Overexpression of Hsp25 in K1735 murine melanoma cells enhances susceptibility to natural killer cytotoxicity

In the present study we used a murine melanoma model to investigate the effect of the 25-kDa heat shock protein (Hsp25) on natural killer (NK) cytotoxicity. The melanoma lines K1735-C123 (low metastatic potential) and K1735-M2 (high metastatic potential) were transfected with hsp25 and a control plasmid. Highly purified interleukin (IL)-2-stimulated DX-5+ NK cells showed enhanced lysis of Hsp25-overexpressing K1735-C123 targets in comparison with controls. In contrast, there was no difference in susceptibility to lysis by purified IL-2-stimulated DX-5+ NK cells between Hsp25-overexpressing and control-transfected K1735-M2 targets. Fluorescence-activated cell sorter analysis revealed that Hsp25 is displayed on the cell surface independently of Hsp25 overexpression and metastatic phenotype. Thus, surface localization of Hsp25 does not correlate with the target cell susceptibility to killing. To sum up, a cytoplasmic overexpression of Hsp25 is associated with an increased susceptibility to lysis by DX-5+ NK cells in the low-metastatic murine melanoma model investigated.     

The combination of glutamate receptor antagonist MK-801 with tamoxifen and its active metabolites potentiates their antiproliferative activity in mouse melanoma K1735-M2 cells

Recent reports suggest that N-methyl-d-aspartate receptor (NMDAR) blockade by MK-801 decreases tumor growth. Thus, we investigated whether other ionotropic glutamate receptor (iGluR) antagonists were also able to modulate the proliferation of melanoma cells. On the other hand, the antiestrogen tamoxifen (TAM) decreases the proliferation of melanoma cells, and is included in combined therapies for melanoma. As the efficacy of TAM is limited by its metabolism, we investigated the effects of the NMDAR antagonist MK-801 in combination with TAM and its active metabolites, 4-hydroxytamoxifen (OHTAM) and endoxifen (EDX). The NMDAR blockers MK-801 and memantine decreased mouse melanoma K1735-M2 cell proliferation. In contrast, the NMDAR competitive antagonist APV and the AMPA and kainate receptor antagonist NBQX did not affect cell proliferation, suggesting that among the iGluR antagonists only the NMDAR channel blockers inhibit melanoma cell proliferation. The combination of antiestrogens with MK-801 potentiated their individual effects on cell biomass due to diminished cell proliferation, since it decreased the cell number and DNA synthesis without increasing cell death. Importantly, TAM metabolites combined with MK-801 promoted cell cycle arrest in G1. Therefore, the data obtained suggest that the activity of MK-801 and antiestrogens in K1735-M2 cells is greatly enhanced when used in combination.     

Association between Tumorigenic Potential and the Fate of Cancer Cells in a Syngeneic Melanoma Model

The self-renewal potential of a cancer cell can be estimated by using particular assays, which include xenotransplantation in immunocompromised animals or culturing in non-adherent serum-free stem-cells media (SCM). However, whether cells with self-renewal potential actually contribute to disease is unknown. Here we investigated the tumorigenic potential and fate of cancer cells in an in-vivo melanoma model. We examined cell lines which were derived from the same parental line: a non-metastatic cell line (K1735/16), a metastatic cell line (K1735/M4) and a cell line which was selected in non-adherent conditions (K1735/16S). All cell lines exhibited similar proliferation kinetics when grown on culture plates. K1735/16 cells grown in soft agar or in suspension non-adherent conditions failed to form colonies or spheroids, whereas the other cell lines showed prominent colonogenicity and spheroid formation capacity. By using sphere limiting dilution analysis (SLDA) in serum-free media, K1735/16S and K1735/M4 cells grown in suspension were capable of forming spheroids even in low frequencies of concentrations, as opposed to K1735/16 cells. The tumorigenic potential of the cell lines was determined in SCID mice using intra footpad injections. Palpable tumors were evident in all mice. In agreement with the in-vitro studies, the K1735/M4 cell line exhibited the highest growth kinetics, followed by the K1735/16S cell line, whereas the K1735/16 cell line had the lowest tumor growth potential (P<0.001). In contrast, when we repeated the experiments in syngeneic C3H/HeN mice, the K1735/16 cell line produced macroscopic tumors 30–100 days after injection, whereas K1735/M4 and K1735/16S derived tumors regressed spontaneously in 90–100% of mice. TUNEL analysis revealed significantly higher number of apoptotic cells in K1735/16S and K1735/M4 cell line-derived tumors compared to K1735/16 tumors (P<0.001). The models we have examined here raised the possibility, that cells with high-tumorigenic activity may be more immunogenic and hence are more susceptible to immune-regulation.     

