Keqin Tang

Overexpression of Platelet-type 12-Lipoxygenase Promotes Tumor Cell Survival by Enhancing {alpha}v{beta}3 and {alpha}v{beta}5 Integrin Expression

Arachidonic acid metabolism leads to the generation of biologically active metabolites that regulate cell growth and proliferation, as well as survival and apoptosis. We have demonstrated previously that platelet-type 12-lipoxygenase (LOX) regulates the growth and survival of a number of cancer cells. In this study, we show that overexpression of platelet-type 12-LOX in prostate cancer PC3 cells or epithelial cancer A431 cells significantly extended their survival and delayed apoptosis when cultured under serum-free conditions. These effects were shown to be a result of enhanced surface integrin expression, resulting in a more spread morphology of the cells in culture. PC3 cells transfected with 12-LOX displayed increased alpha(v)beta(3) and alpha(v)beta(5) integrin expression, whereas other integrins were unaltered. Transfected A431 cells did not express alpha(v)beta(3); however, alpha(v)beta(5) integrin expression was increased. Treatment of both transfected cell lines with monoclonal antibody to alpha(v)beta(5) (and in the case of PC3 cells, anti-alpha(v)beta(3)) resulted in significant apoptosis. In addition, treatment with 100 nM 12(S)-hydroxy-eicosatetraenoic acid, the end product of platelet-type 12-LOX, but not other hydroxy-eicosatetraenoic acids, enhanced the survival of wild-type PC3 and A431 cells and resulted in increased expression of alpha(v)beta(5). Furthermore, Baicalein or N-benzyl-N-hydroxy-5-phenylpentamide, specific 12-LOX inhibitors, significantly decreased alpha(v)beta(5)-mediated adhesion and survival in 12-LOX-overexpressing cells. The results show that 12-LOX regulates cell survival and apoptosis by affecting the expression and localization of the vitronectin receptors, alpha(v)beta(3) and alpha(v)beta(5), in two cancer cell lines.Background—Pulmonary hypertension induced by chronic hypoxia is characterized by thickening of pulmonary artery walls, elevated pulmonary vascular resistance, and right-heart failure. Prostacyclin analogues reduce pulmonary pressures in this condition; raising the possibility that cycloxygenase-2 (COX-2) modulates the response of the pulmonary vasculature to hypoxia. Methods and Results—Sprague-Dawley rats in which pulmonary hypertension was induced by hypobaric hypoxia for 14 days were treated concurrently with the selective COX-2 inhibitor SC236 or vehicle. Mean pulmonary arterial pressure (mPAP) was elevated after hypoxia (28.1±3.2 versus 17.2±3.1 mm Hg; n=8, P<0.01), with thickening of small pulmonary arteries and increased COX-2 expression and prostacyclin formation. Selective inhibition of COX-2 aggravated the increase in mPAP (42.8±5.9 mm Hg; n=8, P<0.05), an effect that was attenuated by the thromboxane (TX) A2/prostaglandin endoperoxide receptor antagonist ifetroban. Urinary TXB2 increased during hypoxia (5.9±0.9 versus 1.2±0.2 ng/mg creatinine; n=6, P<0.01) and was further increased by COX-2 inhibition (8.5±0.7 ng/mg creatinine; n=6, P<0.05). In contrast, urinary excretion of the prostacyclin metabolite 6-ketoprostaglandin F1&agr; decreased with COX-2 inhibition (8.6±3.0 versus 27.0±4.8 ng/mg creatinine; n=6, P<0.05). Platelet activation was enhanced after chronic hypoxia. COX-2 inhibition further reduced the PFA-100 closure time and enhanced platelet deposition in the smaller pulmonary arteries, effects that were attenuated by ifetroban. Mice with targeted disruption of the COX-2 gene exposed to chronic hypoxia had exacerbated right ventricular end-systolic pressure, whereas targeted disruption of COX-1 had no effect. Conclusions—COX-2 expression is increased and regulates platelet activity and intravascular thrombosis in hypoxia-induced pulmonary hypertension.

