Favine/CCDC3 deficiency accelerated atherosclerosis and thrombus formation is associated with decreased MEF2C-KLF2 pathway.

Currently, no mouse models manifest calcification and thrombus formation, which is frequently associated with human atherosclerosis. We demonstrated that lack of Favine/CCDC3 in apoE knockout mice accelerated atherosclerosis accompanied by large cholesterol crystals and calcification, and also promoted thrombus formation in the left ventricle and arteries. Circulating Favine was detectable in WT mouse plasma. RNA-sequencing analysis of aortae in DKO mice showed similar gene expression patterns of human atherosclerosis with unstable and vulnerable plaques. Importantly, human FAVINE mRNA expressions were lower in atheroma plaque than in adjacent intact aortic tissue and decreased with the progression of atherosclerosis. Pathway analysis of aortae in DKO mice suggested the decrease of the MEF2C-KLF2-mediated transcriptional pathway. Favine insufficiency and its attenuated downstream pathways may increase atherosclerosis progression with calcification and thrombus, which have not previously been fully modeled in experimental animals. Favine and its downstream pathways may have therapeutic potential for atherosclerosis.

Molecular evidence of IGFBP-3 dependent and independent VD3 action and its nonlinear response on IGFBP-3 induction in prostate cancer cells.

BACKGROUND: Clinical trials have been conducted to clarify the beneficial effects of VD3 (1α,25-dihydroxy vitamin D3, also known as calcitriol) treatment in prostate cancer. However, the molecular mechanisms underlying these effects are not fully understood. Recent studies on IGFBP-3 have indicated its intracellular functions in cell growth and apoptosis. The aim of this study was to confirm the benefits of low-dose VD3 treatment and clarify the molecular mechanisms underlying these beneficial effects in prostate cancer cells. METHODS: The molecular effects of simultaneous treatment of LNCaP cells and their genetically modified cell lines with low concentration of docetaxel and VD3 were biologically and biochemically analyzed. To further determine the effects of VD3 treatment on IGFBP-3 induction system, cells were temporarily treated with VD3 in combination with a transcriptional inhibitor or protein synthesis inhibitor. Bcl-2 protein and its mRNA behavior were also observed in Igfbp-3 expression-modified LNCaP cells to determine the involvement of IGFBP-3 in the suppression of Bcl-2 by VD3 treatment. RESULTS: Changes in IGFBP-3 expression levels in LNCaP cells indicated that it mediated the inhibition of cell growth induced by VD3 treatment. IGFBP-3 was also found to be a mediator of the enhanced cytotoxicity of prostate cancer cells to VD3 in combination with the anti-cancer drug. We further identified the distinct property of the IGFBP-3 induction system, wherein temporal VD3 stimulation-induced prolonged IGFBP-3 expression and VD3 treatment-induced increase in IGFBP-3 expression were optimized based on the protein concentration rather than the mRNA concentration. Meanwhile, Bcl-2 expression was down-regulated by VD3 treatment in an IGFBP-3-independent manner. CONCLUSION: These findings indicate the molecular mechanisms of IGFBP-3 induction stimulated by VD3 and IGFBP-3 independent Bcl-2 suppression by VD3 treatment in prostate cancer cells. The results could prompt a re-evaluation of VD3 usage in therapy for patients with prostate cancer.

Promotion of malignant phenotype after disruption of the three-dimensional structure of cultured spheroids from colorectal cancer.

Individual and small clusters of cancer cells may detach from the edges of a main tumor and invade vessels, which can act as the origin of metastasis; however, the mechanism for this phenomenon is not well understood. Using cancer tissue-originated spheroids, we studied whether disturbing the 3D architecture of cancer spheroids can provoke the reformation process and progression of malignancy. We developed a mechanical disruption method to achieve homogenous disruption of the spheroids while maintaining cell-cell contact. After the disruption, 9 spheroid lines from 9 patient samples reformed within a few hours, and 3 of the 9 lines exhibited accelerated spheroid growth. Marker expression, spheroid forming capacity, and tumorigenesis indicated that stemness increased after spheroid disruption. In addition, the spheroid forming capacity increased in 6 of 11 spheroid lines. The disruption signature determined by gene expression profiling supported the incidence of remodeling and predicted the prognosis of patients with colorectal cancer. Furthermore, WNT and HER3 signaling were increased in the reformed spheroids, and suppression of these signaling pathways attenuated the increased proliferation and stemness after the disruption. Overall, the disruption and subsequent reformation of cancer spheroids promoted malignancy-related phenotypes through the activation of the WNT and ERBB pathways.