Effects of genistein and daidzein on the cell growth,cell cycle,and differentiation of human and murine melanoma cells(1)

Genistein and daidzein are two major isoflavonoids in dietary soybean that have inhibition effect on the cell growth of different tumor cell lines. We previously reported the anti-tumor activities of genistein and daidzein in human co1on tumor (HCT) cells and their different ability to enhance the activation of murine lymphocytes. In the present study, the effect of genistein and daidzein on the cell growth, cell cycle progression, and differentiation of murine K1735M2 and human WM451 cel1s was investigated. It was found that genistein could inhibit the cell growth of two metastatic melanoma cell lines, murine Kl735M2 and human WM45l in a dose-dependent manner. Flow cytometry showed that genistein could cause arrest of both Kl735M2 and WM45l at G(2)/M phase, while daidzein increased the cell numbers at S phase, decreased the cell numbers at G(1) phase. Detection of melanin and morphological observation showed that genistein can induce Kl735M2 and WM45l to produce dendrite-like structure and produce more melanin by 80%. In contrast, daidzein only retarded the growth of K1735M2 and did not induce differentiation in either K1735M2 or WM451. These results suggest that genistein and daidzein in soybean can inhibit certain malignant phenotype of melanoma via different mechanisms and be potential medical candidates for melanoma cancer therapy.     

Proportion of antigenic variants induced by in vitro UV irradiation differs in clones derived from a single tumor

The purpose of this study was to examine the capacity of different clones derived from the same tumor to generate highly antigenic cells after in vitro exposure to UV radiation. Cells from the metastatic murine melanoma K1735 and clones of K1735 differing in metastatic potential were exposed to UV radiation in vitro, cloned, and tested for antigenic properties in vivo. Approximately half of the clones isolated after UV irradiation of parental K1735 melanoma cells were highly antigenic (five of nine). Similar treatment of cells of a nonmetastatic clone of K1735 generated clones that were all antigenic (nine of nine). In contrast, only one of nine clones derived from UV-irradiated cells of a highly metastatic clone of K1735 were antigenic. Clones derived from unirradiated cultures were not antigenic variants. The increased antigenicity of cells derived from UV-irradiated cultures did not correlate with an increase in expression of cell surface class I major histocompatibility complex antigens. These results demonstrate that the frequency of antigenic variant production after UV irradiation is an intrinsic property of the particular cell line used, and that even cloned cell lines derived from a single tumor differ in their ability to generate antigenic variants after UV irradiation. In addition, they indicate that the increased antigenicity is not necessarily due to a UV-induced increase in expression of cell surface class I histocompatibility antigens.     

Pharmacological characterization of a novel muscarinic partial agonist, YM796, in transfected cells expressing the m1 or m2 muscarinic receptor gene.

To investigate the pharmacological effect of a novel compound YM796, we performed radioligand binding experiments and correlative biochemical experiments using the transfected murine fibroblast B82 cells which expressed the m1 and m2 muscarinic receptor genes (cloned cell lines designated as LK3-3 and M2LKB2-2, respectively). [3H](-)methyl-3-quinuclidinyl benzilate [( 3H](-)MQNB) binding in these transfected cell lines was inhibited by different optical isomers of YM796 and other muscarinic drugs, atropine, pirenzepine, AF-DX 116, as well as selected agonists. (-)YM796, (+)YM796 and (+/-)YM796 inhibited [3H](-)MQNB binding in LK3-3 cells with Ki values of 16.4 microM, 30.1 microM and 21.8 microM and in M2LKB2-2 cells with Ki values of 52.0 microM, 108 microM and 77.1 microM, respectively. From functional assays we found the two isomers, (-)YM796 and (+)YM796 had different intrinsic activities for the M1 and M2 muscarinic receptors. (-)YM796 revealed agonistic activity: stimulation of [3H]IP1 accumulation in LK3-3 cells with an EC50 value of 26.5 microM, which was less efficacious (the Emax value was 5.6 times basal) than carbachol, a full agonist (the Emax value was 17.2 times basal). Interestingly, (-)YM796 did not show significant inhibition of cAMP formation in M2LKB2-2 cells except at extremely high concentrations (greater than 1mM). (+)YM796 exhibited no significant efficacy for the M1 and M2 muscarinic receptors. These results suggest that (-)YM796 represents a muscarinic partial agonist with functional selectivity for the M1 muscarinic receptors whereas (+)YM796 shows no efficacy for either M1 or M2 muscarinic receptors in these transfected cells.     

