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K V Honn

Publications and source records attributed to K V Honn.

At least 73 records · Page 4Linked to original sources
PubMed1995

Immunomorphological characterization and effects of 12-(S)-HETE on a dynamic intracellular pool of the alpha IIb beta 3-integrin in melanoma cells.

In metastatic B16a murine melanoma cells, alpha IIb beta 3 integrin was shown to be one of the key adhesion molecules responsible for matrix adhesion and spreading. Upon stimulation, alpha IIb beta 3 can be upregulated at the cell surface due to translocation of the receptor to the plasma membrane from an intracellular pool. Here we have characterized this integrin pool as a tubulovesicular structure (TVS) corresponding to endosomes. TVS was found to be associated temporarily with microtubules and intermediate filaments especially after protein kinase C (PKC) stimulation with a lipoxygenase metabolite of arachidonic acid, 12-(S)-hydroxyeicosatetraenoic acid [12-(S)-HETE]. After PKC stimulation, the predominantly vesicular TVS became elongated and alpha IIb beta 3 appeared at the apical plasma membrane and microvilli. Disruption of either the microtubules or intermediate filaments prevented the 12-(S)-HETE effect both on vesicular to tubular transition of TVS as well as on surface expression of this integrin. The connection with the Golgi system of the integrin-containing TVS was proved by a Golgi-inhibitor (brefeldin A) pretreatment, which prevented the PKC-stimulation-induced TVS elongation and subsequent receptor-upregulation at the cell surface. After a soluble ligand binding (mAb to the alpha IIb beta 3 complex) the surface receptor endocytosed back to the TVS indicating the presence of a dynamic, cytoskeleton associated integrin pool in melanoma cells.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1995

Transcriptional activation of endothelial cell integrin alpha v by protein kinase C activator 12(S)-HETE.

Previous work demonstrated that 12(S)-HETE [12(S)-hydroxyeicosatetraenic acid], a lipoxygenase metabolite of arachidonic acid, stimulates the surface expression of integrin alpha v beta 3 on mouse lung vascular endothelial cells (CD clone 3) in a post-transcriptional and protein kinase C (PKC)-dependent fashion. In this study we examined the effect of 12(S)-HETE on the expression of integrin receptors alpha v beta 3 and alpha 5 beta 1 in a different clone of a mouse endothelial cell population derived from lung microvasculature (designated CD clone 4). The results indicated that 12(S)-HETE transcriptionally activates the gene expression of integrin alpha v as assessed by quantitative reverse transcription/polymerase chain reaction/Southern hybridization, RNase protection assay, solution hybridization, and northern blotting. The induction of alpha v mRNA occurred within 1 hour, peaked at approximately 4 hours (2- to 4-fold increase), persisted for up to 16 hours, and thereafter gradually declined. The PKC activator phorbol 12-myristate 13-acetate (PMA) induced the alpha v mRNA, in a similar way. 12(S)-HETE treatment did not, in contrast, alter the mRNA levels of integrin subunit alpha 5 or beta 1. The induction of alpha v mRNA appeared to be protein synthesis-independent, since cycloheximide did not alter the 12(S)-HETE effect. 12(S)-HETE also did not appear to alter the mRNA half-life of alpha v. On the other hand, 12(S)-HETE-induced increase in alpha v mRNA levels was PKC-dependent, since pretreatment of CD clone 4 cells with calphostin C significantly inhibited 12(S)-HETE-increased alpha v mRNA. Nuclear runoff experiments revealed that the increase in alpha v mRNA results from an enhanced gene transcription. Facilitated alpha v gene transcription resulted in an increased surface expression of alpha v beta 3 protein, which resulted in an increased cell adhesion to vitronectin. The above observations, in conjunction with our previous experimental data, suggest that 12(S)-HETE may employ diverse mechanisms to stimulate the integrin alpha v beta 3 expression in vascular endothelial cells, which could play important roles in tumor cell adhesion, angiogenesis, hemostasis, and many other vascular events.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1994

Autocrine motility factor induces differential 12-lipoxygenase expression and activity in high- and low-metastatic K1735 melanoma cell variants.