Development of effective tumor immunotherapy using a novel dendritic cell-targeting Toll-like receptor ligand.

Although dendritic cell (DC)-based immunotherapy shows little toxicity, improvements should be necessary to obtain satisfactory clinical outcome. Using interferon-gamma injection along with DCs, we previously obtained significant clinical responses against small or early stage malignant tumors in dogs. However, improvement was necessary to be effective to largely developed or metastatic tumors. To obtain effective methods applicable to those tumors, we herein used a DC-targeting Toll-like receptor ligand, h11c, and examined the therapeutic effects in murine subcutaneous and visceral tumor models and also in the clinical treatment of canine cancers. In murine experiments, most and significant inhibition of tumor growth and extended survival was observed in the group treated with the combination of h11c-activated DCs in combination with interferon-gamma and a cyclooxygenase2 inhibitor. Both monocytic and granulocytic myeloid-derived suppressor cells were significantly reduced by the combined treatment. Following the successful results in mice, the combined treatment was examined against canine cancers, which spontaneously generated like as those in human. The combined treatment elicited significant clinical responses against a nonepithelial malignant tumor and a malignant fibrous histiocytoma. The treatment was also successful against a bone-metastasis of squamous cell carcinoma. In the successful cases, the marked increase of tumor-responding T cells and decrease of myeloid-derived suppressor cells and regulatory T cells was observed in their peripheral blood. Although the combined treatment permitted the growth of lung cancer of renal carcinoma-metastasis, the marked elevated and long-term maintaining of the tumor-responding T cells was observed in the patient dog. Overall, the combined treatment gave rise to emphatic amelioration in DC-based cancer therapy.

Deflection of vascular endothelial growth factor action by SS18-SSX and composite vascular endothelial growth factor- and chemokine (C-X-C motif) receptor 4-targeted therapy in synovial sarcoma.

Synovial sarcoma (SS) is a malignant soft-tissue tumor characterized by the recurrent chromosomal translocation SS18-SSX. Vascular endothelial growth factor (VEGF)-targeting anti-angiogenic therapy has been approved for soft-tissue sarcoma, including SS; however, the mechanism underlying the VEGF signal for sarcomagenesis in SS is unclear. Here, we show that SS18-SSX directs the VEGF signal outcome to cellular growth from differentiation. Synovial sarcoma cells secrete large amounts of VEGF under spheroid culture conditions in autocrine fashion. SS18-SSX knockdown altered the VEGF signaling outcome, from proliferation to tubular differentiation, without affecting VEGF secretion, suggesting that VEGF signaling promoted cell growth in the presence of SS18-SSX. Thus, VEGF inhibitors blocked both host angiogenesis and spheroid growth. Simultaneous treatment with VEGF and chemokine (C-X-C motif) (CXC) ligand 12 and CXC receptor 4 inhibitors and/or ifosfamide effectively suppressed tumor growth both in vitro and in vivo. SS18-SSX directs the VEGF signal outcome from endothelial differentiation to spheroid growth, and VEGF and CXC receptor 4 are critical therapeutic targets for SS.

Development of a dendritic cell-targeting lipopeptide as an immunoadjuvant that inhibits tumor growth without inducing local inflammation.

Materials used for the past 30 years as immunoadjuvants induce suboptimal antitumor immune responses and often cause undesirable local inflammation. Some bacterial lipopeptides that act as Toll-like receptor (TLR) 2 ligands activate immune cells as immunoadjuvants and induce antitumor effects. Here, we developed a new dendritic cell (DC)-targeting lipopeptide, h11c (P2C-ATPEDNGRSFS), which uses the CD11c-binding sequence of intracellular adhesion molecule-1 to selectively and efficiently activate DCs but not other immune cells. Although the h11c lipopeptide activated DCs similarly to an artificial lipopeptide, P2C-SKKKK (P2CSK4), via TLR2 in vitro, h11c induced more effective tumor inhibition than P2CSK4 at low doses in vivo with tumor antigens. Even without tumor antigens, h11c lipopeptide significantly inhibited tumor growth and induced tumor-specific cytotoxic T cells. P2CSK4 was retained subcutaneously at the vaccination site and induced severe local inflammation in in vivo experiments. In contrast, h11c was not retained at the vaccination site and was transported into the tumor within 24 hr. The recruitment of DCs into the tumor was induced by h11c more effectively, while P2CSK4 induced the accumulation of neutrophils leading to severe inflammation at the vaccination site. Because CD11b+ cells, but not CD11c+ cells, produced neutrophil chemotactic factors such as macrophage inflammatory protein (MIP)-2 in response to stimulation with TLR2 ligands, the DC-targeting lipopeptide h11c induced less MIP-2 production by splenocytes than P2CSK4. In this study, we succeeded in developing a novel immunoadjuvant, h11c, which effectively induces antitumor activity without adverse effects such as local inflammation via the selective activation of DCs.

