Alex Zacharek

Elevated 12-lipoxygenase mRNA expression correlates with advanced stage and poor differentiation of human prostate cancer.

OBJECTIVES Prostate cancer is the most frequently diagnosed cancer and the second leading cause of cancer death in males in the United States. The mortality is due mainly to distant metastasis. Therefore, predicting the prognosis of prostate cancer patients is an important clinical problem. Previously, we demonstrated that a 12-lipoxygenase (12-LOX) metabolite of arachidonic acid, 12(S)-hydroxyeicosatetraenoic acid, enhances the invasiveness of prostate cancer cells and that a 12-LOX-selective inhibitor [N-benzyl-N-hydroxy-5-phenylpentanamide] reduces experimental metastasis in animal model systems. In this study, we investigated the potential of 12-LOX as a predictor for the aggressiveness of prostate cancer. METHODS The mRNA expression level of 12-LOX in 122 matching prostate normal and cancerous tissues were measured by quantitative reverse transcription- polymerase chain reaction. Possible association between 12-LOX expression and histologic grade, pathologic and clinical stage, margin positivity, age, and race was analyzed. RESULTS 12-LOX mRNA levels were elevated in cancer cells and the expression associated with poor differentiation and invasiveness of prostate cancer. Overall, 46 (38%) of 122 evaluable patients showed elevated levels of 12-LOX mRNA in prostate cancer tissues compared with the matching normal tissues. A statistically significantly greater number of cases were found to have an elevated level of 12-LOX among T3, high grade, and surgical margin-positive than T2, intermediate, and low grade, and surgical margin-negative prostatic adenocarcinomas. CONCLUSIONS Our data suggest that elevation of 12-LOX mRNA expression occurs more frequently in advanced stage, high-grade prostate cancer and that 12-LOX may serve as an indicator for progression and prognosis of prostate cancer. This enzyme also may be a novel target for the development of anti-invasive and antimetastatic agents.

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.

Role for β3 integrins in human melanoma growth and survival

The role of αIIbβ3 integrin in regulating platelet function is well appreciated, whereas its role in tumor progression and metastasis is not. The purpose of our study was to determine a functional relevance to expression of αIIbβ3 integrin in cells derived from human solid tumors. A study of human melanoma biopsies (n = 24) showed that αIIbβ3 expression increased with tumor thickness, which is indicative of metastatic propensity. Expression of αIIbβ3 was 8% (±1.8), 33% (±10.4) and 62% (±5) in melanomas ranging in thickness from 0–1.5 mm, 1.5–4.0 mm and >4 mm, respectively; αvβ3 was equally high all categories. To determine biological function, we stably transfected αIIbβ3 into human melanoma cells that express αvβ3, but not αIIbβ3. Surface expression of αvβ3 remained unaltered between αIIbβ3 (+) and mock transfected counterparts. The αIIbβ3 (+) cells possessed increased ability to adhere, spread and migrate on fibrinogen. They had decreased ability to attach, spread and migrate on vitronectin. Immunocytochemistry showed that expression of αIIbβ3 displaced αvβ3 from focal contact points. When implanted subcutaneously into SCID mice, the αIIbβ3 (+) cells developed ∼4‐fold larger tumors when compared to their mock counterparts and the level of apoptosis was reduced within the tumors. Results suggest that co‐expression of the 2 β3 integrins, αvβ3 and αIIbβ3, in human melanoma cells enhanced cell survival and promoted growth in vivo.

Thromboxane A2 Receptors in Prostate Carcinoma: Expression and Its Role in Regulating Cell Motility via Small GTPase Rho

