The Inhibitory Effect Of 5-lipoxygenase Inhibitors On Metastasis Of Melanoma Cell A375

Melanoma is a kind of malignant tumor originating from pigment cells of skin andprone to metastasis. So far no specific drug has been aPplied to control the diffusedpathological process. Epidemiological studies show that the content of polyunsaturatedfatty acids in diet were closely associated with incidence of malignant melanoma.Arachidonic acid is the main ingredients of polyunsaturated fatty acids and itslipoxygenase metabolites play a key role in the metastasis process of melanoma cancercells. We conducted experiments trying to stlldy the effects and mechanism of 5-lipoxygenase inhibitors on metastasis of human melanoma cell a375 and to obtain theclue of 5-lipoxygenase inhibitor as poteniial anti-metastatic agents for melanoma.Adhesion of a375 cell to extracellular matrix was determined by optical densityassay. The expression of integrins on cancer cell surface was determined by fiowcytometry ELlSA, flow cytometry and Western blot was used to detect the production oflCAM-l in a375 cell. Northern blot was used to measure the expressing level of ICAM-1mRNA in a375 cell. The 1evels of NF- K B in a375 cell was measured by sandwichELISA.Invasiveness of a375 cells was assayed by using Transwell chamber wth filtermembrane coated with Matrigel. The secretion of active matrix metalloproteinases(MMP-2 and MMP-9) was measured by gelatin zymogram. Western blot was used todetect the production of MMP-2, MMP-9 and tissues inhibitors of metalloproteinase(TIMP-l) and the expressing leveI of MMP-2, MMP-9 and TIMP-l mKNA wasmeasured by Northern blot.The results showed that in control grouP treated with indomethacin, acyclooxygenase inhibitors, adhesion of a375 cells to extracellular matrix was not altered.In the range from 5~20 ll M, lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA),5-lipoxygenase inhibitors AA861 and specific 5-lipoxygenase-activating-prote inhibitorMK886 could all inhibit adhesion of a375 cell to collagen IVin a dose-dependent mannerand had no effect on adhesion to fibronectin or vitronectin. 5-lipoxygenase metabolites(5-HETE) reversed the adhesion behavior of inhibitor-treat cancer cell and leukotrieneshad no effect.Pretreated with 20 ll M of NDGA, AA861 and MK886 remarkably inhibited theA23l87-induced enhancement of a375 cell adhesion to collagen IV respectively andonly partly inhibited the TPA-induced enhancement.A375 cells tfeated with NDGA, AA861 and MK886 in the same concentfationexhibited reduction of the expression of integrin 6 l on the surface of melanoma cell, butnot of integrin Q lIb fl 3'Indomechacin had no effect on the production of ICAM-l protein in a375 cell.NDGA, AA861 and MK886 could reduce the production of ICAM-l protein and itsmRNA of a375 cell and suPpress NF- K B activation in a dose-dependeni manner in therange from 5~20 ll M.No significant inhibition of invasiveness to recombinant basement membrane wasseen when a375 ceIls were treated with indomethacin. In the range from 5~20ll MNDGA, AA861 and MK886 significantly inhibited invasiveness of a375 cell throughMatrigel membrane. 5-HETE could reverse this inhibitory effect and leukotrienes had noeffect.NDGA, AA86l and MK886 could significantly inhibit production of MMP-2 andMMP-9 proteins as well as their mRNA expression level. The inhibition of 5-lipoxygenase activity with the shese inhibitors had no significant effect on production ofTIMP-l protein and mRNA level in a375 ceII.These results suggest that one of the 5-lipoxygenase metabolites, 5-HETE, plays animportant role in the metastatic progress of melanoma. 5-lipoxygenase inhibitors caninhibit the process by suPpressing the expression of adhesion molecule through inhibitingthe nuclear factor activation of a375 cell so as to inhibit a375 cell adhesion toextracel1u1ar matrix and by suPPressing the production of MMP-2 and MMP-9 in a375cell so as to decrease the invasiveness to through recombinant basement membrane.That...