A M(r) 55,000 tumor cell-secreted cytokine has been described which influenced the migration of the producing cells and was called autocrine motility factor (AMF). Activation of the cell surface receptor for AMF (gp78) was shown to stimulate production of a 12-lipoxygenase metabolite of arachidonic acid, 12-(S)-hydroxyeicosatetraenoic acid [12-(S)-HETE], in highly metastatic murine melanoma cells. AMF stimulated the motility of the high-metastatic (K1735-M1) but not the low-metastatic variant (K1735-Cl.11) of the K1735 murine melanoma and increased expression of the 12-lipoxygenase enzyme predominantly in the high-metastatic counterpart. The K1735-M1 cells responded to motile stimulation with increased endogenous 12-(S)-HETE production, and, reciprocally, exogenous 12-(S)-HETE up-regulated surface gp78 and caused gp78 translocation from an intracellular perinuclear pool to tubulovesicles which extended to the cell periphery in the K1735-M1 cells exclusively. These results suggest that differences in AMF responses may be due to alterations in the capacity of low-metastatic cells to transduce signals through 12-lipoxygenase or to involve downstream effector(s) of 12-(S)-HETE after gp78 activation.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1994

12(S)-HETE enhancement of prostate tumor cell invasion: selective role of PKC alpha.

BACKGROUND: Prostate carcinoma has become the second most fatal cancer in American men. In Dunning R3327 rat prostate adenocarcinoma cells, elevated invasiveness positively correlates with metastatic potential. However, the mechanism(s) responsible for regulation of tumor cell motility and invasion is poorly understood. We have reported that a lipoxygenase metabolite of arachidonic acid, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], augments tumor cell metastatic potential through activation of protein kinase C (PKC). PURPOSE: We proposed to determine the effect of 12(S)-HETE on the motility and invasion of low-metastatic rat prostate AT2.1 tumor cells and the effect of 12(S)-HETE activation of specific PKC isoform(s) in these processes. METHODS: The motility of AT2.1 cells was determined by the colloidal gold phagokinetic track assay and the invasiveness measured as their ability to invade through basement membrane Matrigel-coated filters. Expression of PKC isoforms was determined by Western blotting of the whole cell lysate with isoform-specific anti-PKC antibodies. Cytosol and membrane fractions were prepared and the subcellular distribution of PKC was analyzed by Western blotting and activity assay. The effect of 12(S)-HETE on cell proliferation was examined. Data were analyzed for significance of difference with the two-sampled, two-sided Student's t test. RESULTS: 12(S)-HETE increased the motility and invasion of AT2.1 cells, and this 12(S)-HETE-increased motility and invasion were inhibited by a selective PKC inhibitor, calphostin C, as well as a Ca2 chelator, bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/tetra(acetoxy-methyl)ester. AT2.1 cells expressed the PKC isoforms alpha and delta, and 12(S)-HETE increased the membrane association of PKC alpha but not delta. Further, the motility and invasion of AT2.1 cells were increased by thymelea toxin, a selective activator of PKC alpha over PKC delta. CONCLUSION: 12(S)-HETE augments the invasiveness of AT2.1 cells via selective activation of PKC alpha. IMPLICATIONS: 12(S)-HETE modulation of PKC alpha invasiveness may be an important mechanism of action for the regulation of the invasive potential of rat prostate carcinoma cells, and the 12-lipoxygenase enzyme and/or PKC alpha may serve as key targets for the development of anti-invasive agents useful for combating the spread of prostate cancer.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1994

Endogenous 12(S)-HETE production by tumor cells and its role in metastasis.

12(S)-Hydroxyeicosatetraenoic acid [12(S)-HETE] is the 12-lipoxygenase metabolite of arachidonic acid. Previously, we have demonstrated that exogenous 12(S)-HETE can activate protein kinase C, increase cell surface expression of integrins, enhance adhesion, induce endothelial cell retraction, and increase experimental metastasis of tumor cells. Because of these prominent effects of exogenous 12(S)-HETE on tumor cell metastatic potential, it is important to determine whether there is endogenous 12(S)-HETE production by tumor cells. In the present study, mRNAs from human, rat, and mouse platelets as well as human colon carcinoma (Clone A), rat Walker carcinoma (W256), and mouse melanoma (B16a) and lung carcinoma (3LL) were reverse transcribed and amplified by polymerase chain reaction with platelet 12-lipoxygenase specific primers. Identity of the polymerase chain reaction fragments was confirmed by sequencing. 12-Lipoxygenase protein was detected by Western blotting. Tumor cell-derived 12-HETE was determined by reverse phase-high performance liquid chromatography analysis. In addition, the effect of endogenous 12(S)-HETE on tumor cells was studied by using a platelet-type 12-lipoxygenase selective inhibitor (N-benzyl-N-hydroxy-5-phenylpentanamide). Our results suggest that some tumor cells express platelet-type 12-lipoxygenase mRNA, protein and metabolize arachidonic acid to 12(S)-HETE and that endogenous 12(S)-HETE, like the exogenous 12(S)-HETE, may play an important role in tumor cell adhesion to matrix in vitro and lung colonization in vivo.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1994

