Abstract
Pancreatic carcinoma is characterized by a poor prognosis and lack of response to conventional therapy. The incidence of this disease shows no sign of declining. Tins disease is now the fifth leading cause of cancer death in the USA. lire 5-year survival rate is less than 2%, and the median survival time after diagnosis is less than 6 months. Surgical resection is the only effective treatment. However, only about 15% of carcinomas of the head of the pancreas are resectable and few patients survive long-term even after apparent curative resection. Other treatments, such as chemotherapy or radiation therapy, provide limited palliation without improvement of survival in patients with non-resectable pancreatic cancer. While many studies have demonstrated mutations ofK-ras oncogene, pl6 and p53 tumor suppressor genes and the over-expression of a number of growth factors and their receptors inpancreatie cancer, the reason for the aggressiveness and poor prognosis of this cancer is still not well understood. Thus, new targets for pancreatic cancer treatment need to be identified.|The results from epidemiological and animal studies suggest that a high fat consumption is associated with an. increased incidence and growth of tumors in several organs including pancreas, colon, breast and prostate. Diets containing a high proportion of polyunsaturated omega-6 fatty acids are associated with a more advanced disease stage at the time of diagnosis of several kinds of cancer. Linoleic. acid (9,12-Octadecenoic acid) stimulates the progress of human tumors in athymic mice, whereas supplementation of diets, docosahexaenoic acid (cis-4, 7, 10,13,16,19-docosahexaenoic acid) and eicosapentaenoic acid (5, 8, 11, 14,17-eicosapentaenoic acid) exert suppressive effects. Diets with docosahexaenoic acid is accompanied by reduced levels of prostaglandins, and of LOXmetabolites, suggesting that changes in eicosanoid biosynthesis may be responsible for die observed decreases in tumor growth.|Arachidonic acid is a substrate for three different enzymes, cyclooxygenases, lipoxygenases and epoxygenases. Mammalian lipoxygenases insert one molecule oxygen on either carbon 5, carbon 12 or carbon 15 of arachidonic acid and on this basis have been designated as arachidonate 5 -, 12-, or 15-lipoxygenase (5- LOX, 12-LOX, and 15-LOX). The primary product of 5-LQX is 5S-hydroperoxyeicosatetraenoic add (5- HPETE), which can be further reduced by glutathione peroxidase to hydroxy forms (5-HETE). 5-LOX is the only enzyme that can matabolize arachidonic acid to ieukotrien.es. The activity of 5-LOX is dependent on interaction with a second protein, 5-LOX-activating protein (FLAP). Multiple lines of evidence have shown that COX pathways are associated with pancreatic cancer growth while the role of5-LOXin pancreatic cancer cell proliferation is poorly understood. The role of 5-LOX pathway in pancreatic cancer cell growth and survival was investigated in this current study.|Firstly, the extent of expression of 5-LOX in pancreatic cancer cells was investigated. RT-PCR revealed that 5-LOX mJRNA is expressed in both poorly-differentiated arid well-differentiated cell lines. The sequence of the PCR product was identical to that of the 5-I,OX cDNA reported in the gene bank. It was generally considered that human pancreatic cancer is derived from pancreatic ductal cells, and therefore, expression of 5-LOX in normal human pancreatic ductal cells was investigated by RT-PCR. In contrast to pancreatic cancer cells, the expression of 5-LOX mRNA was not detectable in normal human pancreatic ductal cells, indicating that 5-LOX is up-regulated in human pancreatic cancer. Furthermore. She expression of 5- LQX protein in pancreatic cancer cells was also confirmed by western blotting.