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

Abstract

The life threatening characteristics of cancers lie in the uncontrolled cell mitogenic activity and the acquiring of metastatic capability. Therefore, the strategies for cancer therapy are aimed at controlling the cancer cell growth and/or inhibiting the cancer cell invasion/metastasis. Early reports contended that protein tyrosine kinase (PTK) regulatory mechanisms are clearly implicated in the growth of neoplastic cells, and inactivating specific PTKs could retard the growth of tumors. 1-3 Among the PTKs investigated, epidermal growth factor receptor (EGFR) tyrosine kinase has attracted much attention with regard to its role in transducing signal for promoting cell growth. Autophosphorylation of EGFR appears to be a potential target for cancer therapy because that would engender a blockade of the EGFR-mediated signaling. 3 In light of this, it is imperative to explore whether luteinizing hormone-releasing hormone (LHRH) analogues that possess antiproliferative activity could have any direct effects on the autophosphorylation of EGFR and its intrinsic tyrosine kinase activity.More than 3 decades have passed since the isolation and characterization of the hypothalamic LHRH, which controls the anterior pituitary secretion of luteinizing hormone and follicle stimulating hormone. 4 Accumulated data have indicated that LHRH might also present in extra-hypothalamic tissues, such as ovary, testis, kidney and placenta. 5 Currently, more than 3,000 analogues of LHRH have been synthesized. 6 Agonists of LHRH have important clinical applications in gynecology and oncology. 6 Potent antagonists of LHRH, such as Cetrorelix, are also available for clinical use. 6 The actions of LHRH and its analogues are mediated by high-affinity receptors for LHRH found on pituitary gonadotrophs and various tumors. 6 The presence of high affinity binding sites for LHRH and the expression of mRNA for LHRH receptors have been shown in human prostatic, mammary, ovarian and endometrial cancers. These LHRH receptors on tumor cells can mediate direct effects of LHRH agonists and antagonists. Inhibitory effects of LHRH agonists and antagonists on prostatic, mammary, ovarian and endometrial cancer cells have been demonstrated in vitro. 6 -11 Thus, a potent agonist ]LHRH and LHRH antagonist Cetrorelix synthesized in the laboratory of one of us (A.V.S.) 6 was shown to inhibit the growth of human ovarian endometrial and mammary cancer cells 9 and LHRH agonists Zoladex and Buserelin suppressed the proliferation of human prostate cancer lines. 7,10 The effects of LHRH-analogues on mitogenic signal transduction in cancer cells were also the subjects of extenAbbreviations: DMSO, dimethyl sulphoxide; EDTA, ethylenediamine tetraacetic acid; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ICE, interleukin-1␤ converting enzyme; LHRH, luteinizing hormone-releasing hormone; MMP, matrix metalloproteinase; PARP, poly(ADP-ribose) polymerase; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; SDS-PAGE, sodium dodecyl sulphate-p...

Transducer of Cdc42-dependent Actin Assembly Promotes Epidermal Growth Factor-induced Cell Motility and Invasiveness

“…Commercial sources of antibodies included mouse anti-green fluorescent protein (GFP) from Abcam; rabbit anti-p34-Arc (ArpC2) from Millipore; mouse anti-Abi1 (clone 1B9) from Medical and Biological Laboratories Co. Ltd. (MBL); mouse anti-EGFR and mouse anti-active-EGFR (Tyr(P)-EGFR), R-phycoerythrin-conjugated mouse anti-EGFR (clone EGFR.1 and isotype control clone [27][28][29][30][31][32][33][34][35], all from BD Biosciences; mouse anti-Tyr(P) (PY99) from Santa Cruz Biotechnology; mouse anti-tubulin from Sigma-Aldrich; and goat anti¬FBP17 from IMGENEX. Secondary antibodies included Alexa Fluor 488-or Alexa Fluor 633-conjugated goat anti-mouse/rabbit IgG, and Alexa633-conjugated goat antimouse/rabbit IgG (Molecular Probes).…”

“…Several individual colonies for each cell line were recorded at time 0 and 72 h post-EGF treatment using a phasecontrast microscope equipped with a digital camera. Cell invasion assays were performed essentially as previously described (31). Briefly, A431 vector and Toca-1 knockdown cells (1 ϫ 10 5 cells; in triplicate) were placed in the upper chamber of Transwell inserts (8-m pore size) coated with a layer of Matrigel TM (100 l of 1:5 dilution in DMEM).…”

Triptolide induces apoptosis in human adrenal cancer NCI-H295 cells through a mitochondrial-dependent pathway

“…Equal amounts of lysate protein were run on 12% SDS-PAGE and electrophoretically transferred to PVDF membrane (Amersham Pharmacia Biotech, USA) then the blocked blots were incubated with specific primary antibody against cytochrome c, Apaf-1, AIF, Endo G, caspase-9 and caspase-3 overnight and further incubated for 1 h with HRP conjugated secondary antibody (Santa Cruz Biotechnology). Bound antibodies were detected by ECL kit as described previously (18)(19)(20)(21).…”

A novel synthetic 2-(3-methoxyphenyl)-6,7-methylenedioxoquinolin-4-one arrests the G2/M phase arrest via Cdc25c and induces apoptosis through caspase- and mitochondria-dependent pathways in TSGH8301 human bladder cancer cells

“…After blocking, the blots were incubated with specific primary antibody against caspase-3, caspase-8, caspase-9, cytochrome c and TRIAL, Bax, Bcl-X, Bid, AIF, Endo G, Chk2, Chk1, Wee 1, Cdc25c, Cdc2, cyclin A and B1 overnight and further incubated for 1 h with HRP conjugated secondary antibody (Santa Cruz). Bound antibodies were detected by ECL kit (Millipore) as described previously (17)(18)(19)(20).…”