from distinctive signalling cascades that mediate heterophil function. Heterophils from lines A and B were isolated from 1-day-old chickens and total phosphorylated PTK and p38, JNK, ERK, and transcription factor (activator protein 1 (AP-1) and nuclear factor kappa B (NF-κB)) protein levels quantified following interaction with Salmonella Enteritidis (SE). Control and SE-treated heterophils from line A had greater (P≤0.05) PTK phosphorylation compared to line B with increased (P≤0.05) activation of p38. Conversely, line B heterophils activated JNK (P≤0.05). There were no differences in ERK between control and activated heterophils for either line. Defined signalling inhibitors were used to show specificity. The AP-1 and NF-κB transcription factor families were also examined, and c-Jun and p50, respectively, were the only members different between the lines and both were up-regulated in line A compared with line B. These data indicate that increased responsiveness of line A heterophils is mediated, largely, by an increased ability to activate PTKs, the p38 MAPK pathway and specific transcription factors, all of which directly affect the innate immune response.

PP-051 Curcumin suppresses the induction of indoleamine 2,3-dioxygenase by blocking the JAK PKC-δ STAT1 signaling pathway in IFN-γ-stimulated murine dendritic cells

Background: The downstream of kinase (DOK)-1, a 62 kDa adaptor protein, negatively regulates the protein tyrosine kinase (PTK) pathway in mast cells, but the role of DOK-1 in a murine model of asthma has not been defined. Methods: In this study, we have demonstrated a novel regulatory role of DOK-1 in airway inflammation and physiologic responses in a murine model of asthma using lentiviral vector containing DOK-1 cDNA or DOK-1specific ShRNA. The OVA-induced inflammatory cells, airway hyperresponsiveness, Th2 cytokine expression, and mucus response were significantly reduced in DOK-1 overexpressing mice compared to OVA-challenged control mice. Results: The transgenic introduction of DOK-1 significantly stimulates the activation and expression of STAT-4 and T-bet, while impressively inhibits the activation and expression of STAT-6 and GATA-3 in the airway epithelial cells. On the other hand, DOK-1 knockdown mice enhanced STAT-6 expression and its nuclear translocation compared to OVA-challenged control mice. Conclusions: When viewed in combination, our studies demonstrate DOK-1 regulates allergen-induced Th2 immune responses by selective stimulation and inhibition of STAT-4 and STAT-6 signaling pathways, respectively. These studies provide a novel insight on the regulatory role of DOK-1 in allergen-induced Th2 inflammation and airway responses that has therapeutic potential for asthma and other allergic diseases.

Novel KCNA5 mutation implicates tyrosine kinase signaling in human atrial fibrillation

Emerging evidence has strongly implicated hereditary determinants for atrial fibrillation (AF). Loss-of-function mutations in KCNA5 encoding the ultrarapid delayed rectifier potassium current I(Kur) have been identified in AF families. The purpose of this study was to determine the clinical and biophysical phenotypes in a KCNA5 mutation with deletion of 11 amino acids in the N-terminus of the protein, which was identified in patients with lone AF. Patients with AF confirmed by ECG were prospectively enrolled in the Vanderbilt AF Registry, which comprises clinical and genetic databases. A KCNA5 mutation was generated by mutagenesis for electrophysiologic characterization. We identified a novel 33-bp coding region deletion in two Caucasian probands. One proband was part of a kindred that included four other members with AF, and all were mutation carriers. The mutation results in deletion of 11 amino acids in the N-terminus of the protein, a proline-rich region as a binding site for Src homology 3 (SH3) domains associated with Src-family protein tyrosine kinase (TK) pathway. In transfected cells, the mutant caused approximately 60% decreased I(Kur) versus wild-type (WT) (75 +/- 8 pA/pF vs 180 +/- 15 pA/pF, P <.01) and dominant-negative effect on WT current (105 +/- 10 pA/pF, P <.01). Pretreatment with the Src inhibitor PP2 prevented v-Src TK from 90% suppressed WT current. In contrast, the mutant channel displayed no response to v-Src TK. Our data implicate abnormal atrial repolarization control due to variable TK signaling as a mechanism in familial AF and thereby suggest a role for modulation of this pathway in AF and its treatment.