Activation of microvascular endothelium by eicosanoid 12(S)-hydroxyeicosatetraenoic acid leads to enhanced tumor cell adhesion via up-regulation of surface expression of alpha v beta 3 integrin: a posttranscriptional, protein kinase C- and cytoskeleton-dependent process.

Tumor cell interaction with endothelial cells is a crucial step leading to organ-selective metastasis. Adhesion of murine B16 amelanotic melanoma cells (B16a) to murine microvascular endothelial cells (CD3) was enhanced, in a dose- and time-dependent manner, by pretreating CD3 cells with 12(S)-hydroperoxyeicosatetraenoic acid [i.e., 12(S)-HETE], a 12-lipoxygenase metabolite of arachidonic acid. The metabolic precursor of 12(S)-HETE, 12-HPETE (12-hydroperoxyeicosatetraenoic acid) also enhanced B16a cell adhesion to CD3 monolayers, whereas other lipoxygenase products, i.e., 5(S), 11(S), and 15(S)-HETEs were ineffective. 12(S)-HETE-enhanced tumor cell adhesion was blocked by treating endothelial cells with antibodies against the alpha v beta 3 complex or against individual subunits but not with antibodies against alpha 5 beta 1. In contrast, neither of these two integrins appeared to be involved in tumor cell adhesion to unstimulated endothelium. Flow cytometric analysis, immunofluorescent labeling, and image analysis indicated that 12(S)-HETE induced a time- and dose-dependent increase in the surface expression of alpha v beta 3 but not alpha 5 beta 1 on CD3 cells. The increased surface expression of alpha v beta 3 on endothelial cells did not result from an increased transcription or translation of alpha v beta 3 message as confirmed by quantitative reverse transcription-polymerase chain reaction, Northern blotting, and quantitative Western blotting. Instead, subcellular fractionation studies revealed an increased translocation of alpha v beta 3 integrins from the cytosolic pool to the membrane fractions. Pretreatment of endothelial cells with several cytoskeleton-disrupting agents (i.e., cycloheximide or acrylamide to disrupt intermediate filament vimentin, cytochalasin D to disrupt microfilaments, colchicine or Nocodazole to disrupt microtubules) abolished the 12(S)-HETE-enhanced alpha v beta 3 surface expression as well as tumor cell adhesion to endothelial cells. Also, pretreatment of CD3 cells with protein kinase C inhibitor calphostin C, but not with protein kinase A inhibitor H8, blocked 12(S)-HETE-enhanced alpha v beta 3 surface expression and tumor cell adhesion. Collectively, these results suggest that eicosanoid 12(S)-HETE modulates tumor cell interaction with endothelium via protein kinase C- and cytoskeleton-dependent up-regulation of the surface expression of alpha v beta 3 integrin.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1994

Tumor cell-derived 12(S)-hydroxyeicosatetraenoic acid induces microvascular endothelial cell retraction.

