Cathepsin G-Induced Cell Aggregation of Breast Cancer MCF-7 Decreases Doxorubicin Sensitivity in a Hypoxia-Inducible Factor-Independent Mechanism.

Solid tumors habitually harbor regions with insufficient oxygen away from vasculature. Hypoxia is an important factor that confers malignant phenotypes like chemoresistance to tumor cells. We have demonstrated that cathepsin G (CG) stimulates cell aggregation in breast cancer MCF-7 cells by activating insulin-like growth factor-1 signaling. We investigated whether cancer cell aggregates induced by CG acquire hypoxia-dependent chemoresistance. Pimonidazole staining and hypoxia-inducible factor (HIF)-1α and -2α expression indicated that the core of the cell aggregates was hypoxic. Electrophoretic mobility shift and reporter assays showed that the CG-induced cell aggregates displayed transcriptional activity through HIF-responsive elements. Moreover, HIF target genes PGK1 and SLC2A1 demonstrated upregulated expression in CG-induced cell aggregates, indicating that the aggregates expressed functional HIF. Doxorubicin (DXR)-induced cytotoxicity was significantly lower in the cell aggregates induced by CG compared with monolayer cells under normoxia. Unexpectedly, the upregulation of P-glycoprotein expression, which is reported to be a HIF-target gene, and decreasing intracellular accumulation of DXR was not detected in the cell aggregates as opposed to in monolayer cells under normoxia. Additionally, reduction of DXR sensitivity in the aggregates was not suppressed by treatment with the HIF inhibitor, YC-1 and HIF-1α small interfering RNA (siRNA). Therefore, we conclude that cell aggregation induced by CG decreases DXR sensitivity via a HIF-independent mechanism.

Cathepsin G-induced malignant progression of MCF-7 cells involves suppression of PAF signaling through induced expression of PAFAH1B2.

Breast cancer is primarily classified into ductal and lobular types, as well as into noninvasive and invasive cancer. Invasive cancer involves lymphatic and hematogenous metastasis. In breast cancer patients with distant metastases, a neutrophil-derived serine protease; cathepsin G (Cat G), is highly expressed in breast cancer cells. Cat G induces cell migration and multicellular aggregation of MCF-7 human breast cancer cells; however, the mechanism is not clear. Recently, platelet-activating factor (PAF)-acetylhydrolase (PAF-AH), the enzyme responsible for PAF degradation, was reported to be overexpressed in some tumor types, including pancreatic and breast cancers. In this study, we investigated whether PAF-AH is involved in Cat G-induced aggregation and migration of MCF-7 cells. We first showed that Cat G increased PAF-AH activity and elevated PAFAH1B2 expression in MCF-7 cells. The elevated expression of PAFAH1B2 was also observed in human breast cancer tissue specimens by immunohistochemical analysis. Furthermore, knockdown of PAFAH1B2 in MCF-7 cells suppressed the cell migration and aggregation induced by low concentrations, but not high concentrations, of Cat G. Carbamoyl PAF (cPAF), a nonhydrolyzable PAF analog, completely suppressed Cat G-induced migration of MCF-7 cells. In addition, PAF receptor (PAFR) inhibition induced cell migration of MCF-7 cells even in the absence of Cat G, suggesting that Cat G suppresses the activation of PAFR through enhanced PAF degradation due to elevated expression of PAFAH1B2 and thereby induces malignant phenotypes in MCF-7 cells. Our findings may lead to a novel therapeutic modality for treating breast cancer by modulating the activity of Cat G/PAF signaling.

[Unintended Observations Leading to Macrophage Growth and Neutrophil Factor Research].

