CHEMICAL COMPOSITIONS AND BIOLOGICAL PROPERTIES OF ESSENTIAL OILS FROM ZANTHOXYLUM RHETSA (ROXB.) DC AND ZANTHOXYLUM LIMONELLA ALSTON

Background: Zanthoxylum rhetsa (Roxb.) DC and Zanthoxylum limonella Alston are spices for flavouring in indigenous Thai food. They are traditionally used as an aromatic, astringent, antimicrobial, antiseptic and antidiabetic agent. The purpose of this study is to examine their chemical compositions and evaluate antibacterial, antioxidant and anticancer properties of the essential oils. Materials and Methods: The essential oils of Z. rhetsa and Z. limonella were analysed for phytochemical constituents by Gas chromatography–mass spectrometry (GC-MS). The antibacterial activity was determined against several bacteria using the microdilution method. Antioxidant capacity was determined by free radical scavenger 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2-azinobis-3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) methods. The anticancer activity was determined with two breast cancer cell lines (MCF-7 and MDA-MB-231) and the normal African green monkey kidney epithelial (Vero) cell line and using MTT assay. Results: Sabinene (22.51%) and terpinene-4-ol (32.33%) were found to be major components of Z. rhetsa essential oil while limonene (57.94%) and alpha-phelladrene (15.54%) were the major components of Z. limonella essential oil. Essential oil from Z. limonella exhibited broad spectrum antibacterial activity. Z. rhetsa and Z. limonella essential oils exhibited moderate antioxidant activity. The essential oil from Z. rhetsa possessed the ability to inhibit breast cancer cell (MCF-7 and MDA-MB-231) proliferation and cell viability. Conclusion: This study suggest that the essential oils from Z. limonella and Z. rhetsa could be applied as safe antibacterial and antioxidant agents for food and have the potential for further development of new anticancer agents

Comparative proteomic analysis implicates eEF2 as a novel target of PI3Kgamma in the MDA-MB-231 metastatic breast cancer cell line

Cancer cell migration is fundamentally required for breast tumour invasion and metastasis. The insulin-like growth factor 1 tyrosine kinase receptor (IGF-1R) and the chemokine G-protein coupled receptor, CXCR4 have been shown to play an important role in breast cancer metastasis. Our previous study has shown that IGF-1R can transactivate CXCR4 via a physical association in the human MDA-MB-231 metastatic breast cancer cell line and that this plays a key role in IGF-I-induced migration of these cells. In the present study we used pharmacological inhibition and RNAi to identify PI3Kγ as an important migration signalling molecule downstream of receptor transactivation in MDA-MB-231 cells. To identify PI3K?-regulated proteins upon transactivation of CXCR4 by IGF-I, we undertook a comparative proteomics approach using 2-D- Fluorescence Difference Gel Electrophoresis (DIGE) and identified the proteins by mass spectrometry.Meizhi Niu, Manuela Klingler-Hoffmann, Julie A Brazzatti, Briony Forbes, Chareeporn Akekawatchai, Peter Hoffmann and Shaun R McCol

CXCR4/CXCL12 Participate in Extravasation of Metastasizing Breast Cancer Cells within the Liver in a Rat Model

INTRODUCTION: Organ-specific composition of extracellular matrix proteins (ECM) is a determinant of metastatic host organ involvement. The chemokine CXCL12 and its receptor CXCR4 play important roles in the colonization of human breast cancer cells to their metastatic target organs. In this study, we investigated the effects of chemokine stimulation on adhesion and migration of different human breast cancer cell lines in vivo and in vitro with particular focus on the liver as a major metastatic site in breast cancer. METHODS: Time lapse microscopy, in vitro adhesion and migration assays were performed under CXCL12 stimulation. Activation of small GTPases showed chemokine receptor signalling dependence from ECM components. The initial events of hepatic colonisation of MDA-MB-231 and MDA-MB-468 cells were investigated by intravital microscopy of the liver in a rat model and under shRNA inhibition of CXCR4. RESULTS: In vitro, stimulation with CXCL12 induced increased chemotactic cell motility (p,0.05). This effect was dependent on adhesive substrates (type I collagen, fibronectin and laminin) and induced different responses in small GTPases, such as RhoA and Rac-1 activation, and changes in cell morphology. In addition, binding to various ECM components caused redistribution of chemokine receptors at tumour cell surfaces. In vivo, blocking CXCR4 decreased extravasation of highly metastatic MDA-MB-231 cells (p < 0.05), but initial cell adhesion within the liver sinusoids was not affected. In contrast, the less metastatic MDA-MB-468 cells showed reduced cell adhesion but similar migration within the hepatic microcirculation. CONCLUSION: Chemokine-induced extravasation of breast cancer cells along specific ECM components appears to be an important regulator but not a rate-limiting factor of their metastatic organ colonization.Claudia Wendel, André Hemping-Bovenkerk, Julia Krasnyanska, Sören Torge Mees, Marina Kochetkova, Sandra Stoeppeler and Jörg Haie