Our previous work demonstrated that the 12-lipoxygenase metabolite of arachidonic acid, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] induced a nondestructive and reversible retraction of cultured endothelial cells. In the current study we tested the hypothesis that tumor cells produce 12(S)-HETE during their interactions with endothelial cells which in turn induces endothelial cell retraction. Coincubation of Lewis lung carcinoma cells or elutriated B16 amelanotic melanoma (B16a) cells but not 3T3 fibroblasts with microvascular endothelial cells (CD3) resulted in a time- and concentration-dependent retraction of the CD3 monolayers as revealed by quantitative binding assays and phase contrast microscopy. Lewis lung carcinoma cell-induced endothelial cell retraction was blocked by specific lipoxygenase inhibitors but not by cyclooxygenase inhibitors, suggesting the involvement of a lipoxygenase metabolite(s). Radioimmunoassay and high-performance liquid chromatography analysis of tumor cell extracts identified 12(S)-HETE as the major lipoxygenase metabolite of arachidonic acid and tumor cell generation of 12(S)-HETE was specifically blocked by a select 12-lipoxygenase inhibitor N-benzyl-N-hydroxy-5-phenyl-pentamide. The identity and stereochemistry of tumor cell-derived 12-HETE was substantiated by gas chromatography-mass spectrometry analysis and chiral phase high-performance liquid chromatography, respectively. Lewis lung carcinoma cell adhesion to CD3 monolayers was accompanied by an enhanced 12(S)-HETE biosynthesis by tumor cells, which paralleled the tumor cell-induced endothelial cell retraction in a cell number-dependent manner. Pretreatment of tumor cells with N-benzyl-N-hydroxy-5-phenylpentamide inhibited both increased 12(S)-HETE biosynthesis and tumor cell-induced endothelial cell retraction. Highly metastatic variants of elutriated B16a cells which had been shown to produce large quantities of 12(S)-HETE induced significant CD3 cell retraction, while low metastatic subpopulations of B16a cells which synthesized no or little 12(S)-HETE did not induce endothelial cell retraction. These results suggest that 12(S)-HETE synthesis during tumor cell-endothelial cell interactions may represent a key contributory factor in cancer metastasis.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1994

Enhanced endothelial cell retraction mediated by 12(S)-HETE: a proposed mechanism for the role of platelets in tumor cell metastasis.

Platelets have been hypothesized to contribute to tumor cell metastasis, but the underlying mechanism(s) remain unknown. We demonstrate here that one mechanism whereby platelets may facilitate metastasis is by potentiating tumor cell-induced endothelial cell retraction, a prerequisite for the extravasation of most tumor cell types. The integrity of cultured microvascular endothelial cell (CD3 cells) monolayers was perturbed by 12[S]-hydroxyeicosatetraenoic acid (12(S)-HETE), a lipoxygenase metabolite of arachidonic acid, as well as by tumor cells (i.e., Lewis lung carcinoma cells or 3LL). 3LL cells induced a concentration- and time-dependent retraction of the CD3 monolayers, as assessed by quantitative binding assays as well as by phase-contrast microscopy. In contrast, normal murine fibroblasts (3T3) did not induce endothelial cell retraction. 3LL cell-induced endothelial cell retraction was potentiated, in a dose- and time-dependent manner, by homologous murine platelets while platelets alone did not induce endothelial cell retraction. Platelet-enhanced, tumor cell-induced endothelial cell retraction was inhibited by treating either tumor cells or platelets with the lipoxygenase inhibitors nordihydroguaiaretic acid or N-benzyl-N-hydroxy-5-phenylpentanamide (BHPP) as well as by PGI2 or its analogs iloprost and ZK96.480 (cicaprost), but not by the cyclooxygenase inhibitor aspirin (ASA). Tumor cells, upon adhesion to endothelium, initiated 12(S)-HETE biosynthesis, which was inhibited by pretreating tumor cells with BHPP but not with ASA. Additionally, 12(S)-HETE biosynthesis during tumor cell-endothelial cell adhesion was significantly enhanced by the addition of homologous platelets. Collectively, these results suggest that tumor cell-platelet-endothelial cell interactions lead to enhanced biosynthesis of 12(S)-HETE by tumor cells and/or platelets, which in turn induces endothelial cell retraction, thus facilitating tumor cell extravasation and metastasis.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1994

A lipoxygenase metabolite, 12-(S)-HETE, stimulates protein kinase C-mediated release of cathepsin B from malignant cells.