My research area in the pharmaceutical industry is innate immunity, especially in phagocytic cells. First, I studied the heat-stable growth factor of peripheral macrophages in tumorous ascitic fluid and found that lipoproteins are an influencing factor. Later, my colleagues and I found that lipid-containing substances, namely, oxidized low-density lipoprotein, dead neutrophils, or purified lipids that could be scavenged by macrophages, induce their growth. From the series of this study, I concluded that phagocytic substances induce macrophage growth by autocrine stimulation of granulocyte-macrophage colony-stimulating factor (GM-CSF). During the study, we found that neutrophils have growth-inhibitory effects against a variety of cells. Then, I elucidated that the primary factor is a zinc-binding protein, calprotectin, an abundant protein complex in the neutrophil cytosol. I found that calprotectin induces apoptosis in many cell types, including tumor cells and normal fibroblasts, and that the zinc-binding capacity is essential for its activity. Microscopic observations revealed that neutrophil extract contains factor-inducing three-dimensional cell aggregation of human mammary carcinoma, MCF-7. I elucidated that cathepsin G is responsible for this activity and that its effect is dependent on the activation of insulin-like growth factor-1. I believe that this modest, albeit novel, observation was crucial to my thirty-nine-year-long career researching phagocytic cells.

Effect of antipsychotics on breast tumors by analysis of the Japanese Adverse Drug Event Report database and cell-based experiments.

BACKGROUND: Since antipsychotics induce hyperprolactinemia via the dopamine D2 receptor, long-term administration may be a risk factor for developing breast tumors, including breast cancer. On the other hand, some antipsychotic drugs have been reported to suppress the growth of breast cancer cells in vitro. Thus, it is not clear whether the use of antipsychotics actually increases the risk of developing or exacerbating breast tumors. The purpose of this study was to clarify the effects of antipsychotic drugs on the onset and progression of breast tumors by analyzing an adverse event spontaneous reporting database and evaluating the proliferation ability of breast cancer cells. METHODS: Japanese Adverse Drug Event Report database (JADER) reports from April 2004 to April 2019 were obtained from the Pharmaceuticals and Medical Devices Agency (PMDA) website. Reports of females only were analyzed. Adverse events included in the analysis were hyperprolactinemia and 60 breast tumor-related preferred terms. The reporting odds ratio (ROR), proportional reporting ratio (PRR), and information component (IC) were used to detect signals. Furthermore, MCF-7 cells were treated with haloperidol, risperidone, paliperidone, sulpiride, olanzapine and blonanserin, and cell proliferation was evaluated by WST-8 assay. RESULTS: In the JADER analysis, the IC signals of hyperprolactinemia were detected with sulpiride (IC, 3.73; 95% CI: 1.81-5.65), risperidone (IC, 3.69; 95% CI: 1.71-5.61), and paliperidone (IC, 4.54; 95% CI: 2.96-6.12). However, the IC signal of breast tumors was not observed with any antipsychotics. In cell-based experiments, MCF-7 cells were treated with six antipsychotics at concentrations of 2 and 32 µM, and none of the drugs showed any growth-promoting effects on MCF-7 cells. On the other hand, blonanserin markedly suppressed the growth of MCF-7 cells at a concentration of 32 µM, and the effect was concentration dependent. CONCLUSIONS: Analysis of the JADER using the IC did not show breast tumor signals due to antipsychotic drugs. In in vitro experiments, antipsychotics did not promote MCF-7 cell proliferation whereas blonanserin suppressed MCF-7 cell growth. Further research on the effects of blonanserin on the onset and progression of breast tumor is expected.

Cathepsin G-Induced Insulin-Like Growth Factor (IGF) Elevation in MCF-7 Medium Is Caused by Proteolysis of IGF Binding Protein (IGFBP)-2 but Not of IGF-1.

Cathepsin G (CG), a neutrophil serine protease, induces cell migration and multicellular aggregation of human breast cancer MCF-7 cells. It has been suggested that tumor cell aggregates are associated with tumor embolism, thus CG-induced cell aggregation may promote tumor metastasis. We have revealed that cell aggregation is caused by elevated free insulin-like growth factor (IGF)-1 in the medium, followed by activation of IGF-1 receptor (IGF-1R). However, the molecular mechanism underlying IGF-1 elevation induced by CG remains unclear. Here, we aimed to elucidate the mechanism by examining the degradative effects of CG on IGF-1, and the IGF binding proteins (IGFBPs), which interfere with the binding of IGF-1 to its receptor. CG specifically evoked MCF-7 cell aggregation at less than 1 nM in a dose-dependent manner, however, neutrophil elastase (NE), chymotrypsin, and trypsin did not. Free IGF-1 concentration was continuously elevated in the medium of cells treated with CG, whereas treatments with other serine proteases resulted in only a transient or slight increase. IGFBP-2, the predominant IGFBP in MCF-7 cells, was gradually digested by CG. CG did not cleave IGF-1 for at least 48 h, whereas other proteases completely digested it. Moreover, CG induced continuous phosphorylation of IGF-1R and Akt, whereas NE-induced phosphorylation was transient, possibly due to insulin receptor substrate (IRS)-1 digestion. These results indicated that CG-specific IGF-1 elevation in the medium is caused by digestion of IGFBP-2, not IGF-1. Hence, this study clarifies the molecular mechanism of CG-specific cell aggregation.

