Daotai Nie

Cyclooxygenase, lipoxygenase and tumor angiogenesis

Abstract. Arachidonic acid metabolism through cyclooxygenase (COX) and lipoxygenase (LOX) pathways generates various biologically active lipids that play important roles in inflammation, thrombosis and tumor progression. Angiogenesis, the formation of new capillary vessels from preexisting ones, underpins a number of physiological processes and participates in the development of several pathological conditions such as arthritis, cancer and various eye diseases. The formation of new capillary vessels is a multistep process that involves endothelial cell proliferation, migration and tube formation. In the present review, we survey the literature on the regulation of angiogenesis by arachidonate metabolites, especially those from the COX and 12-LOX pathways in the context of tumor growth, and put forward some unanswered but important questions for future studies.

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.

Expression, subcellular localization and putative function of platelet‐type 12‐lipoxygenase in human prostate cancer cell lines of different metastatic potential

The involvement of 12‐lipoxygenase (12‐LOX) expression and function in tumor metastasis has been demonstrated in several murine tumor cell lines. In addition, 12‐LOX expression was detected in human prostatic tumors and correlated to the clinical stage of disease. Here we provide data that human prostate cancer cell lines express the platelet‐type isoform of 12‐LOX at both the mRNA and protein levels, and immunohistochemistry revealed 12‐LOX expression in human prostate tumors. The enzyme was localized to the plasma membrane, cytoplasmic organelles and nucleus in non‐metastatic cells (PC‐3 nm) and to the cytoskeleton and nucleus in metastatic cells (DU‐145). After orthotopic/intraprostatic injection of tumor cells into SCID mice, the metastatic prostate carcinoma cells (DU‐145) expressed 12‐LOX at a significantly higher level compared with the non‐metastatic counterparts, PC‐3nm. The functional involvement of 12‐LOX in the metastatic process was demonstrated when DU‐145 cells were pretreated in vitro with the 12‐LOX inhibitors N‐benzyl‐N‐hydroxy‐5‐phenylpentamide (BHPP) or baicalein, the use of which significantly inhibited lung colonization. These data suggest a potential involvement of 12‐LOX in the progression of human prostate cancer. Int. J. Cancer 87:37–43, 2000.

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.

Platelet-type 12-lipoxygenase activates NF-κB in prostate cancer cells

Abstract Platelet-type arachidonate 12-lipoxygenase (12-LOX) is highly expressed in many types of cancers and plays an important role in cancer pathophysiology. Arachidonic acid metabolism by 12-LOX results in the stable end product 12( S )-hydroxy eicosatetraenoic acid (12( S )-HETE), which is a signaling molecule with effects on cell proliferation, motility, invasiveness, angiogenesis, and inhibition of apoptosis. The myriad biological activities manifested by 12( S )-HETE appear to be mediated, at least in part, by the activation of NF-κB. Overexpression of the 12-LOX in PC-3 prostate cancer cells resulted in the constitutive activation of the transcription factor. The enzymatic product of arachidonic acid metabolism, 12( S )-HETE, mediates the activation of NF-κB by the 12-LOX. 12( S )-HETE treatment of PC-3 cells induced the degradation of IκB by the S6 proteasomal pathway and the activated NF-κB translocated to the nucleus causing κB-induced transcription. Specificity of the NF-κB activation by 12( S )-HETE was established by the use of a 12-LOX-specific inhibitor and 13( S )-HODE, a known 12( S )-HETE antagonist. Considering the known involvement of MAP kinase pathway in NF-κB activation and that of 12( S )-HETE in MAP kinase pathway, 12-LOX present in prostate cancer tissues may contribute to the constitutive activation of NF-κB in prostate cancer cells.