Spheroid culture of primary lung cancer cells with neuregulin 1/HER3 pathway activation.

INTRODUCTION: Primary culture of cancer cells is expected to be useful for investigating the biology of cancer and predicting chemosensitivity for individual patients, yet has been hampered by technical difficulties. We recently developed the cancer tissue-originated spheroid (CTOS) method for the primary culture of colorectal cancer cells. In the present study, we applied this system to the primary culture of non-small-cell lung cancer. METHODS: We used 125 surgical specimens and 18 pleural effusions for CTOS preparation. Partially digested tumor fragments were cultured in a medium for embryonic stem cells. CTOSs were subjected to sensitivity assay and signal transduction assay for the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) erlotinib. We also investigated the effects of growth factors in culturing lung cancer CTOS. RESULTS: The success rate of CTOS preparation from surgical specimens was 80.0%. The CTOS method was also suitable for culturing tumor spheroids from pleural effusions. CTOSs from lung cancer consisted mostly of pure cancer cells. CTOSs and CTOS-derived xenografts retained the characteristics of the original tumors. In vitro assay results showed that EGFR mutation status and expression levels corresponded with erlotinib sensitivity, confirming previous clinical findings. Furthermore, we found that neuregulin 1, a ligand of HER3, potently induced CTOS growth. CONCLUSIONS: The CTOS method enables us to obtain primary lung tumor cells of high viability and purity. CTOS could be a new platform for studying lung cancer biology.

Hypoxia inducible factor-1α is necessary for invasive phenotype in Vegf-deleted islet cell tumors.

In the mouse model of pancreas endocrine tumor, loss of Vegf (VKO) results in dramatically decreased tumor progression; however, the residual microscopic lesions show increased invasion into surrounding exocrine tissue. Double KO mice of Vegf and hypoxia inducible factor-1α (Hif-1α) showed increased life span and suppressed tumor growth due to increased apoptosis. The increased invasiveness of tumors in VKO mice was diminished in DKO mice to the levels of wild-type mice. Compared to VKO mice, DKO mice also exhibited smaller changes in the expression levels of adhesion molecules, including E-cadherin, N-cadherin, and NCAM. These changes of adhesion molecules were not observed in the primary culture of the tumor cells under hypoxic conditions. Thus, the invasive phenotype observed under angiogenesis inhibition requires Hif-1α, but is not directly caused by acute hypoxia.

Role of Meis1 in mitochondrial gene transcription of pancreatic cancer cells.

Pre-B-cell leukemia transcription factor (PBX)/three-amino-acid loop extension (TALE) class transcription factors [PBX1-4, Meis homeobox (Meis) 1-3, pbx/knotted 1 homeobox (Prep) 1, 2] are involved in tumorigenesis and metastasis. To investigate further the function of PBX/TALE class transcription factors, mRNA expression profile after downregulation of each mRNA expression by siRNA transfection in pancreatic cancer cell line, Panc-1, was examined. Downregulation of Meis1 resulted in downregulation of mitochondrial genes, but those of PBX1 and PBX2 did not. Quantitative reverse transcription polymerase chain reaction confirmed downregulation of mitochondrial genes by Meis1 siRNA transfection. Chromatin immunoprecipitation assay revealed the binding of Meis1 to the mitochondrial promoter region that contained the putative Meis1 binding site. Luciferase reporter assay showed the increase of luciferase activity of a construct containing the Meis1 binding site compared with that with shorter fragment without Meis1 binding region. These findings indicate that Meis1 works as a transcription factor for mitochondrial genes in pancreatic cancer cells.

Possible mechanism of growth inhibition by Scutellaria baicalensis in an estrogen-responsive mouse tumor cell line.