Sensitive Analysis of Sialic Acid and Related Compound by Hydrophilic Interaction Liquid Chromatography Using Fluorescence Detection after Derivatization with DBD-PZ.

N-Acetylneuraminic acid (NANA) has been reported to react with hydrogen peroxide in vitro to produce 4-(acetylamino)-2,4-dideoxy-D-glycero-D-galacto-octonic acid (ADOA). We labeled NANA and ADOA with 4-(N,N-dimethylaminosulfonyl)-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ) for simultaneous detection. The derivatized NANA and ADOA were separated using hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection. The calibration curves of DBD-PZ-derivatized NANA and ADOA showed good linearity in the range of 221 fmol to 1.5 nmol, and 44 fmol to 1.5 nmol, respectively. This analytical method has high specificity and is useful for the detection of NANA and ADOA in saliva and serum.

Insulin-like growth factor-1 signaling is responsible for cathepsin G-induced aggregation of breast cancer MCF-7 cells.

Cathepsin G (CG), a neutrophil serine protease, induces cell migration and multicellular aggregation of human breast cancer MCF-7 cells in a process that is dependent on E-cadherin and CG enzymatic activity. While these tumor cell aggregates can cause tumor emboli that could represent intravascular growth and extravasation into the surrounding tissues, resulting in metastasis, the molecular mechanism underlying this process remains poorly characterized. In this study, we aimed to identify the signaling pathway that is triggered during CG-mediated stimulation of cell aggregation. Screening of a library of compounds containing approximately 90 molecular-targeting drugs revealed that this process was suppressed by the insulin-like growth factor-1 (IGF-1) receptor (IGF-1R)-specific kinase inhibitor OSI-906, as well as the multikinase inhibitors axitinib and sunitinib. Antibody array analysis, which is capable of detecting tyrosine phosphorylation of 49 distinct receptor tyrosine kinases, and the results of immunoprecipitation studies indicated that IGF-1R is phosphorylated in response to CG treatment. Notably, IGF-1R neutralization via treatment with a specific antibody or silencing of IGF-1R expression through siRNA transfection suppressed cell aggregation. Furthermore, CG treatment of MCF-7 cells resulted in increased release of IGF-1 into the medium for 24 h, while antibody-mediated IGF-1 neutralization partially prevented CG-induced cell aggregation. These results demonstrate that autocrine IGF-1 signaling is partly responsible for the cell aggregation induced by CG.

Inhibitory effects of several saturated fatty acids and their related fatty alcohols on the growth of Candida albicans.

We examined the effect of 5 saturated fatty acids and their related alcohols on the growth of Candida albicans. The inhibitory effects of these compounds against the yeast and hyphal growth forms of C. albicans were examined using the modified NCCLS method and crystal violet staining, respectively. Among these compounds, capric acid inhibited both types of growth at the lowest concentration. The IC(80), i.e., the concentration at which the compounds reduced the growth of C. albicans by 80% in comparison with the growth of control cells, of capric acid for the hyphal growth of this fungus, which is indispensable for its mucosal invasion, was 16.7 µM. These fatty acids, including capric acid, have an unpleasant smell, which may limit their therapeutic use. To test them at reduced concentrations, the combined effect of these fatty acids and oligonol, a depolymerized polyphenol, was evaluated in vitro. These combinations showed potent synergistic inhibition of hyphal growth [fractional inhibitory concentration (FIC) index = 0.319]. Our results demonstrated that capric acid combined with oligonol could be used as an effective anti-Candida compound. It may be a candidate prophylactic or therapeutic tool against mucosal Candida infection.

