Overexpression of 5-lipoxygenase in colon polyps and cancer and the effect of 5-LOX inhibitors in vitro and in a murine model.

PURPOSE: Arachidonic acid metabolism via the cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) pathways modulates cell growth and apoptosis. Many studies have examined the effects of COX inhibitors on human colorectal cancer, but the role of 5-LOX in colonic cancer development has not been well studied. The purpose of this study was to evaluate the expression of 5-LOX in colonic polyps and cancer and the effect of 5-LOX inhibition on colon cancer cell proliferation. EXPERIMENTAL DESIGN: Colonic polyps, cancer, and normal mucosa were evaluated for 5-LOX expression by immunohistochemistry. Reverse transcription-PCR was used to establish 5-LOX expression in colon cancer cells. Thymidine incorporation and cell counts were used to determine the effect of the nonspecific LOX inhibitor Nordihydroguaiaretic Acid and the 5-LOX inhibitor Rev5901 on DNA synthesis. A heterotopic xenograft model in athymic mice using HT29 and LoVo human colon cancer cells was used to evaluate the effect of the 5-LOX inhibitor zileuton on tumor growth. RESULTS: 5-LOX is overexpressed in adenomatous polyps and cancer compared with that of normal colonic mucosa. LOX inhibition and 5-LOX inhibition decreased DNA synthesis in a concentration- and time-dependent manner in the Lovo cell line (P < 0.05). Inhibition of 5-LOX in an in vivo colon cancer xenograft model inhibited tumor growth compared with that of controls (P < 0.05). CONCLUSIONS: This study showed that 5-LOX is up-regulated in adenomatous colon polyps and cancer compared with normal colonic mucosa. The blockade of 5-LOX inhibits colon cancer cell proliferation both in vitro and in vivo and may prove a beneficial chemopreventive therapy in colon cancer.

Leukotriene B4 receptor antagonist LY293111 inhibits proliferation and induces apoptosis in human pancreatic cancer cells.

PURPOSE: The effects of leukotriene (LT) B4 and its receptor antagonist LY293111 on proliferation and apoptosis of human pancreatic cancer cells were investigated, both in vitro and in vivo. EXPERIMENTAL DESIGN: Six human pancreatic cancer cell lines (MiaPaCa-2, HPAC, Capan-1, Capan-2, PANC-1, and AsPC-1) were used. Expression of LTB4 receptors, BLT1 and BLT2, was measured by reverse transcription-PCR. Cell proliferation was measured by [methyl-(3)H]thymidine incorporation and cell number counting. Extracellular signal-regulated kinase (ERK) 1/2 activation was measured by Western blotting. Apoptosis was assessed by morphology, terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay, and poly(ADP-ribose) polymerase cleavage. The effect of LY293111 on growth of AsPC-1 and HPAC cell xenografts was assessed in BALB/c nu/nu athymic mice. RESULTS: Both LTB4 receptor types were found to be expressed in human pancreatic cancer cells. The LTB4 receptor antagonist LY293111 caused both time- and concentration-dependent inhibition of proliferation of all six human pancreatic cancer cell lines studied. In contrast, LTB4 stimulated proliferation of these cell lines and induced ERK1/2 phosphorylation. The growth-stimulatory effect and ERK1/2 phosphorylation induced by LTB4 were inhibited by LY293111. Coincident with growth inhibition, LY293111 induced apoptosis in these pancreatic cancer cell lines, as indicated by morphology, TUNEL assay, and poly(ADP-ribose) polymerase cleavage. In studies using AsPC-1 and HPAC cell xenografts in athymic mice, LY293111 treatment markedly inhibited tumor growth over a 24-day treatment period, as measured by both tumor volume and tumor weight. In situ tissue TUNEL assay showed massive apoptosis in LY293111-treated tumor tissues. CONCLUSIONS: LTB4 can directly regulate the growth of human pancreatic cancer cells and control their survival. Additional studies will clarify the underlying mechanisms of LTB4-regulated pancreatic cancer cell growth and apoptosis. LTB4 receptor blockade and inhibition of the downstream signal pathway are likely to be valuable for the treatment of human pancreatic cancer.