Protein tyrosine kinase pathway-derived ROS/NO productions contribute to G2/M cell cycle arrest in evodiamine-treated human cervix carcinoma HeLa cells

A previous study indicated that reactive oxygen species (ROS) and nitric oxide (NO) played pivotal roles in mediating cytotoxicity of evodiamine in human cervix carcinoma HeLa cells. This study suggested that G2/M cell cycle arrest was triggered by ROS/NO productions with regulations of p53, p21, cell division cycle 25C (Cdc25C), Cdc2 and cyclin B1, which were able to be prevented by protein tyrosine kinase (PTK) activity inhibitor genistein or JNK inhibitor SP600125. The decreased JNK phosphorylation by addition of Ras or Raf inhibitor, as well as the increased cell viability by addition of insulin-like growth factor-1 receptor (IGF-1R), Ras, Raf or c-Jun N-terminal kinase (JNK) inhibitor, further demonstrated that the Ras-Raf-JNK pathway was responsible for this PTK-mediated signalling. These observations provide a distinct look at PTK pathway for its suppressive effect on G2/M transition by inductions of ROS/NO generations.

The novel role of downstream of kinase-1 in the ovalbumin-induced murine model of asthma (141.4)

Abstract The downstream of kinase (DOK)-1, a 62 kDa adaptor protein negatively regulates the protein tyrosine kinase (PTK) pathway in mast cells, but the role of DOK-1 in a murine model of asthma has not been defined. This study provides experimental evidence demonstrating that DOK-1 is a key regulator for aiway inflammation in a murine model of asthma. Here, we have found that the markable reduction of pulmonary CD4+ T cell population and Th2 cytokines leads to the inhibition of pulmonary CD4+ T cell differentiation into Th2 cells in DOK-1 overexpression mice. DOK-1 overexpression in STAT-6 knockout (KO) mice increased the expression of STAT-4 and T-bet, which is transcription factor for Th1 differentiation. Conversely, Th2 regulatory molecules, STAT-6 and GATA-3 are impaired by DOK-1 overexpression in STAT-4 KO mice. Taken together, DOK-1 regulates Th1/Th2 immune balance through STAT4- and STAT6-dependent pathways, our findings provide additional insight into the immunological function of DOK-1 in Th2 inflammation and suggest a new potential therapeutic target for the treatment of asthmatic patients.

Prolactin induces chitotriosidase expression in human macrophages through PTK, PI3‐K, and MAPK pathways

We previously reported that prolactin (PRL) induces chitotriosidase (CHIT-1) mRNA expression in human macrophages. In this investigation we determined the signaling pathways involved in CHIT-1 induction in response to PRL. The CHIT-1 induction PRL-mediated was reduced by wortmannin and LY-294002, inhibitors of phosphatidylinositol 3-kinase (PI3-K) and by genistein an inhibitor of protein tyrosine kinase (PTK). Pre-treatment of macrophages with SB203580, a specific inhibitor of the mitogen-activated kinases (MAPK) p38, or with U0126, an inhibitor of MAPK p44/42, prevented both basal and exogenous PRL-mediated CHIT-1 expression. No significant effects on CHIT-1 induction PRL-mediated were observed with a protein kinase C inhibitor (PKC), rottlerin, or with an Src inhibitor, PP2, or with JAK2 inhibitor, AG490. In addition, PRL induced a phosphorylation of AKT that was prevented both by the two MAPK inhibitors SB203580 and U0126 and by the PI3-K inhibitors wortmannin and LY-294002. In conclusion, our results indicate that PRL up-regulated CHIT-1 expression via PTK, PI3-K, MAPK, and signaling transduction components.