Role of Eicosanoids in Prostate Cancer Progression

Metabolism of arachidonic acid through cyclooxygenase, lipoxygenase, or P450 epoxygenase pathways leads to the formation of various bioactive eicosanoids. In this review, we discuss alterations in expression pattern of eicosanoid-generating enzymes found during prostate tumor progression and expound upon their involvement in tumor cell proliferation, apoptosis, motility, and tumor angiogenesis. The expression of cyclooxygenase-2, 12-lipoxygenase, and 15-lipoxygenase-l are up-regulated during prostate cancer progression. It has been demonstrated that inhibitors of cyclooxygenase-2, 5-lipoxygenase and 12-lipoxygenase cause tumor cell apoptosis, reduce tumor cell motility and invasiveness, or decrease tumor angiogenesis and growth. The eicosanoid product of 12-lipoxygenase, 12(S)-hydroeicosatetraenoic acid, is found to activate Erkl/2 kinases in LNCaP cells and PKCα in rat prostate AT2.1 tumor cells. Overexpression of 12-lipoxygenase and 15-lipoxygenase-l in prostate cancer cells stimulate prostate tumor angiogenesis and growth, suggesting a facilitative role for 12-lipoxygenase and 15-lipoxygenase-l in prostate tumor progression. The expression of 15-lipoxygenase-2 is found frequently to be lost during the initiation and progression of prostate tumors. 15(S)-hydroxyeicosatetraenoic acid, the product of 15-lipoxygenase-2, inhibits proliferation and causes apoptosis in human prostate cancer cells, suggesting an inhibitory role for 15-lipoxygenase-2 in prostate tumor progression. The regulation of prostate cancer progression by eicosanoids, in either positive or negative ways, provides an exciting possibility for management of this disease.

Mechanisms regulating tumor angiogenesis by 12-lipoxygenase in prostate cancer cells

12-Lipoxygenase utilizes arachidonic acid to synthesize 12(S)-hydroperoxyeicosatetraenoic acid, which is converted to the end product 12(S)-hydroxyeicosatetraenoic acid, an eicosanoid that promotes tumorigenesis and metastasis. Increased expression of 12-lipoxygenase has been documented in a number of carcinomas. When overexpressed in human prostate or breast cancer, 12-lipoxygenase promotes tumor angiogenesis and growth in vivo. The present study was undertaken to delineate the mechanisms by which 12-lipoxygenase enhances angiogenesis. Herein we report that nordihydroguaiaretic acid, a pan inhibitor of lipoxygenases and baicalein, a selective inhibitor of 12-lipoxygenase, reduced VEGF expression in human prostate cancer PC-3 cells. Overexpression of 12-lipoxygenase in PC-3 cells resulted in a 3-fold increase in VEGF protein level when compared with vector control cells. An increase in PI 3-kinase activity was found in 12-LOX-transfected PC-3 cells and inhibition of PI 3-kinase by LY294002 significantly reduced VEGF expression. Northern blot and real time PCR analyses revealed an elevated VEGF transcript level in PC-3 cells transfected with a 12-lipoxygenase expression construct. Using a VEGF promoter luciferase construct (-1176/+54), we found a 10-fold increase in VEGF promoter activity in 12-lipoxygenase-transfected PC-3 cells. The region located between -88 and -66 of the VEGF promoter was identified as 12-lipoxygenase responsive using VEGF promoter-based luciferase assays. Further analysis with mutant constructs indicated Sp1 as a transcription factor required for 12-lipoxygenase stimulation of VEGF. Neutralization of VEGF by a function-blocking antibody significantly decreased the ability of 12-lipoxygenase-transfected PC-3 cells to stimulate endothelial cell migration, suggesting VEGF as an important effector for 12-lipoxygenase-mediated stimulation of tumor angiogenesis.

Differential Expression of Thromboxane Synthase in Prostate Carcinoma Role in Tumor Cell Motility

Arachidonic acid metabolism through cyclooxygenase, lipoxygenase, or P-450 epoxygenase pathways can generate a variety of eicosanoids. Thromboxane synthase (TxS) metabolizes the cyclooxygenase product, prostanglandin H(2), into thromboxane A(2) (TXA(2)), which can cause vessel constriction, platelet activation, and aggregation. Here we demonstrate that human prostate cancer (PCa) cells express enzymatically active TxS and that this enzyme is involved in cell motility. In human PCa cell lines, PC-3, PC-3M, and ML-2 cells expressed higher levels of TxS than normal prostate epithelial cells or other established PCa cell lines such as DU145, LNCaP, or PPC-1. We cloned and sequenced the full-length TxS cDNA from PC-3 cells and found two changes in the amino acid residues. Immunohistochemical analysis of tumor specimens revealed that expression of TxS is weak or absent in normal differentiated luminal, or secretory cells, significantly elevated in less differentiated or advanced prostate tumors, and markedly increased in tumors with perineural invasion. TxS expressed in PC-3 cells was enzymatically active and susceptible to carboxyheptal imidazole, an inhibitor of TxS. The biosynthesis of TXA(2) in PC-3 cells was dependent on COX-2, and to a lesser extent, COX-1. Treatment of PC-3 cells with a COX-1 selective inhibitor, piroxicam, reduced TXA(2) synthesis by approximately 40%, while the COX-2 specific inhibitor NS398 reduced TXA(2) production by approximately 80%. Inhibition of TxS activity or blockade of TXA(2) function reduced PC-3 cell migration on fibronectin, while having minimal effects on cell cycle progression or survival. Finally, increased expression of TxS in DU145 cells increased cell motility. Our data suggest that human PCa cells express TxS and that this enzyme may contribute to PCa progression through modulating cell motility.