Prostaglandin E2 receptor heterogeneity and dysfunction in mammary tumor cells

We have reported previously that murine mammary tumor cell subpopulations isolated from one spontaneous adenocarcinoma are heterogenous in terms of prostaglandin E2 (PGE2) synthetic capacity. We have also shown that tumor-PGE2 contributes to the ability of these cells to grow and metastasize in vivo (Fulton and Heppner: Cancer Research 45:4779-4784, 1985). In the present study, we have asked whether exogenous PGE2 has direct effects on the proliferation of these cells in vitro and if such responses can be attributed to the capacity of these cells to 1) bind PGE2 and 2) activate adenylate cyclase via the PGE2 receptor. We report that PGE2, at concentrations below 1 x 10(-5) M, does not affect the proliferation rate of these cells. This unresponsiveness is not due to the absence of receptors for PGE2. However, marked heterogeneity in receptor binding and function was detected in these closely related cell lines. Two metastatic lines (66 and 410.4) have high-affinity receptors for PGE2 (average Kd = 4.3 x 10(-9) M/L and 4.2 x 10(-9) M/L, respectively) and similar binding capacities (4.1 x 10(-4) and 2.9 x 10(4) binding sites, respectively). Two nonmetastatic lines, 410 and 67, have receptors with lower affinity (Kd = 8.3 x 10(-9) M/L and 1.6 x 10(-7) M/L, respectively) and binding capacities of 2.8 x 10(5)/410 cell or 7.3 x 10(4)/67 cell. A third nonmetastatic line (168) exhibits no specific binding. PGE2 receptor stimulation leads to elevated intracellular cAMP in lines 66, 410, and 67. Line 410.4 cells appear to have a functional lesion in the PGE2 receptor resulting in a failure to elevate cAMP in response to receptor occupancy. Adenylate cyclase can, however, be activated in these cells by cholera toxin, NaF, or forskolin. In comparison to the other cell lines, line 168 cells respond poorly to all cAMP-stimulating agents. Thus, we have found that PGE2 binding is a heterogenous property for these cells, and, in addition, we have identified an apparent uncoupling of PGE2 receptor to the adenylate cyclase system in one cell line.     

Autocrine/paracrine regulation of apoptosis in epithelial cells by prostaglandin E2

This study was designed to investigate the effect of IL-1alpha-induced up-regulation of cyclooxygenase-2 (COX-2) on prostaglandin E(2) (PGE(2)) secretion and the subsequent phenotypic effects of PGE(2) on epithelial cells. The effect of IL-1alpha on COX-2 expression was investigated in the T24 bladder epithelial cell line following treatment with 0, 0.05, 0.5, 1 or 10 ng/ml IL-1alpha for 1, 2, 4 or 6 h. Quantitative PCR confirmed up-regulation of expression of COX-2 with maximal expression observed following treatment with 0.5 ng/ml IL-1alpha for 1 h. Co-treatment of the cells with 0.5 ng/ml IL-1alpha in the presence or absence of 100 ng/ml IL-1 receptor antagonist (RA) abolished the up-regulation in COX-2 expression confirming that the effect of IL-1alpha is mediated via its membrane-bound receptors. Treatment with 0.5 ng/ml IL-1alpha resulted in a time-dependent increase in PGE(2) secretion with maximal secretion detected at 24 and 48 h after stimulation with IL-1alpha. Co-treatment of the cells with IL-1alpha and IL-1RA or the COX-2 enzyme inhibitor NS398 abolished the IL-1alpha mediated secretion of PGE(2). Treatment of T24 cells with 100 nM PGE(2) resulted in a significant elevation in cAMP generation confirming the expression of functional PGE(2) receptors. Finally, the effect of exogenous treatment with PGE(2) on apoptosis of T24 cells was assessed using cell death detection ELISA. T24 cells were treated with camptothecin to induce apoptosis in the presence or absence of 50 or 100 nM PGE(2) or 10 microM forskolin. Treatment of T24 cells with increasing doses of camptothecin alone resulted in a significant increase in the induction of apoptosis (P<0.01). However, co-treatment of the cells with 50 or 100 nM PGE(2) or 10 microM forskolin resulted in the inhibition of induction of the apoptotic pathway by camptothecin. These data demonstrate that PGE(2) inhibits apoptosis of epithelial cells possibly via cAMP-dependent pathway.     