Alterations in Protein Tyrosine Kinase Pathways in Rat Liver Following Normothermic Ischemia-Reperfusion

The phosphoregulation of signal transduction pathways is a complex series of reactions that modulate the cellular response to ischemia-reperfusion (I-R). The aim of this study was to evaluate the effect of normothermic liver I-R on protein tyrosine phosphorylation, production of angiogenic growth factors, and activation of signal proteins in tyrosine kinase pathways. A segmental normothermic ischemia of the liver was induced in rats by occluding the blood vessels (including the bile duct) to the median and left lateral lobes for 120 minutes. Liver extracts from either ischemic or nonischemic lobes were prepared at 0, 1, 3, and 6 hours after reperfusion. Liver tyrosine phosphorylation of proteins was examined by Western blot analysis, whereas vascular endothelial growth factor (VEGF) mRNA was analyzed by Northern blot. In ischemic liver lobes, VEGF mRNA and total protein levels increased at 1 and 3 hours after reperfusion. Tyrosine phosphorylation of the VEGF receptor Flk-1 and the platelet-derived growth factor receptor (PDGF-R) was increased only at 1 hour after reperfusion, while c-Src tyrosine phosphorylation remained increased at 3 hours and remained up to 6 hours after reperfusion. In conclusion, 1-R led to alterations in protein tyrosine phosphorylation and increased expression of VEGF in rat liver.

Epithelial cells treated with genistein inhibit adhesion and endocytosis of Paracoccidioides brasiliensis

Paracoccidioidomycosis is caused by Paracoccidioides brasiliensis, which although not formally considered an intracellular pathogen, can be internalized by epithelial cells in vitro and in vivo. The mechanisms used by P. brasiliensis to adhere to and invade non-professional phagocytes have not been identified. The signal-transduction networks, involving protein tyrosine kinase (PTK) and protein phosphatase activities, can modulate crucial events during fungal infections. In this study, the involvement of PTK has been investigated in P. brasiliensis adherence and invasion in mammalian epithelial cells. A significant inhibition of the fungal invasion occurred after the pre-treatment of the epithelial cells with genistein, a specific tyrosine kinase inhibitor, indicating that the tyrosine kinase pathway is involved in P. brasiliensis internalization. In contrast, when the fungus was treated, a slight (not significant) inhibition of PTK was observed, suggesting that PTK might not be the fungus' transduction signal pathway during the invasion process of epithelial cells. An intense PTK immunofluorescence labeling was observed in the periphery of the P. brasiliensis infected cells, little PTK labeling was found in both uninfected cells and yeast cells, at later infection times (8 and 24 h). Moreover, when the epithelial cells were treated with genistein and infected with P. brasiliensis, no labeling was observed, suggesting the importance of the PTK in the infectious process. These results suggest that PTK pathway participates in the transduction signal during the initial events of the adhesion and invasion processes of P. brasiliensis to mammalian epithelial cells.

Protein tyrosine kinase and p38 MAP kinase pathways are involved in stimulation of matrix metalloproteinase-9 by TNF-α in human monocytes

Matrix metalloproteinase-9 (MMP-9), through its catalytic and non-catalytic activities, plays critical roles in inflammation, tumor invasion and angiogenesis. Human monocytes actively involved in inflammatory and tumoral states secrete proMMP-9 (92 kDa). Endogenous TNF-α stimulates MMP-9 gene transcription in monocytes through NF-κB activation. In this study, we investigated the intracellular signaling pathways underlying TNF-α/NF-κB-dependent expression of MMP-9 in monocytes using chemical inhibitors that specifically inhibit distinct kinase pathways. We confirmed the expression of MMP-9 by reverse transcription chain reaction (RT-PCR), ELISA and gelatin zymography. PGE2/cAMP inhibitor indomethacin, PI-3K inhibitor wortmannin, PKC inhibitor bisindolylmaleimide and PKA inhibitor H-89 did not affect the levels of released MMP-9. In contrast, MMP-9 mRNA and protein expression was down-regulated by p38 MAPK inhibitor SB203580 and protein tyrosine kinase (PTK) inhibitor tyrphostin 25. These inhibitors increased IκB-α levels, which correlate with decreased NF-κB activation. Although SB203580 induced a decrease in TNF-α release, addition of exogenous TNF-α did not reverse the inhibitory effect of SB203580 toward MMP-9 thus suggesting that SB203580 could modulate down-stream effects of TNF-α. In parallel, TIMP-1 levels decreased in the presence of SB203580. Both kinase inhibitors did not influence the maturation pathway of monocytes. Our results indicate that these two inhibitors of p38 MAPK and PTK pathways could be used as combined targets for inhibiting MMP-9 expression in inflamed tissues.