Increased metastatic potential in human prostate carcinoma cells by overexpression of arachidonate 12-lipoxygenase

Arachidonate 12-lipoxygenase (LOX) converts arachidonic acid to 12(S)-hydroxyeicosatetraenoic acid (HETE), a bioactive lipid implicated in tumor angiogenesis, growth, and metastasis. Alteration in 12-LOX expression or activity has been reported in various carcinomas including prostate carcinoma. However, little is known about the impact of the altered expression or activity of 12-LOX on tumor metastasis. In the present study, we examined whether or not an increase in 12-LOX expression in human prostate carcinoma cells can modulate their metastatic potential. We report that increased expression of 12-LOX in PC-3 cells caused a significant change in cell adhesiveness, spreading, motility, and invasiveness. Specifically 12-LOX transfected PC-3 cells were more adhesive toward vitronectin, type I and IV collagen, but not to fibronectin or laminin, than cells transfected with control vector. Increased spreading on vitronectin, fibronectin, collagen type I and IV also was observed in 12-LOX transfected PC-3 cells when compared to control PC-3 cells. The increased spreading of 12-LOX transfected PC-3 cells was blocked by treatment with 12-LOX inhibitors, baicalein and CDC. 12-LOX transfected PC-3 cells were more invasive through Matrigel than cells transfected with control vector. In vivo, tumor cell invasion to surrounding muscle or fat tissues was more frequent in nude mice bearing s.c. tumors from 12-LOX transfected PC-3 cells than in those from control vector transfected cells. When injected via the tail vein into SCID mice with implanted human bone fragments, there was an increase in tumor metastasis to human bone by 12-LOX transfected PC-3 cells in comparison to control vector transfected cells. Taken together, our data suggest that an increase in 12-LOX expression enhances the metastatic potential of human prostate cancer cells.

12(S)-HETE in Cancer Metastasis

Metastasis is a complex process composed of sequential events involving host celltumor cell interactions, which enable tumor cells to disseminate from the primary site to distant locations (1). To successfully establish a secondary metastatic colony, tumor cells must be able to overcome all of the steps in the metastatic cascade, including detachment, intravasation, arrest, extravasation and proliferation. Ample cell-host interactions, such as tumor cell-platelet, tumor cell-endothelial cell and tumor cell-matrix protein interaction, are influenced by positive and negative regulatory factors. Eicosanoids and other bioactive lipids have been shown to be involved in various aspects of neoplasia including cell transformation, proliferation, apoptosis, invasion and metastasis. Platelets and endothelial cells with which tumor cells interaction during hematogenous metastasis are capable of producing a vast array of lipid mediators by either direct or transcellular metabolism of precursors. Platelets and some tumor cells are capable of converting arachidonic acid (AA) through the 12-lipoxygenase (12-LOX) pathway into 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). A large collection of experimental data suggest that 12(S)-HETE plays a pivotal role in multiple steps of the metastatic cascade encompassing tumor cell-vasculature interaction, tumor cell motility, proteolysis, invasion, and angiogenesis. In this review we summarize the effects of 12(S)-HETE in modulating tumor metastasis.

Eicosanoid Regulation of Angiogenesis in Human Prostate Carcinoma and Its Therapeutic Implications

Abstract: Cancer of the prostate is the most commonly diagnosed cancer in America. There are several lines of evidence implicating the involvement of arachidonate 12‐lipoxygenase, an enzyme metabolizing arachidonic acid to form 12(S)‐hydroxyeicosatetraenoic acid (HETE), in prostate cancer progression. First, as prostate cancer reaches a more advanced stage, the level of 12‐lipoxygenase expression is increased. Second, overexpression of 12‐lipoxygenase in human prostate cancer cells stimulates angiogenesis and tumor growth. Third, an inhibitor of 12‐lipoxygenase has been found effective against metastatic prostate tumor growth, and the inhibition of 12‐lipoxygenase is related with the reduction of tumor angiogenesis. Collectively, these studies suggest that 12‐lipoxygenase regulates tumor angiogenesis in prostate cancer and that inhibition of 12‐lipoxygenase is a novel therapeutic approach for the treatment of prostate cancers.