Inhibition of Ca2+-activated and voltage-dependent K+ currents by 2-mercaptophenyl-1,4-naphthoquinone in pituitary GH3 cells: contribution to its antiproliferative effect

Quinones have been shown to possess antineoplastic activity; however, their effects on ionic currents remain unclear. The effects of 2-mercaptophenyl-1,4-naphthoquinone (2-MPNQ), menadione (MD) and 1,4-naphthoquinone (1,4 NQ) on cell proliferation and ionic currents in pituitary GH3 lactotrophs were investigated in this study. 2-MPNQ was more potent than menadione or 1,4-naphthoquinone in inhibiting the growth of GH3 cells. 2-MPNQ decreased cell proliferation in a concentration-dependent manner with an IC50 value of 3 microM. In whole-cell recording experiments, 2-MPNQ reversibly caused an inhibition of Ca2+-activated K+ current (I(K(Ca)) in a concentration-dependent manner. The IC50 value for 2-MPNQ-induced inhibition of I(K(Ca)) was 7 microM. In the inside-out configuration of single channel recording, 2-MPNQ (30 microM) applied intracellularly suppressed the activity of large-conductance Ca2+-activated K+ (BK(Ca)) channels but did not modify single channel conductance. Menadione (30 microM) had no effect on the channel activity, whereas 1,4-naphthoquinone (30 microM) suppressed it by about 26%. Both 2-MPNQ and thimerosal suppressed the dithiothreitol-stimulated channel activity. 2-MPNQ also blocked voltage-dependent K+ currents, but it produced a slight reduction of L-type Ca2+ inward current. However, unlike E-4031, 2-MPNQ (30 microM) did not suppress inwardly rectifying K+ current present in GH3 cells. Under the current clamp configuration, the presence of 2-MPNQ (30 microM) depolarized the cells, and increased the frequency and duration of spontaneous action potentials. The 2-MPNQ-mediated inhibition of K+ currents would affect hormone secretion and cell excitability. The blockade of these ionic channels by 2-MPNQ may partly explain its inhibitory effect on the proliferation of GH3 cells.     

Erythromyeloid tumor cells (K562) induce PGE synthesis in human peripheral blood monocytes

Human peripheral blood monocytes were found to spontaneously produce prostaglandin of the E series (PGE) in culture medium (0.5 ng to 3.0 ng/7.5 X 10(5) cells), and the addition of K562 tumor cells enhanced the production by five- to 15-fold after 18 hr of incubation. PGE2 (10(-6) M) inhibited the cytolytic activity of freshly isolated peripheral blood monocytes against K562 target cells by 50%. The PGE production was inhibited by inhibitors of cyclo-oxygenase (indomethacin, aspirin, and ETYA) when present during the incubation. However, pretreatment of monocytes with these cyclo-oxygenase inhibitors was ineffective in preventing PGE production. Kinetic experiments showed that appreciable stimulation of PGE production occurred only after 6 hr of co-culture. Other human tumor cell lines (HSB, SB, and CEM) enhanced PGE production upon co-culture with monocytes but to a lesser extent (twofold to threefold). Monocytes treated with 0.4% formaldehyde or heat (56 degrees C) were not capable of producing PGE when cultured alone or with K526 tumor cells. In contrast, formaldehyde-treated, but not heat-treated, K562 tumor cells were able to induce monocytes to produce PGE. By using a single cell conjugation assay, K562 tumor cells were found to bind equally well to treated or untreated monocytes. In contrast, the lytic activity of treated monocytes against K562 target cells was abolished. The presence of protein synthesis inhibitor, cycloheximide, was found to inhibit PGE production by monocytes cultured alone or with K562 tumor cells. Supernatants from K562 tumor cell cultures were also capable of inducing monocytes to produce PGE, and their effect on PGE production from monocytes was suppressed by cycloheximide. In addition, pretreatment of either K562 tumor cells or monocytes with an irreversible protein synthesis inhibitor, emetine, also suppressed the production of PGE upon co-culture with the untreated counterpart. The production of PGE by monocytes in response to exposure to tumor cells may represent a mechanism whereby tumor cells subvert host immune defense against them.     