Insulin-like growth factor - Oestradiol crosstalk and mammary gland tumourigenesis

Development and differentiation of the mammary gland are dependent on the appropriate temporal expression of both systemically acting hormones and locally produced growth factors. A large body of evidence suggests that molecular crosstalk between these hormonal and growth factor axes is crucial for appropriate cell and tissue function. Two of the most important trophic factors involved in this process are the oestrogen (E) and insulin-like growth factor (IGF) molecular axes. The reciprocal crosstalk that exists between these pathways occurs at transcriptional/post-transcriptional and translational/post-translational levels regulate the expression and activity of genes involved in this process. In a clinical context an important consequence of such crosstalk in the mammary gland is the role which it may play in the aetiology, maintenance and development of breast tumours. Although oestradiol (E2) acting through oestrogen receptors α and β (ERα/β) is important for normal mammary gland function it can also provide a mitogenic drive to ER+ breast tumours. Therefore over several years anti-oestrogen therapeutic regimens in the form of selective oestrogen receptor modulators (SERMs - e.g. tamoxifen), aromatase inhibitors (AI e.g. anastrozole) or selective oestrogen receptor down regulators (SERDs - e.g. fulvestrant) have been used in an adjuvant setting to control tumour growth. Although initial response is usually encouraging, large cohorts of patients eventually develop resistance to these treatments leading to tumour recurrence and poor prognosis. There are potentially many routes by which breast cancer (BC) cells could escape anti-oestrogen based therapeutic strategies and one of the most studied is the possible growth factor mediated activation of ER(s). Because of this, growth factor modulation of ER activity has been an intensively studied route of molecular crosstalk in the mammary gland. The insulin-like growth factors (IGF-1 and -2) are amongst the most potent mitogens for mammary epithelial cells and there is accumulating evidence that they interact with the E2 axis to regulate mitogenesis, apoptosis, adhesion, migration and differentiation of mammary epithelial cells. Such interactions are bi-directional and E2 has been shown to regulate the expression and activity of IGF axis genes with the general effect of sensitising breast epithelial cells to the actions of IGFs and insulin. In this short review we discuss the evidence for the involvement of crosstalk between the insulin-like growth factor (IGF) and oestrogen axes in the mammary gland and comment on the relevance of such studies in the aetiology and treatment of BC

Cross-talk between CXCR4 and IGF-1R signal transduction pathways in a metastatic breast cancer cell line

"The present study investigated the expression and function of IGF-1R, CXCR4 and CCR7, in metastatic MDA-MB-231 and non-metastatic MCF-7 cells."Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 200

Chemokines and chemokine receptors in cancer progression

Directed cell migration is a fundamental component of numerous biological systems and is critical to the pathology of many diseases. Although the importance of chemokines in providing navigational cues to migrating cells bearing specific receptors is well-established, how chemokine function is regulated is not so well understood and may be of key importance to the design of new therapeutics for numerous human diseases, particularly for the control of cancer growth and metastasis, diseases in which chemokines have recently been implicated. In this review, we discuss the general views on the role of specific chemokines in these pathological processes. In addition, we discuss two novel aspects of chemokine cancer biology; cross-talk between chemokine and growth factor receptors, and refractory chemokine receptors.Chareeporn Akekawatchai, Marina Kochetkova, Jane Holland and Shaun R McCol

Transactivation of CXCR4 by the insulin-like growth factor-1 receptor (IGF-1R) in human MDA-MB-231 breast cancer epithelial cells