Convergence of eicosanoid and integrin biology: 12-lipoxygenase seeks a partner.

BackgroundIntegrins and enzymes of the eicosanoid pathway are both well-established contributors to cancer. However, this is the first report of the interdependence of the two signaling systems. In a screen for proteins that interacted with, and thereby potentially regulated, the human platelet-type 12-lipoxygenase (12-LOX, ALOX12), we identified the integrin β4 (ITGB4).MethodsUsing a cultured mammalian cell model, we have demonstrated that ITGB4 stimulation leads to recruitment of 12-LOX from the cytosol to the membrane where it physically interacts with the integrin to become enzymatically active to produce 12(S)-HETE, a known bioactive lipid metabolite that regulates numerous cancer phenotypes.ResultsThe net effect of the interaction was the prevention of cell death in response to starvation. Additionally, regulation of β4-mediated, EGF-stimulated invasion was shown to be dependent on 12-LOX, and downstream Erk signaling in response to ITGB4 activation also required 12-LOX.ConclusionsThis is the first report of an enzyme of the eicosanoid pathway being recruited to and regulated by activated β4 integrin. Integrin β4 has recently been shown to induce expansion of prostate tumor progenitors and there is a strong correlation between stage/grade of prostate cancer and 12-LOX expression. The 12-LOX enzymatic product, 12(S)-HETE, regulates angiogenesis and cell migration in many cancer types. Therefore, disruption of integrin β4-12LOX interaction could reduce the pro-inflammatory oncogenic activity of 12-LOX. This report on the consequences of 12-LOX and ITGB4 interaction sets a precedent for the linkage of integrin and eicosanoid biology through direct protein-protein association.

Overexpression of leukocyte-type 12-lipoxygenase promotes W256 tumor cell survival by enhancing αvβ5 expression

The metabolism of arachidonic acid (AA) leads to the generation of biologically active metabolites that have been implicated in cell growth and proliferation, as well as survival and apoptosis. We have previously demonstrated that rat Walker 256 (W256) carcinosarcoma cells express the platelet‐type 12‐lipoxygenase (12‐LOX) and synthesize 12(S)‐ and 15(S)‐HETE as their major LOX metabolites. Here we show that Walker 256 cells also express leukocyte‐type 12‐LOX and that its overexpression in these cells significantly extends their survival and delays apoptosis when cells are cultured under serum‐free conditions. Under serum‐free conditions, the expression of leukocyte‐type 12‐LOX is upregulated. 12‐LOX‐transfected W256 cells had a more spread morphology in culture compared with wild‐type or mock‐transfected cells. Examination of W256 cells showed that the cells expressed a number of integrins on their surface. Overexpression of 12‐LOX enhanced the surface expression and focal adhesion localization of integrin αvβ5, while not affecting other integrins. Also, the 12‐LOX‐transfected W256 cells exhibited higher levels of microfilament content. Treatment of cells with monoclonal antibody to αvβ5 or cytochalasin B (a microfilament‐disrupting agent), but not antibodies to other integrin receptors, resulted in significant apoptosis, characterized by rapid rounding up and detachment from the substratum. These results show that the 12‐LOX pathway is a regulator of cell survival and apoptosis, by affecting the expression and localization of the αvβ5 integrin and actin microfilaments in Walker 256 cells.

The Role of Eicosanoids in Tumor Growth and Metastasis

Mobilization of esterified arachidonic acid (AA) from membrane glycerolipid pools represents the key regulatory step in cellular responses to various stimuli, such as growth factors, cytokines, chemokines and circulating hormones. Released AA is metabolized via the cyclo-oxygenase (COX 1 and COX 2), lipoxygenase (LOX) or P450-dependent epoxygenase pathways to generate eicosanoids. In addition to their normal biological activities, such as stimulation of mitogenesis and cellular motility, eicosanoids have also been postulated to contribute to tumorigenesis and to the progression of certain tumor cells. Various AA metabolites have been implicated in a variety of growth-related signaling pathways involving ras (Han et al. 1991), interferon-α, epithelial growth factor (EGF), cyclic adenosine monophosphate, protein kinase C (PKC; Hannigan and Williams 1991; Peppelenbosch et al. 1993; Tang et al. 1995a), mitogen-activated kinases (Rao et al. 1988) and fos (Danesch et al. 1994). Numerous studies have demonstrated a strong correlation between growth-factor-promoted cell proliferation and generation of various COX products, primarily prostaglandins (Nolan et al. 1988).