Neutrophil cathepsin G, but not elastase, induces aggregation of MCF-7 mammary carcinoma cells by a protease activity-dependent cell-oriented mechanism.

We previously found that a neutrophil serine protease, cathepsin G, weakens adherence to culture substrates and induces E-cadherin-dependent aggregation of MCF-7 human breast cancer cells through its protease activity. In this study, we examined whether aggregation is caused by degradation of adhesion molecules on the culture substrates or through an unidentified mechanism. We compared the effect of treatment with cathepsin G and other proteases, including neutrophil elastase against fibronectin- (FN-) coated substrates. Cathepsin G and elastase potently degraded FN on the substrates and induced aggregation of MCF-7 cells that had been subsequently seeded onto the substrate. However, substrate-bound cathepsin G and elastase may have caused cell aggregation. After inhibiting the proteases on the culture substrates using the irreversible inhibitor phenylmethylsulfonyl fluoride (PMSF), we examined whether aggregation of MCF-7 cells was suppressed. PMSF attenuated cell aggregation on cathepsin G-treated substrates, but the effect was weak in cells pretreated with high concentrations of cathepsin G. In contrast, PMSF did not suppress cell aggregation on elastase-treated FN. Moreover, cathepsin G, but not elastase, induced aggregation on poly-L-lysine substrates which are not decomposed by these enzymes, and the action of cathepsin G was nearly completely attenuated by PMSF. These results suggest that cathepsin G induces MCF-7 aggregation through a cell-oriented mechanism.

Development of a fluorescence analysis method for N-acetylneuraminic acid and its oxidized product ADOA.

N-acetylneuraminic acid (NANA) consumes toxic hydrogen peroxide (H2O2) under physiological conditions and is oxidized by an equimolar amount of H2O2 to yield its decarboxylated product 4-(acetylamino)-2,4-dideoxy-d-glycero-d-galacto-octonic acid (ADOA). Highly sensitive analytical methods are required to detect ADOA in the human body. We labeled NANA and ADOA with 4-(N,N-dimethylaminosulfonyl)-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DBD-ED) to enable their fluorometric detection, and developed a method using HPLC with fluorometric detection (HPLC-FD) for the simultaneous determination of the derivatized NANA and ADOA. The derivatized NANA and ADOA were separated by a hydrophilic interaction liquid chromatography (HILIC) column using an H2O/CH3CN/HCOOH (10/90/0.35) mobile phase. Fluorescence was monitored at excitation and emission wavelengths of 450nm and 560nm, respectively. Both intra- and inter-day (n=6) repeat determinations of the DBD-ED-derivatized NANA and ADOA gave relative standard deviations of less than 5%. The calibration curves for standard solutions of DBD-ED-derivatized NANA and ADOA were linear over the ranges from 576fmol to 2.0nmol and 556fmol to 2.0nmol, respectively. The method developed was highly specific and sensitive for NANA and ADOA. The presence of ADOA in biological samples was revealed for the first time using this method.

Cathepsin G induces cell aggregation of human breast cancer MCF-7 cells via a 2-step mechanism: catalytic site-independent binding to the cell surface and enzymatic activity-dependent induction of the cell aggregation.

Neutrophils often invade various tumor tissues and affect tumor progression and metastasis. Cathepsin G (CG) is a serine protease secreted from activated neutrophils. Previously, we have shown that CG induces the formation of E-cadherin-mediated multicellular spheroids of human breast cancer MCF-7 cells; however, the molecular mechanisms involved in this process are unknown. In this study, we investigated whether CG required its enzymatic activity to induce MCF-7 cell aggregation. The cell aggregation-inducing activity of CG was inhibited by pretreatment of CG with the serine protease inhibitors chymostatin and phenylmethylsulfonyl fluoride. In addition, an enzymatically inactive S195G (chymotrypsinogen numbering) CG did not induce cell aggregation. Furthermore, CG specifically bound to the cell surface of MCF-7 cells via a catalytic site-independent mechanism because the binding was not affected by pretreatment of CG with serine protease inhibitors, and cell surface binding was also detected with S195G CG. Therefore, we propose that the CG-induced aggregation of MCF-7 cells occurs via a 2-step process, in which CG binds to the cell surface, independently of its catalytic site, and then induces cell aggregation, which is dependent on its enzymatic activity.