The process of tumor cell invasion of the basement membrane is proposed to consist of three steps: attachment, local proteolysis and migration. 12-(S)-HETE, a 12-lipoxygenase metabolite of arachidonic acid, upregulates surface expression of integrin cytoadhesins and an autocrine motility factor receptor, suggesting that this metabolite may play an important regulatory function in tumor cell invasion. In the present study, we determined whether 12-(S)-HETE affects surface expression and/or release of cathepsin B, a cysteine protease that has been implicated in focal degradation of basement membrane. Secretion and distribution of cathepsin B was evaluated in two model systems for various stages of neoplastic progression: (i) murine B16 melanoma lines of low (B16-F1) and high (B16a) lung colonization potential, and (ii) immortalized and ras-transfected MCF-10 human breast epithelial cells that differ in their invasive capacities in vitro. In the B16a cells, 12-(S)-HETE induced release of native and latent cathepsin B activity and concomitantly reduced cell-associated cathepsin B immunoreactivity. In contrast, 12-(S)-HETE did not induce the release of cathepsin B from B16-F1 cells, suggesting that there may be an enhanced response to 12-(S)-HETE in more malignant cells. This was confirmed in the MCF-10 system, in which 12-(S)-HETE was able to induce the release of cathepsin B from the ras-transfected cells, but not from the immortal cells. A simultaneous reduction in staining for cathepsin B was observed in the ras-transfected cells, but not in their immortal counterparts. The release of cathepsin B may be mediated by PKC as pretreatment of B16a cells with the selective PKC inhibitor calphostin C, but not with the PKA inhibitor H8, prevented the stimulated release of cathepsin B. In B16a cells, the release of cathepsin B was accompanied by a translocation toward the cell periphery of vesicles staining for cathepsin B, resulting in focal areas of accumulation of cathepsin B. After 12-(S)-HETE stimulation of the ras-transfected MCF-10 cells, cathepsin B was distributed homogeneously on the apical surface. Thus, 12-(S)-HETE can upregulate the surface expression on tumor cells of proteins able to mediate each of the three steps of tumor cell invasion: adhesion, degradation, and migration.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1994

Prostacyclin and its analogues: antimetastatic effects and mechanisms of action.

More than a decade ago, prostacyclin, a dienoic bicyclic eicosanoid derived from the metabolism of arachidnoic acid, was found to possess potent inhibitory effects on tumor cell metastasis. Thereafter, several laboratories demonstrated the metastasis-suppressive activity of prostacyclin in a wide spectrum of tumor types. Due to the short half-life of prostacyclin, researchers have focused on looking for stable prostacyclin analogues which have extended half lives and increased bioavailabilities. Cicaprost, among other prostacyclin analogues tested, has been demonstrated, like prostacyclin, to effectively inhibit metastasis in several different animal models (i.e., both experimental and spontaneous metastasis models). Prostacyclin as well as cicaprost prevent not only hematogenous, but also lymphatic metastasis. Furthermore, these compounds also inhibit the growth of established micrometastases after removal of the primary tumors. Mechanistic studies revealed that the antimetastatic effects of prostacyclin and its analogues are more related to their interference with tumor cell-host interactions (such as tumor cell induced platelet aggregation, tumor cell adhesion to endothelial cells and subendothelial matrix, tumor cell induced endothelial cell retraction, etc.) than their direct inhibition of the growth of primary tumors. The potent and widespread metastasis-retarding effects of prostacyclin and its stable analogues in animal tumor models warrant their clinical trial in treating human cancer patients and preventing metastasis.

Animals↗
PubMed1994

12-lipoxygenases and 12(S)-HETE: role in cancer metastasis.

Arachidonic acid metabolites have been implicated in multiple steps of carcinogenesis. Their role in tumor cell metastasis, the ultimate challenge for the treatment of cancer patients, are however not well-documented. Arachidonic acid is primarily metabolized through three pathways, i.e., cyclooxygenase, lipoxygenase, and P450-dependent monooxygenase. In this review we focus our attention on one specific lipoxygenase, i.e., 12-lipoxygenase, and its potential role in modulating the metastatic process. In mammalian cells there exist three types of 12-lipoxygenases which differ in tissue distribution, preferential substrates, and profile of their metabolites. Most of these 12-lipoxygenases have been cloned and sequenced, and the molecular and biochemical determinants responsible for catalysis of specific substrates characterized. Solid tumor cells express 12-lipoxygenase mRNA, possess 12-lipoxygenase protein, and biosynthesize 12(S)-HETE [12(S)-hydroxyeicosatetraenoic acid], as revealed by numerous experimental approaches. The ability of tumor cells to generate 12(S)-HETE is positively correlated to their metastatic potential. A large collection of experimental data suggest that 12(S)-HETE is a crucial intracellular signaling molecule that activates protein kinase C and mediates the biological functions of many growth factors and cytokines such as bFGF, PDGF, EGF, and AMF. 12(S)-HETE plays a pivotal role in multiple steps of the metastatic 'cascade' encompassing tumor cell-vasculature interactions, tumor cell motility, proteolysis, invasion, and angiogenesis. The fact that 12-lipoxygenase is expressed in a wide diversity of tumor cell lines and 12(S)-HETE is a key modulatory molecule in metastasis provides the rationale for targeting these molecules in anti-cancer and anti-metastasis therapeutic protocols.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1994