12-Lipoxygenase and the regulation of hypoxia-inducible factor in prostate cancer cells

12-Lipoxygenase, an arachidonic acid metabolizing enzyme of the lipoxygenase pathway, has been implicated as a major factor in promoting prostate cancer progression and metastasis. The ability of 12-LOX to aggravate the disease was linked to its proangiogenic role. Recent studies clearly demonstrated that 12-LOX enhances the expression and secretion of the angiogenic factor, vascular endothelial growth factor (VEGF) thus providing a direct link between this enzyme and its angiogenic properties. In the present study we have investigated the relationship between 12-LOX and hypoxia inducible factor-1alpha (HIF-1alpha), a transcription factor involved in the regulation of VEGF expression under hypoxic conditions in solid tumors. Our findings have revealed that HIF-1 is one of the target transcription factors regulated by 12-LOX and 12(S)-HETE, in hypoxic tumor cells of the prostate. Regulation of HIF-1alpha by 12-LOX adds to the complexity of pathways mediated by this enzyme in promoting prostate cancer angiogenesis and metastasis. We have evidence that 12-LOX increases the protein level, mRNA, and functional activity of HIF-1alpha under hypoxic conditions, one of the mechanisms by which it upregulates VEGF secretion and activity.

RETINOIC ACID TREATMENT ELEVATES MATRIX METALLOPROTEINASE-2 PROTEIN AND MRNA LEVELS IN AVIAN GROWTH PLATE CHONDROCYTE CULTURES

Matrix metalloproteinases (MMPs) play a crucial role in tissue remodeling. In growth plate (GP) cartilage, extensive remodeling occurs at the calcification front. To study the potential involvement of MMPs in retinoic acid (RA) regulation of skeletal development, we studied the effect of all‐trans‐RA on MMPs levels in mineralizing chicken epiphyseal chondrocyte primary cultures. When treated for 4 day periods on days 10 and 17, RA increased levels of an ∼70 kDa gelatinase activity. The N‐terminal sequence of the first 20 amino acid residues of the purified enzyme was identical to that deduced from chicken MMP‐2 cDNA. Time‐course studies indicated that RA elevated MMP‐2 activity levels in the cultures within 16 h. This increase was inhibited by cycloheximide and was enhanced by forskolin. The increase in MMP‐2 activity induced by RA was accompanied by an increase in MMP‐2 mRNA levels and was abolished by treatment with cycloheximide. This upregulation of MMP levels by RA in GP chondrocytes is consistent with its effects on osteoblasts and osteosarcoma cells and opposite its inhibitory effects on fibroblasts and endothelial cells. It may well be related to the breakdown of the extracellular matrix in the GP and would be governed by the availability of RA at the calcification front where extensive vascularization also occurs. J. Cell. Biochem. 68:90–99, 1998.

Retinoic acid stimulates matrix calcification and initiates type I collagen synthesis in primary cultures of avian weight-bearing growth plate chondrocytes.

The effect of retinoic acid (RA) on primary cultures of growth plate chondrocytes obtained from weight‐bearing joints was examined. Chondrocytes were isolated from the tibial epiphysis of 6‐ to 8‐week‐old broiler‐strain chickens and cultured in either serum‐containing or serum‐free media. RA was administered at low levels either transiently or continuously after the cells had become established in culture. Effects of RA on cellular protein levels, alkaline phosphatase (AP) activity, synthesis of proteoglycan (PG), matrix calcification, cellular morphology, synthesis of tissue‐specific types of collagen, and level of matrix metalloproteinase (MMP) activity were explored. RA treatment generally increased AP activity, and stimulated mineral deposition, especially if present continuously. RA also caused a shift in cell morphology from spherical/polygonal to spindle‐like. This occurred in conjunction with a change in the type of collagen synthesized: type X and II collagens were decreased, while synthesis of type I collagen was increased. There was also a marked increase in the activity of MMP. Contrasting effects of continuous RA treatment on cellular protein levels were seen: they were enhanced in serum‐containing media, but decreased in serum‐free HL‐1 media. Levels of RA as low as 10 nM significantly inhibited PG synthesis and caused depletion in the levels of PG in the medium and cell‐matrix layer. Thus, in these appendicular chondrocytes, RA suppressed chondrocytic (PG, cartilage‐specific collagens) and enhanced osteoblastic phenotype (cell morphology, type I collagen, alkaline phosphatase, and mineralization). J. Cell. Biochem. 65:209–230.

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.