Thromboxane A(2) (TxA(2)) is a prostanoid formed by thromboxane synthase using the cyclooxygenase product prostaglandin H(2) as the substrate. Previously, increased expression of thromboxane synthase was found in prostate tumors, and tumor cell motility was attenuated by inhibitors of thromboxane synthase. This study was undertaken to elucidate how tumor motility is regulated by TxA(2). Here, we report that human prostate cancer cells express functional receptors for TxA(2) (TP). Ligand binding assay found that PC-3 cells binded to SQ29548, a high-affinity TP antagonist, in a saturable manner with K(d) of 3.64 nmol/L and B(max) of 120.4 fmol per million cells. Treatment of PC-3 cells by U46619, a TP agonist, induced PC-3 cell contraction, which was blocked by pretreatment with the TP antagonist SQ29548 or pinane TxA(2). The migration of prostate cancer cells was significantly inhibited either by sustained activation of TP or by blockade of TP activation, suggesting that TP activation must be tightly controlled during cell migration. Further studies found that small GTPase RhoA was activated by TP activation, and pretreatment of PC-3 cells with Y27632, a Rho kinase (ROCK) inhibitor, blocked U46619-induced cell contraction. A dominant-negative mutant of RhoA also blocked U46619-induced cell contraction. Taken together, the data suggest that TPs are expressed in prostate cancer and activation of TPs regulates prostate cancer cell motility and cytoskeleton reorganization through activation of Rho.

12-Lipoxygenase in Lewis lung carcinoma cells: Molecular identity, intracellular distribution of activity and protein, and Ca2+-dependent translocation from cytosol to membranes

Recently we demonstrated that Lewis lung (3LL) tumor cells express 12-lipoxygenase (12-LOX) mRNA and protein, respectively. In this study we partially sequenced the 12-LOX cDNA after reverse-transcription polymerase chain reaction amplification of 12-LOX mRNA from cultured 3LL cells. Comparison with platelet and leukocyte 12-LOX indicates that 3LL 12-LOX is identical with the platelet-type enzyme at least within the sequenced region. Further, we investigated the intracellular distribution of both 12-LOX enzyme protein and its activity which are prerequisites for understanding 12-LOX regulation. 12-LOX activity was monitored via the production of 12-hyroxyeicosatetraenoic acid from 3LL cells and their subcellular fractions using reverse-phase high performance liquid chromatography. 12-LOX protein was measured by direct slot blot and by Western Blotting. In 3LL cells, both 12-LOX activity and 12-LOX protein were predominantly localized in the cytosol. This 12-LOX activity was optimal at 37 degrees C. However at 24 degrees C and 10 degrees C, it showed 87% and 61% of this activity, respectively, thus differing distinctly from 12-LOX in platelets or rat basophilic leukemia cells. Incubation of 3LL cell homogenates with 0-100 microM free Ca2+ and subsequent separate analyses of cytosol and membrane fractions indicated that, as in platelets, an increase in intracellular free Ca2+ caused a loss of cytosolic 12-LOX activity. However, no significant Ca(2+)-induced increase in membrane-associated 12-LOX activity was observed under these conditions in 3LL cells. In contrast, at the 12-LOX protein level we observed a Ca(2+)-dependent loss in the cytosol and a concomitant increase in the membrane fraction. Thus, we suggest that 12-LOX in 3LL cells undergoes rapid translocation from cytosol to membrane in a Ca(2+)-dependent manner, but is no longer active or becomes inactivated at the membrane site.

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.

Eicosanoids, cancer metastasis, and gene regulation: an overview.

Eicosanoids are a group of oxygenated arachidonic acid (AA) metabolites including prostaglandins (PGs), thromboxanes (TXs), leukotrienes (LTs), lipoxins (LXs), and various hydroperoxy and hydroxy fatty acids (1–3). AA is released from phospholipids mainly by the action of phospholipase A2 and is the substrate for lipoxygenases (LOXs), cyclooxygenase (COX) and cytochrome P-450 monooxygenase (also called epoxygenase). AA is metabolized to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) by 5-lipoxygenase (5-LOX), to 12-HPETE by 12-lipoxygenase (12-LOX; EC 1.13.11.31) and to 15-HPETE by 15-lipoxygenase (15-LOX). Then, 5-HPETE can be converted to 5-hydroxyeicosatetraenoic acid (5-HETE) and LTs, 12-HPETE to 12-HETE and hepoxilins (HXs), 15-HPETE to 15-HETE and LXs. AA can also be metabolized to PGs, prostacyclin and TXs by COX, or to epoxides and diols by cytochrome P-450. Both the LOX and COX pathways have been implicated in several aspects of cancer from carcinogenesis to metastasis (4–11).