We have studied the effects of Saiboku-to, a traditional Chinese medicine having suppressive activities for leukotriene production and release, on the proliferation of the estrogen-responsive mouse Leydig tumor cell line B-1F. In our previous reports, it is shown that Saiboku-to promotes, but Scutellaria baicalensis, one of the components (herbs) of Saiboku-to, significantly inhibits the proliferation of B-1F cells in vitro and in vivo, and induces DNA fragmentation and morphological changes such as nuclear aggregation and fragmentation. In this study, we examined telomerase activity, cell cycle, polyunsaturated fatty acid metabolism and expression of nuclear factor κB (NF-κB) in order to determine the mechanism of growth inhibition in B-1F cells treated with Scutellaria baicalensis. Telomerase activity was decreased in a dose-dependent manner in treated B-1F cells. Cellular populations in the sub-G0/G1 and G2/M phases were increased, but those in M phase had no change. Although cyclin D1 mRNA was highly expressed in the presence of estradiol (E2), cyclin A and E mRNA levels did not significantly change. When B-1F cells were treated with Scutellaria baicalensis, expression of cyclin D1 was suppressed and that of p21 was inversely increased. Moreover, Scutellaria baicalensis influenced arachidonic and linoleic acid metabolism, and increased production of 13(S)-HODE. In the presence of E2 Scutellaria baicalensis decreased expression of NF-κB p65 to 0.71-fold in B-1F cells. These results show that Scutellaria baicalensis might induce cell cycle arrest at G1 phase and apoptosis via inhibition of telomerase activity, changes of enzymatic activities in polyunsaturated fatty acid metabolism and suppression of NF-κB.

Clinical utility of apparent diffusion coefficient (ADC) values in patients with prostate cancer: can ADC values contribute to assess the aggressiveness of prostate cancer?

PURPOSE: To retrospectively evaluate the relationship between apparent diffusion coefficient (ADC) values and Gleason score (GS) in prostate cancer. METHODS: A total of 60 patients who underwent radical prostatectomy for clinically localized prostate cancer were selected for this study. Diffusion-weighted magnetic resonance (MR) images were obtained using a 1.5 T system. ADC values were analyzed between three groups: GS of 6 or less (n = 7); GS of 7 (n = 37); and GS of 8 or higher (n = 16). ADC values of the three GS groups were statistically analyzed in order to determine the relationship with GS. In the 37 patients with GS = 7 the difference in ADC values between GS 3+4 and GS 4+3 was analyzed. RESULTS: Median ADC values (10⁻³ mm² /s) of the three GS groups were 1.04 (GS = 6 or less), 0.867 (GS = 7), and 0.729 (GS = 8 or higher). Although there was considerable overlap among the groups, the differences in ADC were statistically significant (P < 0.0001). There was a significant inverse correlation between GS and ADC values (z = -0.437, P < 0.0005). Median ADC values (10⁻³ mm² /s) of GS 3+4 and GS 4+3 patients were 0.88 and 0.814, respectively (P < 0.05). CONCLUSION: ADC values showed a negative correlation with GS. Pathologically, however, there was considerable intrasubject heterogeneity.

The pure anti-androgen bicalutamide inhibits cyclin A expression both in androgen-dependent and -independent cell lines.

We investigated the effects of testosterone and the pure anti-androgen, bicalutamide, on DNA synthesis and cell cycle in androgen-sensitive or -insensitive human and mouse cell lines by 3H-thymidine incorporation, flow cytometry, RT-PCR and Western blotting. In androgen-dependent mouse SC-3 cells, testosterone induced DNA synthesis, shift of cell cycle distribution from G0/G1 to S/G2/M and expression of cyclin A. The induction was preceded by that of fibroblast growth factor 8 (FGF-8), and completely blocked by monoclonal antibody to FGF-8. Dihydrotestosterone (DHT) induced cyclin A expression in androgen-sensitive human prostate cancer cells, but not in androgen-independent cell lines. Bicalutamide almost completely inhibited these androgen-dependent effects both in LNCaP and SC-3 cells, but had no or limited effect on androgen-independent or FGF-8-induced DNA synthesis, and FGF-8 induced cyclin A expression. Interestingly, bicalutamide inhibited both DNA synthesis and the cyclin A expression in androgen-independent human cell lines in serum-free condition. A MEK1/2 inhibitor U0126 blocked both androgen- and rFGF-8-induced DNA synthesis. Overall, bicalutamide inhibits the cyclin A expression possibly by inhibiting FGF-8 mRNA expression and FGF-8 protein secretion but not by inhibiting FGF receptor (FGFR) signalling in androgen-dependent cell lines, and by other mechanisms in androgen-independent cell lines. The results suggest that combination with compounds such as FGFR signalling inhibitors may provide additional benefits to anti-androgens. It is also suggested that cyclin A could be a sensitive marker for androgen-induced cancer growth and for the growth inhibitory effects of anti-androgen.