The definitive version can be found at http://www.jbc.org/In the multimolecular environment in tissues and organs, crosstalk between growth factor and G protein-coupled receptors is likely to play an important role in both normal and pathological responses. In this report, we demonstrate transactivation of the chemokine receptor CXCR4 by the growth factor insulin-like growth factor (IGF)-1 is required for IGF-1-induced cell migration in metastatic MDA-MB-231 cells. The induction of chemotaxis in MDAMB- 231 cells by IGF-1 was inhibited by pretreatment of the cells with pertussis toxin (PTX) and by RNAi-mediated knockdown of CXCR4. Transactivation of the CXCR4 pathway by IGF-1 occurred independently of CXCL12, the chemokine ligand of CXCR4. Neither CXCR4 knockdown nor PTX had any effect on the ability of IGF-1 to activate IGF-1R, suggesting thatCXCR4andGproteins are activated subsequent to, or independently of, phosphorylation of IGF-1R by IGF-1. Coprecipitation studies revealed the presence of a constitutive complex containing IGF-1R, CXCR4, and theGprotein subunits, Giα2 and Gß, and stimulation of MDA-MB-231 cells with IGF-1 led to the release of Giα2 and Gß from CXCR4. Based on our findings, we propose that CXCR4 constitutively forms a complex with IGF-1R in MDA-MB-231 cells, and that this interaction allows IGF-1 to activate migrational signaling pathways through CXCR4, Giα2 and Gß.Chareeporn Akekawatchai, Jane D. Holland, Marina Kochetkova, John C. Wallace and Shaun R. McCol

Differential functional activation of chemokine receptor CXCR4 is mediated by G proteins in breast cancer cells

© 2006 American Association for Cancer ResearchCXCR4 is a G protein–coupled receptor of considerable biological significance, and among its numerous functions, it is suggested to play a critical role in cancer metastasis. We have investigated the expression and function of CXCR4 in a range of breast cancer cell lines covering a spectrum of invasive phenotypes and found that, while surface levels of CXCR4 were uniform across the entire panel, only highly invasive cells that are metastatic in immunocompromised mice expressed functional receptors. CXCL12/SDF-1 induced cellular responses such as calcium mobilization, actin polymerization, and chemotaxis in metastatic cells, whereas noninvasive cells were unresponsive. Moreover, CXCL12 activated multiple signaling pathways downstream of G proteins in highly invasive cells but failed to activate any of the examined kinase cascades in noninvasive cell lines. This blockade in nonmetastatic cell lines seems to be due to the inability of G protein A and B subunits to form a heterotrimeric complex with CXCR4. GA and GB were able to bind to CXCR4 independently in all cell lines, but the association of G protein AB; heterotrimers with the receptor, a prerequisite for signal transduction downstream from G protein–coupled receptors, was only observed in the highly invasive cell lines. Our findings show, for the first time, that CXCR4 function is subject to complex and potentially tightly controlled regulation in breast cancer cells via differential G protein–receptor complex formation, and this regulation may play a role in the transition from nonmetastatic to malignant tumors.Jane D. Holland, Marina Kochetkova, Chareeporn Akekawatchai, Mara Dottore, Angel Lopez and Shaun R. McCol

Protean agonism of the lysophosphatidic acid receptor-1 with Ki16425 reduces nerve growth factor-induced neurite outgrowth in pheochromocytoma 12 cells

We report here a novel role for the constitutively active lysophosphatidic acid receptor-1 (LPA(1)) receptor in providing Gbetagamma subunits for use by the Trk A receptor. This enhances the ability of nerve growth factor (NGF) to promote signalling and cell response. These conclusions were based on three lines of evidence. Firstly, the LPA(1) receptor was co-immunoprecipitated with the Trk A receptor from lysates, suggesting that these proteins form a complex. Secondly, Ki16425, a selective protean agonist of the LPA(1) receptor, decreased constitutive basal and LPA-induced LPA(1) receptor-stimulated GTPgammaS binding. Ki16425 reduced the LPA-induced activation of p42/p44 mitogen activated protein kinase (MAPK), while acting as a weak stimulator of p42/p44 MAPK on its own, properties typical of a protean agonist. Significantly, Ki16425 also reduced the NGF-induced stimulation of p42/p44 MAPK and inhibited NGF-stimulated neurite outgrowth. Thirdly, the over-expression of the C-terminal GRK-2 peptide, which sequesters Gbetagamma subunits, reduced the NGF-induced activation of p42/p44 MAPK. In contrast, the stimulation of PC12 cells with LPA leads to a predominant G(i)alpha2-mediated Trk A-independent activation of p42/p44 MAPK, where Gbetagamma subunits play a diminished role. These findings suggest a novel role for the constitutively active LPA(1) receptor in regulating NGF-induced neuronal differentiation