Genistein suppresses the invasive potential of human breast cancer cells through transcriptional regulation of metalloproteinases and their tissue inhibitors

Progression of breast cancer implicates the degradation of extracellular matrix (ECM) by metallo-proteinases (MMPs), a process with important consequences on the growth and invasiveness of cancer cells in adjacent and distant sites. The isoflavone, genistein--a natural inhibitor of protein tyrosine kinase pathway--inhibits the growth of a wide range of cancer cells in vitro. The aim of this study was to investigate: i) the expression of mRNAs encoded for MMPs and their endogenous inhibitors (TIMPs) associated with pathogenesis and metastatic potential of breast cancer cells; and ii) the effect of genistein on the transcription of MMPs and TIMPs and the invasive potential of breast cancer cells. Gene expression at transcriptional level was examined in cell cultures of two epithelial breast cancer cell lines, the high invasive (ER-negative) MDA-MB-231 and the low invasive (ER-positive) MCF-7, as well as the normal mammary cells (MCF-12A) following RNA isolation and reversed transcriptase polymerase chain reaction (RT-PCR). The inhibitory effect of genistein on functional invasiveness was examined by a cell invasion assay. Cell cycle distribution showed that genistein arrested breast cancer MDA-MB-231, MCF-7 and BT-20 cells in the G2/M phase. Both normal and breast cancer cell lines express the genes of MMP-2, -9, MT1-, MT2-, MT3-MMP and TIMP-1, -2 and -3. MCF-7 express notably less MMPs than MDA-MB-231 cell line. The addition of genistein resulted in down-regulation of the transcription of all MMP genes in MDA-MB-231 and most of MMPs in MCF-7 cells. The inhibitory effect of genistein on MMPs was functionally confirmed, since it significantly reduced the invasion properties of cancer cells in vitro. The obtained results indicate that genistein may be of great value in prevention of breast cancer cell metastasis, since it represents both a transcriptional modulator of genes involved in this pathogenetic process and a suppressor of breast cancer cell invasiveness.

Signal Transduction of ET-1 in Contraction of Cerebral Arteries

The signaling pathways of endothelin-1-induced contraction, including the role of protein tyrosine kinase (PTK), mitogen-activated protein kinase (MAPK), protein kinase C (PKC) and RhoA/Rho-kinase were studied using rabbit basilar arteries by isometric tension and Western blot. The following results were observed: (1) endothelin-1 produced phosphorylation of MAPK and RhoA and contraction by activation of endothelin-A but not endothelin-B receptors; (2) MAPK inhibitors, PD 98059 and U0126, PTK inhibitor, genistein, Src kinase inhibitor, damnacanthal, and Janus tyrosine kinase (JAK2) inhibitor, AG-490, abolished endothelin-1-induced contraction and MAPK immunoreactivity; (3) PTK inhibitor, staurosporine, and phosphatidylinositol 3-kinase (PI- 3K) inhibitor wortmannin abolished endothelin-1 induced contraction but not MAPK immunoreactivity; (4) Rho-kinase inhibitor, Y-27632, reduced endothelin-1-induced contraction; (5) PI-3K inhibitor, wortmannin, but not PKC and PTK inhibitors, reduced endothelin-1-induced RhoA activation; (6) endothelin-1 increased the level of myosin light chain (MLC) phosphorylation, and Rho-kinase inhibitor, Y-27632, reduced the effect of endothelin- 1 on MLC phosphorylation. This study demonstrated that three signaling pathways Src-JAK2-PTK-MAPK, PI-3K-RhoA-Rhokinase- MLC and PKC all contribute to endothelin-1-induced contraction in the rabbit basilar artery. MAPK is downstream of PTK, Src and JAK pathways. PI-3 kinase and MLC might be the upstream and downstream factors of RhoA activation.