Inhibition of radiation-enhanced expression of integrin and metastatic potential in B16 melanoma cells by a lipoxygenase inhibitor.

Low-dose gamma radiation stimulates expression of phenotypic characteristics in B16 melanoma cells which regulate metastatic potential. A transient increase in the expression of an integrin receptor (alpha IIb beta 3) was observed after exposure of B16 melanoma cells to 0.25 to 2.0 Gy of gamma radiation. This increased receptor expression resulted in enhanced adhesion of tumor cells to fibronectin in vitro and increased experimentally induced metastasis in vivo. In this report, we determined a role for the 12-lipoxygenase metabolite, 12-HETE, in radiation-enhanced metastasis. A significant increase in biosynthesis of 12-HETE in B16 melanoma cells was detected < 5 min after exposure to 0.5 Gy gamma radiation. We then determined that radiation-enhanced expression of alpha IIb beta 3 integrin and adhesion of B16 melanoma cells to fibronectin in vitro and metastasis in vivo were reduced by treatment of the cells with the lipoxygenase inhibitor NDGA prior to irradiation. These findings suggest that low-dose radiation, at levels comparable to those used in fractionated or hyper-fractionated radiotherapy, increases the metastatic potential of surviving tumor cells via a rapid and transient alteration in lipoxygenase metabolism of arachidonic acid and surface expression of an integrin receptor.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1994

Biosynthesis of 12(S)-hydroxyeicosatetraenoic acid by B16 amelanotic melanoma cells is a determinant of their metastatic potential.

BACKGROUND: We have previously demonstrated that the metastatic potential of tumor cells can be increased by treatment with exogenous 12(S)hydroxyeicosatetraenoic acid [12(S)-HETE], a lipoxygenase metabolite of arachidonic acid. However, the biosynthesis of the authentic lipid mediator by tumor cells, and especially the correlation of its biosynthesis to tumor cell metastatic capacity have not been characterized. In addition, a role for other mono HETEs in influencing tumor cell metastatic behavior has been suggested, but conclusive evidence is lacking. In this study, we analyzed the biosynthesis of mono HETEs from arachidonic acid in tumor cells of different metastatic ability and correlated biosynthesis to metastatic potential. EXPERIMENTAL DESIGN: The biosynthesis of mono HETEs by low and high metastatic subpopulations of B16 amelanotic melanoma (B16a) cells was analyzed by high performance liquid chromatography (HPLC). The identity of biosynthetic 12-HETE was confirmed by gas chromatography/mass spectrometry (GC/MS) and its stereochemical structure assigned by chiral phase HPLC. The effect of a lipoxygenase inhibitor on the biosynthesis of mono HETEs and its effect on metastatic behavior was examined. RESULTS: HPLC analysis revealed that low (LM180) and high (HM340) metastatic B16a cells exhibited different profiles and efficiencies for conversion of arachidonic acid to mono HETEs. LM180 cells produced equal quantities of 12-HETE and 5-HETE. In contrast, HM340 cells synthesized predominantly 12-HETE and small amounts of 15-, 11- and 5-HETEs. At equal concentrations of substrate, four times more 12-HETE was synthesized by HM340 cells than by LM180 cells. The identity of biosynthetic 12-HETE was confirmed by gas chromatography/mass spectrometry and chiral phase HPLC demonstrated that it was the S enantiomer. The biosynthesis of 12(S)-HETE, but not other HETEs, was significantly inhibited by a lipoxygenase inhibitor, N-benzyl-N-hydroxy-5-phenylpentanamide. N-benzyl-N-hydroxy-5-phenylpentanamide, in a dose-dependent manner, decreased the adhesion of HM340 cells to murine pulmonary microvessel endothelium in vitro and lung colony formation in vivo. Furthermore, re-introduction of 12(S)-HETE, but not other mono HETEs, to HM340 cells pretreated with N-benzyl-N-hydroxy-5-phenylpentanamide, increased their adhesion to endothelium. CONCLUSIONS: Biosynthesis of 12(S)-HETE by tumor cells is a determinant of their metastatic potential and inhibition of 12(S)-HETE biosynthesis in tumor cells may be a crucial target for intervening in metastasis.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1993