Lipoxygenase inhibitors attenuate growth of human pancreatic cancer xenografts and induce apoptosis through the mitochondrial pathway.

Several studies have suggested that high dietary fat intake, particularly essential fatty acids, is associated with pancreatic cancer development and growth. Our previous studies have demonstrated that blockade of either the 5-lipoxygenase (LOX) or 12-LOX pathway of arachidonic acid metabolism inhibited pancreatic cancer cell proliferation and induced apoptosis. This study investigated the underlying mechanisms for LOX inhibitor-induced apoptosis and the potential of LOX inhibitors as antipancreatic cancer agents using the athymic mice xenograft model. Apoptosis of pancreatic cancer cells induced by LOX inhibitors (including the nonselective LOX inhibitor nordihydroguaiaretic acid, the 5-LOX inhibitor Rev-5901, and the 12-LOX inhibitor baicalein) was confirmed by growth inhibition, annexin V binding, and terminal deoxynucleotidyl transferase-mediated nick end labeling assay in MiaPaCa-2 and AsPC-1 human pancreatic cancer cells. Expression of the antiapoptotic proteins Bcl-2 and Mcl-1 was significantly decreased after LOX inhibitor treatment while that of the proapoptotic protein bax was increased. LOX inhibitors also markedly induced the release of cytochrome c from mitochondria into the cytosol. Caspase-9, caspase-7, and caspase-3 but not caspase-8 were activated after treatment, concomitant with cleavage of the capase-3 substrate poly(ADP-ribose) polymerase. In vivo studies in the athymic mice xenograft model also confirmed the growth inhibitory effect and induction of apoptosis by these LOX inhibitors in pancreatic cancer. In conclusion, LOX inhibitors block pancreatic cancer cell proliferation and induce apoptosis through the mitochondrial pathway both in vivo and in vitro. LOX inhibitors are likely to be valuable for the treatment of human pancreatic cancer.

Effect of LY293111 in combination with gemcitabine in colonic cancer.

New adjuvant therapies are needed for the treatment of stage III colon cancer. The essential fatty acids, linoleic and arachidonic acid enhance tumorigenesis through the cyclooxygenase and lipoxygenase pathways. Leukotriene B4 (LTB4) is a product of 5-lipoxygenase (5-LOX) which has tumor-promoting effects. The LTB4 receptor antagonist, LY293111 inhibited tumor growth and induced apoptosis in vitro. The effectiveness of LY293111, alone and in combination with gemcitabine was investigated in a heterotopic xenograft model in athymic mice using HT29 and LoVo human colonic cancer cells. The combined therapy markedly inhibited tumor growth and could warrant consideration as a new therapeutic option.

Differential effects of calpain inhibitors on hypertrophy of cardiomyocytes.

Two inhibitors of the calcium-dependent cysteine protease, calpain, have markedly different effects on the extent of hypertrophy induced by the alpha-adrenergic agonist, phenylephrine, of cultured neonatal rat ventricular myocytes. E64c, an inhibitor of calpain and other cysteine proteases, stimulated the hypertrophy by 59%. PD 150606, a specific calpain inhibitor, reduced the hypertrophy by 38%. Phenylephrine decreased the proteolysis of a calpain substrate by the cells 1-2 h after its addition but not at 24 h. PD 150606 inhibited proteolytic activity at all times, and the combination of phenylephrine and PD 150606 did not give greater inhibition. This suggests that cysteine proteases of the papain sub-family are involved with the hypertrophic response at two points, promoting hypertrophy at the first and limiting it at the second. Calpain appears to be the protease involved at the first point, and there may be another cysteine protease acting at the second site.