Indomethacin decreases EP2 prostanoid receptor expression in colon cancer cells

Nonsteroidal anti-inflammatory drugs (NSAIDs) can decrease the risk of colorectal cancer; however, it has not been established if this effect is solely through their ability to inhibit cyclooxygenase (COX). In this study the effects of indomethacin, a potent NSAID and nonselective COX inhibitor, was examined in LS174T human colon cancer cells. These cells were found to express EP2 prostanoid receptors, but not the EP1, EP3 or EP4 subtypes. Pretreatment of LS174T cells with indomethacin produced a complete inhibition of prostaglandin E(2) (PGE(2)) stimulated cyclic AMP (cAMP) formation in a dose dependent manner with an IC(50) of 21 microM. Interestingly, the inhibition of PGE(2)-stimulated cAMP formation by indomethacin was accompanied by a decrease in EP2 mRNA expression and by a decrease in the whole cell specific binding of [(3)H]PGE(2). Thus, treatment of LS174T cells with indomethacin causes a down regulation of EP2 prostanoid receptors expression that may be independent of COX inhibition.     

Anti-apoptotic roles of prostaglandin E2 and F2alpha in bovine luteal steroidogenic cells

Production of prostaglandins (PGs) and expression of their receptors have been demonstrated in bovine corpus luteum (CL). The aim of the present study was to determine whether PGE2 and PGF2alpha have roles in bovine luteal steroidogenic cell (LSC) apoptosis. Cultured bovine LSCs obtained at the midluteal stage (Days 8-12 of the cycle) were treated for 24 h with PGE2 (0.001-1 microM) and PGF2alpha (0.001-1 microM). Prostaglandin E2 (1 microM) and PGF2alpha (1 microM) significantly stimulated progesterone (P4) production and reduced the levels of cell death in the cells cultured with or without tumor necrosis factor alpha (TNF)/interferon gamma (IFNG), in the presence and absence of FAS ligand (P < 0.05). Furthermore, DNA fragmentation induced by TNF/IFNG was observed to be suppressed by PGE2 and PGF2alpha. Prostaglandin E2 and PGF2alpha also attenuated mRNA expression of caspase 3 and caspase 8, as well as caspase 3 activity (P < 0.05) in TNF/IFNG-treated cells. FAS mRNA and protein expression were decreased only by PGF2alpha (P < 0.05). A specific P4 receptor antagonist (onapristone) attenuated the apoptosis-inhibitory effects of PGE2 and PGF2alpha in the absence of TNF/IFNG (P < 0.05). A PG synthesis inhibitor (indomethacin) reduced cell viability in PGE2- and PGF2alpha-treated cells (P < 0.05). A specific inhibitor of cyclooxygenase (PTGS), PTGS2 (NS-398), also reduced cell viability, whereas an inhibitor of PTGS1 (FR122047) did not affect it. The overall results suggest that PGE2 and PGF2alpha locally play luteoprotective roles in bovine CL by suppressing apoptosis of LSCs.     

PGE2-induced apoptotic cell death in K562 human leukaemia cells.

Prostaglandin-E2 (PGE2) is known to trigger suicidal death of nucleated cells (apoptosis) and enucleated erythrocytes (eryptosis). In erythrocytes PGE2 induced suicidal cell death involves activation of nonselective cation channels leading to Ca2+ entry followed by cell shrinkage and triggering of Ca2+ sensitive cell membrane scrambling with phosphatidylserine (PS) exposure at the cell surface. The present study was performed to explore whether PGE2 induces apoptosis of nucleated cells similarly through cation channel activation and to possibly disclose the molecular identity of the cation channels involved. To this end, Ca2+ activity was estimated from Fluo3 fluorescence, mitochondrial potential from DePsipher fluorescence, phosphatidylserine exposure from annexin binding, caspase activation from caspAce fluorescence, cell volume from FACS forward scatter, and DNA fragmentation utilizing a photometric enzyme immunoassay. Stimulation of K562 human leukaemia cells with PGE2 (50 microM) increased cytosolic Ca2+ activity, decreased forward scatter, depolarized the mitochondrial potential, increased annexin binding, led to caspase activation and resulted in DNA fragmentation. Gene silencing of the Ca2+-permeable transient receptor potential cation channel TRPC7 significantly blunted PGE2-induced triggering of PS exposure and DNA fragmentation. In conclusion, K562 cells express Ca2+-permeable TRPC7 channels, which are activated by PGE2 and participate in the triggering of apoptosis.     

Identification of distinct signalling pathways for somatostatin receptors SSTR1 and SSTR2 as revealed by microphysiometry.