Proliferation of estrogen-responsive mouse tumor cell line B-1F stimulated by Saiboku-to, but inhibited by Scutellaria baicalensis, a component of Saiboku-to.

We have demonstrated that the proliferation of estrogen-responsive mouse Leydig tumor cell line B-1F is induced via suppression of 5-lipoxygenase activity followed by decrease of leukotrienes (LTs). Additionally, it has been reported that LTD4 induces apoptosis in B-1F cells. In this study, we examined effects of Saiboku-to, a traditional Chinese medicine having suppressive activities for LT production and release, on the proliferation. Saiboku-to promoted, but Scutellaria baicalensis, one of components (herbs) of Saiboku-to, significantly inhibited the proliferation of B-1F cells in vitro and in vivo. The action of Scutellaria baicalensis in B-1F cells was studied in more detail. Although Scutellaria baicalensis consists of flavonoids, iridoids, volatile oils and others, it and its major constituents had no direct effect on estrogen binding sites in B-1F cells. B-1F cells treated with Scutellaria baicalensis showed morphological changes such as nuclear aggregation and fragmentation. DNA fragmentation was also observed, indicating that Scutellaria baicalensis induces apoptosis in B-1F cells and that it or its constituents might be a good resource for searching new drugs, especially anti-cancer drugs. Moreover, Saiboku-to promoted B-1F cell proliferation, but Scutellaria baicalensis inhibited it, showing complexity of action of traditional Chinese medicines.

Adiponectin deficiency enhances colorectal carcinogenesis and liver tumor formation induced by azoxymethane in mice.

AIM: To investigate the causal relationship between hypoadiponectinemia and colorectal carcinogenesis in in vivo experimental model, and to determine the contribution of adiponectin deficiency to colorectal cancer development and proliferation. METHODS: We examined the influence of adiponectin deficiency on colorectal carcinogenesis induced by the administration of azoxymethane (AOM) (7.5 mg/kg, intraperitoneal injection once a week for 8 wk), by using adiponectin-knockout (KO) mice. RESULTS: At 53 wk after the first AOM treatment, KO mice developed larger and histologically more progressive colorectal tumors with greater frequency compared with wild-type (WT) mice, although the tumor incidence was not different between WT and KO mice. KO mice showed increased cell proliferation of colorectal tumor cells, which correlated with the expression levels of cyclooxygenase-2 (COX-2) in the colorectal tumors. In addition, KO mice showed higher incidence and frequency of liver tumors after AOM treatment. Thirteen percent of WT mice developed liver tumors, and these WT mice had only a single tumor. In contrast, 50% of KO mice developed liver tumors, and 58% of these KO mice had multiple tumors. CONCLUSION: Adiponectin deficiency enhances colorectal carcinogenesis and liver tumor formation induced by AOM in mice. This study strongly suggests that hypoadiponectinemia could be involved in the pathogenesis for colorectal cancer and liver tumor in human subjects.

Methylcobalamin inhibits fibroblast growth factor-8 stimulated proliferation and induces apoptosis in Shionogi carcinoma cells.

SC-3 cells, an androgen-dependent mouse mammary carcinoma cell line, in response to androgen stimuli, induces the secretion of fibroblast growth factor (FGF-8), which in turn increases the proliferation of these cells. We have shown previously that methylcobalamin (MeCbl) decreases the levels of FGF-8 mRNA in SC-3 cells stimulated by testosterone, inhibiting the proliferation of SC-3 cells and inducing apoptosis. In the present study, we analyzed the effects of MeCbl on SC-3 cell proliferation in response to exogenous addition of FGF-8. Thymidine incorporation showed a significant decrease in SC-3 cells cultured with MeCbl. Immunocytochemistry for single-stranded DNA (ssDNA) and DNA fragmentation analysis demonstrated that MeCbl induced apoptosis in SC-3 cells, even in the presence of FGF-8. These results show that the addition of FGF-8 stimulates the proliferation of SC-3 cells under the androgen-depleted condition and that MeCbl might be able to interfere with FGF-8 action.