Stimulatory effect of PG-KII, an NK 3 tachykinin receptor agonist, on isolated pancreatic acini: species-related differences

More information is needed on the physiological role of the tachykinins (TKs), especially neurokinin 3-receptor (NK 3) agonists, in the pancreas. In this paper we investigated and compared the effect of PG-KII (10 −9 to 10 −6 M), a natural NK 3-receptor agonist, with that of the known secretagogues substance P (10 −9 to 10 −6 M), caerulein (10 −11 to 10 −8 M) and carbachol (10 −8 to 10 −5 M), on amylase secretion from dispersed pancreatic acini of the guinea pig and rat. PG-KII (10 −7 M) significantly increased basal amylase release from guinea pig pancreatic acini (from 5.4±0.9% to 11.3±0.5%, P<0.05) but left basal release in the rat unchanged (6.5±0.5%). The stimulant effect of PG-KII on guinea pig acini was significantly reduced by the NK 3-receptor antagonist, SR 142801 (5×10 −7 M), and left unchanged by the NK 1-receptor antagonist, SR 140333 (5×10 −7 M). Conversely, substance P (10 −7 M) significantly stimulated amylase secretion from rat and guinea pig acini (12.6±0.6% and 12.1±0.7%, P<0.05). This stimulated effect of substance P was antagonized by the NK 1-receptor antagonist (5×10 −7 M), but not by the NK 3-receptor antagonist (5×10 −7 M). The PG-KII- and substance P-evoked maximal responses were lower than those evoked by caerulein (10 −9 M) (guinea pig, 19.1±1.3%; rat, 18.2±0.9%, P<0.01) and carbachol (10 −5 M) (guinea pig, 23.3±1.2%; rat, 24.0±1.1%, P<0.01). The inhibitors of phospholipase C U-73122 (10 −5 M), phospholipase A 2 quinacrine (10 −5 M), and protein tyrosine kinase genistein (10 −4 M), partly but significantly inhibited PG-KII, as well as carbachol-stimulated amylase release. Coincubation of PG-KII 10 −7 M with submaximal doses of caerulein (10 −11 to 10 −10 M) and carbachol (10 −7 to 10 −6 M) had an additive effect on amylase release. Pre-incubation with PG-KII (10 −7 M) for 30 min significantly reduced the subsequent amylase response to PG-KII, whereas pre-incubation with caerulein 10 −10 M or carbachol 10 −6 M did not. These findings suggest that PG-KII directly contributes to pancreatic exocrine secretion by interacting with acinar NK 3 receptors of the guinea pig but not of the rat. PG-KII signal transduction involves the intracellular phospholipase C, phospholipase A 2 and protein tyrosine kinase pathways. The NK 3 receptor system cooperates with the other known secretagogues in regulating guinea pig exocrine pancreatic secretion and undergoes rapid homologous desensitization.

The effect of mycobacterial virulence and viability on MAP kinase signalling and TNF α production by human monocytes

Setting: The success of Mycobacterium tuberculosis as a human pathogen depends on its ability to tolerate and perhaps manipulate host defense mechanisms. Objective: To determine the induction of tumour necrosis factor-alpha (TNF α), a central mediator of immunity, by human monocytes infected with virulent M. tuberculosis, M. leprae and attenuated M. bovis BCG. Design: Mycobacteria-induced cellular activation pathways of TNF α production was investigated using an inhibitor of protein tyrosine kinase (PTKs) and an inhibitor of mitogen-activated protein (MAP) kinases. Results:TNF α production was significantly lower during infection with virulent M. tuberculosis than with BCG and this differential response was independent of mycobacterial viability. TNF α production involved the PTK and MAP kinase pathways. Reduced TNF α induction by M. tuberculosis was associated with a reduction in the extent and duration of phosphorylation of extracellular-signal regulated kinases (ERK 1/2). Infection with M. leprae triggered low and transient ERK 1/2 activation as well as low TNF α production. Conclusion: Maintenance of the differential response in both live and heat-killed preparations suggests that the reduced TNF α response associated with virulent mycobacteria is due to differences in the presence of components capable of triggering host pattern recognition receptors, rather than events associated with phagosome trafficking or the active release of intracellular modulators.