Anti-oxidant effects of kiwi fruit in vitro and in vivo.

We previously reported that kiwi fruit is rich in polyphenols and has immunostimulatory activity. Polyphenols are widely known for having anti-oxidant effects. We also revealed potential anti-oxidant effects of kiwi fruit in vivo by oral administration to mice. Here, we compared the anti-oxidant effects of kiwi fruit with those of other fruits in vitro. Then, we examined the inhibitory effects of kiwi fruit on oxidation in the human body. There are two varieties of kiwi fruit, green kiwi and gold kiwi. We also examined variation between these varieties. Comparison of the anti-oxidant effects in vitro demonstrated that kiwi fruit had stronger anti-oxidant effects than orange and grapefruit, which are rich in vitamin C; gold kiwi had the strongest anti-oxidant effects. Kiwi fruit inhibited oxidation of biological substances in the human body. In particular, kiwi fruit may inhibit early lipid oxidation. In this study, kiwi fruit had strong anti-oxidant effects and may prevent the development and deterioration of diseases caused by oxidative stress.

Cathepsin G, a neutrophil protease, induces compact cell-cell adhesion in MCF-7 human breast cancer cells.

Cathepsin G is a serine protease secreted by activated neutrophils that play a role in the inflammatory response. Because neutrophils are known to be invading leukocytes in various tumors, their products may influence the characteristics of tumor cells such as the growth state, motility, and the adhesiveness between cells or the extracellular matrix. Here, we demonstrate that cathepsin G induces cell-cell adhesion of MCF-7 human breast cancer cells resulting from the contact inhibition of cell movement on fibronectin but not on type IV collagen. Cathepsin G subsequently induced cell condensation, a very compact cell colony, resulting due to the increased strength of E-cadherin-mediated cell-cell adhesion. Cathepsin G action is protease activity-dependent and was inhibited by the presence of serine protease inhibitors. Cathepsin G promotes E-cadherin/catenin complex formation and Rap1 activation in MCF-7 cells, which reportedly regulates E-cadherin-based cell-cell junctions. Cathepsin G also promotes E-cadherin/protein kinase D1 (PKD1) complex formation, and Go6976, the selective PKD1 inhibitor, suppressed the cathepsin G-induced cell condensation. Our findings provide the first evidence that cathepsin G regulates E-cadherin function, suggesting that cathepsin G has a novel modulatory role against tumor cell-cell adhesion.

Steroidal glycosides from Agave utahensis and their cytotoxic activity.

Eight new spirostanol saponins (1-8) and three new furostanol saponins (9-11) were isolated from the whole plants of Agave utahensis. The structures of 1-11 were determined by analysis of extensive spectroscopic data. The saponins were evaluated for their cytotoxic activity against HL-60 human promyelocytic leukemia cells. Compound 1 showed cytotoxicity against HL-60 cells with an IC(50) value of 4.9 microg/mL, induced apoptosis in HL-60 cells, and markedly activated caspase-3.

The S100A8/A9 heterodimer amplifies proinflammatory cytokine production by macrophages via activation of nuclear factor kappa B and p38 mitogen-activated protein kinase in rheumatoid arthritis.

