SC-41661A

The 5-lipoxygenase inhibitors ETYA, SC41661A and MK886 reduced the proliferation and viability of Panc-1 human pancreatic cancer cells.The extent of inhibition depended upon drug concentration and with continued culture, cells detached and stained with trypan blue.Although results from flow cytometry were consistent with programmed cell death, despite repeated attempts, no DNA laddering characteristic of its later stages was detected, and studies with the TUNEL assay were neg.Light and electron microscopy of cells cultured with SC41661A provided morphol. evidence of an incompletely expressed type 1 programmed cell death.Cells cultured with MK886 exhibited an unusual cytoplasmic mode of cell death characterized by vacuolization and widely separated smooth internal membranes without diagnostic nuclear changes.The incomplete destruction of chromatin and absence of a fully expressed type 1 response in SC41661A-treated Panc-1 cells implies a defective initiation and/or implementation of the enzymic program for cellular suicide by these agents.The response of Panc-1 cells to MK886 suggests the expression of a variant cytoplasmic form of non-necrotic cell death.In a European clin. trial, gamma linolenic acid, a polyunsaturated fatty acid that generates free radicals, has been combined with 5-fluorouracil as chemotherapy for pancreatic cancer.Panc-1 cell proliferation was insensitive to inhibition by several chemotherapeutic agents employed clin., including 5-fluorouracil, cisplatin or gemcitabine and only somewhat sensitive to gamma linolenic acid.When gamma linolenic acid was combined with MK886, the more effective of the two 5-lipoxygenase inhibitors, a synergistic reduction in Panc-1 cell number and viability occurred.

Products of the arachidonic acid-metabolizing enzyme, 5-lipoxygenase, stimulate the growth of several cell types. Selective inhibitors of the enzyme, including SC41661A and MK886, reduce PC-3 prostate cell proliferation. With continued culture, cells die, but the mode of death, necrotic or nonnecrotic, has not been established.

Flow cytometry, laddering after agarose electrophoresis of DNA from inhibitor-treated cells, and light and electron microscopy were employed to examine the type of death in PC-3 prostate cells cultured with either 5-lipoxygenase inhibitor.

The inhibitors induced nonnecrotic, programmed cell death. SC41661A-treated cells exhibited "foamy," vacuolated cytoplasm and mitochondria with disrupted cristae and limiting membranes, while some cells contained numerous polysomes and extended hypertrophic Golgi and secretory cisternal networks. A proportion of the treated cells detached and the nuclei of these cells were characteristic of type 1 "apoptotic" programmed cell death. MK886, a 5-lipoxygenase-inhibitor with a different mechanism of action, induced nonnecrotic changes largely confined to the cytoplasm, most consistent with type 2 "autophagic" programmed cell death. In preliminary studies of mechanism, we demonstrated that PC-3 cells express mRNA for 5-lipoxygenase and for 5-lipoxygenase-activating protein. The less active inhibitor, SC45662 neither reduced proliferation nor induced DNA laddering. The antioxidant, N-acetyl-l-cysteine but not butylated hydroxy toluene or alpha tocopherol, partially reduced the inhibition of proliferation from SC41661A.

SC41661A and MK886 inhibit PC-3 cell proliferation and induce a form of type 1 or type 2 programmed cell death, respectively. PC-3 cells contain messenger RNA for 5-lipoxygenase and 5-lipoxygenase-activating proteins. Drug-induced changes included altered redox potential, inferred from the increased survival due to the antioxidant and glutathione precursor, N-acetyl-l-cysteine. PC-3 cells are an appropriate model for studying the mechanism responsible for 5-lipoxygenase inhibitor-induced cellular suicide.