Somatostatin receptors (SSTRs) are known to mediate diverse cellular responses. Most target cell express more than one SSTR isoform, making it difficult to define the signalling pathway used by individual receptor subtypes. Thus, we have expressed SSTR1 or SSTR2 in rat pituitary F4C1 cells which lack endogenous SSTRs. Using a silicon-based biosensor system, the Cytosensor microphysiometer, which measures the extracellular acidification rate (ECAR) in real time, we have studied the responses to SS mediated by either SSTR1 or SSTR2. In control F4C1 cells, SS had no effect on the basal ECAR. In transfected cells expressing only SSTR1, SS caused a unique decrease in ECAR in a concentration-dependent manner. Receptor-mediated decreases in ECAR have not been reported previously. In F4C1 cells expressing only SSTR2, SS induced a bidirectional ECAR response, a rapid increase followed by a decrease below basal. Two SS analogues, MK678 and CH275, induced characteristic ECAR responses with the expected receptor selectivities for SSTR1 or SSTR2. Pretreatment of F4C1 cells with pertussis toxin abolished the decreases in ECAR mediated by both SSTR1 and SSTR2, but only partially reduced the increase in ECAR mediated by SSTR2. The decrease in ECAR did not depend on a decrease in intracellular cAMP. The ECAR responses to SS were modestly attenuated by methylisobutylamiloride (MIA), an inhibitor of the ubiquitous Na(+)-H+ exchanger NHE1. Removal of extracellular Na+ greatly inhibited the ECAR responses to SS, demonstrating a role for both amiloride-sensitive and -insensitive Na(+)-dependent acid transport mechanisms in SS-induced extracellular acidification. In conclusion, we have identified and characterized different signalling pathways for SSTR1 and SSTR2 in pituitary cells as measured by microphysiometry.     

Desensitization of the human V2 vasopressin receptor. Homologous effects in the absence of heterologous desensitization.

Three main pathways have been implicated in desensitization of receptors that stimulate adenylylcyclase (AC): cAMP-mediated phosphorylation; cAMP-independent phosphorylation, and receptor internalization. Cell lines derived from the murine Ltk- cell were found useful in exploring the contribution of cAMP-dependent phosphorylation in V2 vasopressin receptor desensitization. The HTB-2 cell expresses the human V2 vasopressin receptor, introduced by transfection of human genomic DNA, and the prostaglandin E1 (PGE1) receptor, endogenous to the Ltk- cell. The A7 cell expresses the hamster beta 2-adrenoceptor, which undergoes the above-mentioned desensitization processes. Treatment of HTB-2 cells with arginine-vasopressin (AVP) had no effect on AC responsiveness to PGE1, but promoted desensitization of the AVP response. This was seen as a 5-6-fold right shift in the dose-response curves for AVP action (cAMP accumulation in intact cells and AC stimulation in homogenates and isolated membranes) and in a decrease in the maximum effect of AVP on these parameters. AVP treatment caused a decrease in cell surface receptors to approximately 75% of control without changes in KD, as determined by Scatchard analysis. When cAMP was increased by treatment with 10 microM PGE1 and isobutylmethylxanthine, desensitization of the PGE1 receptor was observed but not of the AVP receptor. In A7 cells the same treatment caused, as expected, a 3-fold right shift in the dose-response curve for AC stimulation by isoproterenol, indicating that L cells can mediate heterologous desensitization. These data demonstrate that the V2 vasopressin and the PGE1 receptors undergo homologous desensitization in the absence of cAMP-mediated phosphorylation and that this component is not required for vasopressin receptor internalization.     

Exogenous prostaglandin E2 inhibits TPA induced matrix metalloproteinase-9 production in MCF-7 cells

Elevated levels of prostaglandin E2 (PGE2) have been reported in many high metastatic human breast cancers, but no relationship between exogenous PGE2 activity, expression of matrix metalloproteinases (MMPs) and metastasis in human tumor cells has been reported. The poorly invasive human breast cancer cell line MCF-7 was cultured for 24h in the presence of both phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, 50 nM) and PGE2 (1 microM) and the activity of MMP-9, one of the MMPs involved in metastasis, was measured, in growth medium by gelatin substrate zymography. TPA induced a strong production of MMP-9 while exogenous PGE2 had no effect on the basal MMP-9 level, but inhibited the TPA induced enzyme expression and matrigel invasiveness. We showed that MCF-7 cells expressed EP2, EP3 and EP4 receptors for PGE2 and that its action was probably mediated by EP4 receptor and adenylyl cyclase activation while cAMP dependent PKA was not involved in the process of inhibition of MMP-9 production. These findings suggest a possible inhibitory role for exogenous PGE2 in the metastatic process development.