S100A8 and S100A9, two Ca2+-binding proteins of the S100 family, are secreted as a heterodimeric complex (S100A8/A9) from neutrophils and monocytes/macrophages. Serum and synovial fluid levels of S100A8, S100A9, and S100A8/A9 were all higher in patients with rheumatoid arthritis (RA) than in patients with osteoarthritis (OA), with the S100A8/A9 heterodimer being prevalent. By two-color immunofluorescence labeling, S100A8/A9 antigens were found to be expressed mainly by infiltrating CD68+ macrophages in RA synovial tissue (ST). Isolated ST cells from patients with RA spontaneously released larger amounts of S100A8/A9 protein than did the cells from patients with OA. S100A8/A9 complexes, as well as S100A9 homodimers, stimulated the production of proinflammatory cytokines, such as tumor necrosis factor alpha, by purified monocytes and in vitro-differentiated macrophages. S100A8/A9-mediated cytokine production was suppressed significantly by p38 mitogen-activated protein kinase (MAPK) inhibitors and almost completely by nuclear factor kappa B (NF-kappaB) inhibitors. NF-kappaB activation was induced in S100A8/A9-stimulated monocytes, but this activity was not inhibited by p38 MAPK inhibitors. These results indicate that the S100A8/A9 heterodimer, secreted extracellularly from activated tissue macrophages, may amplify proinflammatory cytokine responses through activation of NF-kappaB and p38 MAPK pathways in RA.

Regulation of S100A8/A9 (calprotectin) binding to tumor cells by zinc ion and its implication for apoptosis-inducing activity.

S100A8/A9 (calprotectin), which is released by neutrophils under inflammatory conditions, has the capacity to induce apoptosis in various cells. We previously reported that S100A8/A9 induces apoptosis of EL-4 lymphoma cells via the uptake of extracellular zinc in a manner similar to DTPA, a membrane-impermeable zinc chelator. In this study, S100A8/A9-induced apoptosis was examined in several cell lines that are weakly sensitive to DTPA, suggesting S100A8/A9 is directly responsible for apoptosis in these cells. Since zinc inhibits apoptosis of MM46, one of these cells, the regulation by zinc of the capacity of S100A8/A9 to bind MM46 cells was studied. When MM46 cells were incubated with S100A8/A9 in standard or zinc-depleted medium, the amounts of S100A8/A9 bound to cells was markedly lower at 3 h than at 1 h. In contrast, when MM46 cells were incubated with S100A8/A9 in the presence of high levels of zinc, binding to cells was the same at 1 and 3 h. When the cells were permeabilized with saponin prior to analysis, a larger amount of cell-associated S100A8/A9 was detected at 3 h. The amount was further increased in cells treated with chloroquine, suggesting that S100A8/A9 was internalized and degraded in lysosomes. Although it has been reported that S100A8/A9 binds to heparan sulfate on cell membranes, the amount of S100A8/A9 bound to MM46 cells was not reduced by heparinase treatment, but was reduced by trypsin treatment. These results suggest that S100A8/A9 induces apoptosis by direct binding to MM46 cells, and that this activity is regulated by zinc.

Induction of multicellular 3-D spheroids of MCF-7 breast carcinoma cells by neutrophil-derived cathepsin G and elastase.

In tumor metastasis, multicellular aggregates of tumor cells form and disseminate into the blood or lymph vessels from the tumor mass, following the formation of tumor cell emboli in distant vessels. However, the mechanism by which aggregates form in the tumor mass is unknown. Neutrophils often exist in tumors and are considered to affect tumor development. We observed that neutrophils had the capacity to induce the aggregation of MCF-7 human breast carcinoma cells adhering to culture substrates. When MCF-7 cells were cultured with rat inflammatory neutrophils, the soluble fraction of their lysate, and the conditioned medium of neutrophils stimulated with N-formyl-Met-Leu-Phe plus cytochalasin B, multicellular aggregates formed within 16 h, and tightly aggregated 3-D spheroids formed when the cultures were prolonged. The spheroid-inducing reaction was reversible and energy-dependent. The MCF-7 cells induced to aggregate by the neutrophil extract showed growth potential, although the growth rate of the cells was slightly reduced. The aggregation was dependent on E-cadherin, because the spheroids dispersed into isolated cells on incubation with EGTA or anti-E-cadherin antibody following pipetting. The aggregation-inducing activity in neutrophils was completely inhibited by soybean trypsin-chymotrypsin inhibitor. Moreover, the commercially available human neutrophil elastase and cathepsin G induced the aggregation of MCF-7 cells and formation of spheroids. The proteases secreted by infiltrated neutrophils in tumors are implicated in the dissemination of tumor aggregates from primary tumor sites.

Characterization of the growth-inhibitory and apoptosis-inducing activities of a triterpene saponin, securioside B against BAC1.2F5 macrophages.