Blocking CD147 induces cell death in cancer cells through impairment of glycolytic energy metabolism.

CD147 is a multifunctional transmembrane protein and promotes cancer progression. We found that the anti-human CD147 mouse monoclonal antibody MEM-M6/1 strongly induces necrosis-like cell death in LoVo, HT-29, WiDr, and SW620 colon cancer cells and A2058 melanoma cells, but not in WI-38 and TIG-113 normal fibroblasts. Silencing or overexpression of CD147 in LoVo cells enhanced or decreased the MEM-M6/1 induced cell death, respectively. CD147 is known to form complex with proton-linked monocarboxylate transporters (MCTs), which is critical for lactate transport and intracellular pH (pHi) homeostasis. In LoVo cells, CD147 and MCT-1 co-localized on the cell surface, and MEM-M6/1 inhibited the association of these molecules. MEM-M6/1 inhibited lactate uptake, lactate release, and reduced pHi. Further, the induction of acidification was parallel to the decrease of the glycolytic flux and intracellular ATP levels. These effects were not found in the normal fibroblasts. As cancer cells depend on glycolysis for their energy production, CD147 inhibition might induce cell death specific to cancer cells.

Stat4 suppresses the proliferation of connective tissue-type mast cells.

Mast cells are the progeny of hematopoietic stem cells, and murine mast cells are usually divided into two distinct populations, mucosal mast cells (MMCs) and connective tissue-type mast cells (CTMCs). We previously reported that CTMCs expressed signal transducer and activator of transcription (Stat) 4, but MMCs did not. Stat4 is also expressed in T cells and plays important roles in their homeostasis. In the present study, we show that Stat4 is involved in the homeostasis of CTMCs. The number of skin CTMCs increased in Stat4-deficient Balb/c mice, but that of gastric MMCs did not, when compared to those in control Balb/c(+/+) mice. The comparison between cultured Stat4-deficient CTMCs and cultured Balb/c(+/+) CTMCs revealed that cell cycle progression and cyclin D3 expression in the cultured Stat4-deficient CTMCs were enhanced in a Stat3 activation-dependent manner. This phenotype was explained by upregulation of KitL-induced interleukin (IL)-6 acting in an autocrine manner in cultured Stat4-deficient CTMCs. These results show that Stat4 suppresses the proliferation of CTMCs by controlling IL-6 via an autocrine mechanism.

Expression of p21Cip1/Waf1 and p27Kip1 in small cell neuroendocrine carcinoma of the uterine cervix.

Small-cell neuroendocrine carcinoma of the uterine cervix (SCCC), a rare but malignant cervical neoplasm, has a highly aggressive phenotype that requires more intensive treatment than other cervical tumors. Immunohistochemical methods were used to compare the expression of p21Cip1/Waf1 and p27Kip1 in SCCC and squamous cell carcinoma, the most common type of cervical cancer. In SCCC, p21 expression was significantly reduced compared with squamous cell carcinoma, whereas expression of p27 was similar in both carcinomas. Reduced expression of p21 could be a helpful diagnostic marker and may contribute to the invasive phenotype of SCCC.

Establishment of a progesterone-sensitive cell line from human lung cancer.

For deveplopment and function of the lung, progesterone (Prog) fulfils important roles. In a recent report, immunolocalization of Prog and estrogen receptors in non-small cell lung carcinomas were examined and it was shown that the Prog receptor might be a potent prognostic factor. In the present study, a cell line with the sensitivity to Prog was established from a human lung cancer and the growth mechanism was analyzed. The proliferation of established SN96-42 cells was sensitive to Prog and antiprogesterone RU38486 inhibited their proliferation stimulated by Prog. Exposure of these cells to Prog resulted in a decreased formation of leukotriene (LT). The 5-lipoxygenase inhibitor (5-LOX), AA861, effectively stimulated SN96-42 cell proliferation and 5-LOX-catalyzed product(s), especially LTC4, inhibited SN96-42 cell proliferation caused by Prog. Prog-sensitive enhancement of SN96-42 cell proliferation is at least partly mediated through an inhibition of LT formation and these data suggest that 5-LOX and LTs play important roles in SN96-42 cell proliferation stimulated by Prog.