Regulation of melanoma-cell motility by the lipoxygenase metabolite 12-(S)-HETE.

Cellular motility, a prerequisite for metastasis of tumor cells, is affected by a 55-kDa tumor-cell-secreted cytokine which influences the migration of the producing cells and is called autocrine motility factor (AMF). Previous studies indicated that AMF stimulates motility by binding to its receptor, a cell-surface glycoprotein of 78 kDa (gp78), inducing its phosphorylation, activating a pertussis toxin (PT)-sensitive G-protein, and stimulating inositol metabolism. However, the intracellular signaling mechanisms which transduce and regulate the AMF motility response remain largely unknown. 12-(S)-HETE, a lipoxygenase metabolite of arachidonic acid which affects the cytoskeletal architecture of murine melanoma cells, also stimulates cell motility independently of PT-sensitive G-proteins and up-regulates gp78 surface expression. 12-(S)-HETE induces the phosphorylation of gp78 in a manner analogous to AMF and the motility response of these murine melanoma cells to both AMF and 12-(S)-HETE is inhibited by protein kinase C inhibitors. Furthermore, perturbation of the AMF receptor stimulated endogenous biosynthesis of 12(S)HETE. These results suggest the existence of an "autocrine motility cycle" which influences melanoma cell motility by gp78 activation, and production of second messengers which affect the cytoskeletal architecture and expression of the AMF receptor itself.

Animals↗
PubMed1993

Identification of PECAM-1 in solid tumor cells and its potential involvement in tumor cell adhesion to endothelium.

PECAM-1 (CD31/EndoCAM) is an adhesion molecule in the immunoglobulin supergene family that is expressed on endothelial cells, platelets, and some hematopoietic lineage cells. In this paper, using several polyclonal and monoclonal antibodies against PECAM-1, we identified PECAM-1 molecules on human, rat, and murine solid tumor cell lines. Immunocytochemical labeling and flow cytometric analysis using either polyclonal, monoclonal, or Fab portion of the antibodies against PECAM-1 detected a distinct distribution on tumor cell surface. Immunoblotting revealed proteins ranging from 120 to 130 kDa in tumor cells derived from different species. Immunoprecipitation and subcellular fractionation studies indicated that PECAM-1 is constitutively expressed on the surface of human tumor cells (i.e. colon adenocarcinoma). The specificity of a major polyclonal anti-PECAM-1 used in the current study (i.e. SEW-3) was confirmed by the preabsorption studies. PECAM-1 molecules on tumor cells appear to bear terminal carbohydrate moieties (i.e. sialic acid residues) different from those on platelets, since neuraminidase treatment of tumor cells, unlike platelets, did not result in a mobility shift. Polymerase chain reaction (PCR) analysis of genomic DNA derived from tumor cell lines of different species revealed the presence of PECAM-1 gene in the genome. The mRNAs of PECAM-1 in tumor cells were detected by reverse transcription-PCR followed by Southern hybridization. Screening of more than 20 human, rat, and murine solid tumor cell lines indicated that PECAM-1 is widely expressed, although the level of expression varies considerably among different cell lines. The expression of PECAM-1 message in tumor cells was confirmed by Northern blotting. DNA sequencing of the PCR fragment revealed that human tumor cell PECAM-1 matches 100% to the human endothelial cell counterpart. Finally, it was demonstrated that tumor cell PECAM-1 is involved in mediating tumor cell adhesion to endothelium, as evidenced by the ability of anti-PECAM-1 antibodies to decrease the adhesion of unstimulated tumor cells to microvascular endothelial cells.

Amino Acid Sequence↗