BACKGROUND: Since the growth state of macrophages in local pathological sites is considered a factor that regulates the processes of many disease, such as tumors, inflammation, and atherosclerosis, the substances that regulate macrophage growth or survival may be useful for disease control. We previously reported that securiosides A and B, novel triterpene saponins, exerted macrophage-oriented cytotoxicity in the presence of a L-cell-conditioned medium containing macrophage colony-stimulating factor (M-CSF), while the compounds did not exhibit an effect on macrophages in the absence of the growth-stimulating factors. AIM: This study was undertaken to characterize the growth-inhibitory and the apoptosis-inducing activities of securioside B, focusing on the effects of the macrophage-growth factor(s), and to examine the implication of a mitochondria pathway in apoptosis induction. METHODS: The effect of securioside B on a murine macrophage cell line (BAC1.2F5) was examined by MTT assay and lactose dehydrogenase release assay in the presence of L-cell-conditioned medium, M-CSF, or granulocyte-macrophage CSF (GM-CSF). RESULT: Securioside B inhibited the growth of the cells stimulated by recombinant M-CSF or GM-CSF, but it scarcely induced cytolysis of the cells under the same conditions. Moreover, securioside B did not induce cell death when the compound only was added to the cells. On the other hand, the compound extensively induced apoptotic cell death in the presence of L-cell-conditioned medium, suggesting that apoptosis induction by securioside B requires the additional factor(s) present in L-cell-conditioned medium. Securioside B plus L-cell-conditioned medium induced the activation of caspase-3 and caspase-9, but not caspase-8. In addition, cytochrome c release from the mitochondria into the cytosol, and disrupted mitochondria membrane potential, was also observed in the apoptotic BAC1.2F5 cells. CONCLUSION: These data suggest that securioside B has growth-inhibitory activity against growth factor-stimulated macrophages, and that it induces apoptotic macrophage death through the activation of a mitochondrial pathway in the presence of L-cell-conditioned medium.

Calprotectin (S100A8/S100A9), an inflammatory protein complex from neutrophils with a broad apoptosis-inducing activity.

Calprotectin, a complex of two calcium-binding proteins that belong to the S100 protein family, is abundant in the cytosolic fraction of neutrophils. A high level of calprotectin reportedly exists in extracellular fluid during various inflammatory conditions, such as rheumatoid arthritis, cystic fibrosis and abscesses. However, the exact biological role(s) of the factor is now under investigation. We recently observed that neutrophils contain a factor that shows growth-inhibitory and apoptosis-inducing activities against various cell types including tumor cells and normal fibroblasts, and we identified that factor as calprotectin. The findings suggest that calprotectin exerts a regulatory activity in inflammatory processes through its effect on the survival or growth states of cells participating in the inflammatory reaction. It is also possible that calprotectin, at a high concentration, might have a deleterious effect on fibroblasts and influence the recovery of inflammatory tissue. Therefore, the protein factor may be a new drug target to control inflammatory reactions. We found that a few of the Amaryllidaceae alkaloids effectively inhibited the growth-inhibitory and apoptosis-inducing activities of calprotectin. In this article, we focus on the biological functions of calprotectin in extracellular fluids, focusing on its apoptosis-inducing activity.

Enterolosaponins A and B, novel triterpene bisdesmosides from Enterolobium contortisiliquum, and evaluation for their macrophage-oriented cytotoxic activity.

Two novel triterpene bisdesmosides, designated as enterolosaponin A (1) and B (2), were isolated from Enterolobium contortisiliquum. The chemical structures of 1 and 2 were determined by analysis of their extensive spectroscopic data, as well as hydrolysis followed by chromatographic study. Enterolosaponins have a 2-amino-2-deoxy-D-glucosyl unit (D-glucosamine) as one of the monosaccharides constituting their oligosaccharide moieties, which have been rarely found in natural product research. Enterolosaponin A (1) exhibited a highly selective cytotoxicity against BAC1.2F5 mouse macrophages, and it should be notable that the macrophage death caused by 1 was shown to be neither necrotic nor due to induction of apoptosis from morphology of the died cells, whose cytosol occurred in vacuolation.