|Secondly, the potential effect ofLOX inhibition on pancreatic cancer cell proliferation and survival was investigated. Blockade of 5-LOX with pharmacological LOX inhibitors, NDGA and Rev5901, abolished in vitro pancreatic cancer cell proliferation, as measured by both DMA synthesis and cell number, in multiple pancreatic cancer ceil lines. Also, the FLAP inhibitor, MK886, which indirectly blocks 5-LQX activity, inhibited pancreatic cancer cell proliferation. Treatment of pancreatic cancer cells with 5-LOX antisense oligonucleotide significantly decreased proliferation of pancreatic cancer cells Blockade of 5-LOX enzyme also induced apoptosis in pancreatic cancer cells, as documented by propidium iodide DNA staining, TUNEL assay and DNA fragmentation. The inhibitory effect of the LOX inhibitors on pancreatic cancer cell proliferation was reversed by the 5-LOX metabolite, 5-HETE, providing farther evidence of the importance of 5 -LOX in controlling pancreatic cancer cell proliferation.|Thirdly, the direct effect of the 5-LOX metabolite, 5(S)~HETE on pancreatic cancer cell proliferation was investigated. The results show that 5(S)-HE'fE can directly stimulate pancreatic cancer cell proliferation, as determined by both [3H]-methylthymidine incorporation and celi counting in vitro. Subsequent studies revealed that both intracellular tyrosine kinases and MEK/ERK. i/2 are involved in 5(S)-HETE-stiniu!ated pancreatic cancer cel! proliferation. Treatment of pancreatic cancer cells with 5(S)-HETE stimulated tyrosine phosphorylation of multiple cellular proteins with different molecular sizes, in a time-dependent manner. Treatment of pancreatic cancer cells with genestein abolished the mitogenic effect of 5(S)-HETE, suggesting the involvement of genestein-sensitive tyrosine kinases in 5(S)-HETE-stixnulated pancreatic cancer cell proliferation. Both ERK1 /2 and MEK were activated by 5(S)-HETE, as shown by erihanced phosphorylation. Furthermore, the activation of the MEK/ERK cascade appeals to be involved in 5(S)-HETE-stimulated pancreatic cancer cell proliferation, since blockade of the MEK/ERK 1/2 cascade using two structurally unrelated MEK inhibitors, PD098059 and U0256, blocked 5(S)-HETE-mduced pancreatic cancer cel! proliferation. In contrast, the results suggest that PKC, P'38 MAP kinase and JNK'SAPK are not involved in the mitogenic effect of 5(S)-HETE on pancreatic cancer cells. On the other hand, the PIS kinase/AKT cascade is activated and is involved in 5(S)-HETE-stimulated pancreatic cancer cell proliferation. Treatment of pancreatic cancer cells with 5(S)-HETE induced a significant phosphorylation of AKT/PKB while the PI3 kinase inhibitor, wortmannin, inhibited 5( S i-induced cell proliferation as well as AKT phosphorylation.|Finally, since EGF receptor ligands and the EGF receptors are over-expressed in pancreatic cancer, the interaction of the 5-LOX pathway with EGF-mediated ERK activation and pancreatic cancer ceil proliferation was investigated. The 5-LOX inhibitor, Rev5901, abolished EGF-induced pancreatic cancer cell proliferation. The phosphorylation and activation of ERK1/2 induced by EGF was also inhibited by Rev5901. Further studies showed that the actin cytoskeleten reorganization induced by EGF was blocked by Rev5901. On the other hand, EGF enhanced expression of both 5-LOX mRNA and protein. Up-regulation of 5-LOX expressionby EGF is, at least in part, through activating 5 -LOX promoter and inducing transcription since the 5-LOX promoter reporter assay showed that EGF significantly enhances 5-L;OX promoter activity.|In summary, the resuits indicate that 5-LOX activity is crucial in regulating pancreatic cancer cell proliferation and survival, and involved in EGF-mediated signal cascade and cell proliferation. Further studies which could reveal the therapeutic potential of 5-LOX inhibition in pancreatic cancer should focus on the mechanism of 5-LOX inhibition-induced apoptosis and in vivo growth of pancreatic cancer and interaction|between 5-LOX and EGF-mediated signaling.