Oncostatin M stimulates proliferation,induces collagen production and inhibits apoptosis of human lung fibroblasts

1. Oncostatin M (OSM), a member of the interleukin-6 (IL-6) cytokine family, acts on a variety of cells and elicits diversified biological responses, suggesting potential roles in the regulation of cell survival, differentiation and proliferation. 2. We have examined the effect of OSM on the regulation of human lung fibroblast proliferation, collagen production and spontaneous apoptosis. The proliferative effects of OSM (0.5 - 100 ng ml(-1)) were assessed using a MTS assay as well as [(3)H]-thymidine incorporation and cell counts at 24 and 48 h. Hydroxyproline was measured as an index of procollagen production by high pressure liquid chromotography (HPLC). Apoptosis was determined by annexin staining. 3. OSM enhanced the mitotic activity of lung fibroblasts in a time and dose dependent manner. Maximum proliferation of 57% above control was observed after incubation for 48 h with 2 ng ml(-1) OSM (P<0.05). 4. Incubation with the mitogen activated protein kinase (MAPK) kinase inhibitor, PD98059 or the tyrosine kinase inhibitor, genestein both significantly reduced the mitogenic effect of OSM (P<0.05). 5. In contrast, proliferation in response to OSM was not regulated by induction of cyclo-oxygenase and subsequent prostaglandin E(2) (PGE(2)) release or by IL-6. 6. OSM also stimulated fibroblasts to synthesize pro-collagen by a maximum of 35% above control levels after 48 h (P<0.05). 7. OSM significantly inhibited the spontaneous apoptosis of fibroblasts at 24 and 48 h. 8. These results provide evidence that OSM has pro-fibrotic properties and suggest that it may play a role in normal lung wound repair and fibrosis.     

Inhibition of HgCl2-induced mitogen-activated protein kinase activation by LL-Z1640-2 in CCRF-CEM cells

Exposure of HgCl2 to CCRF-CEM human lymphoblastoid cells induced phosphorylation of mitogen-activated protein kinases (MAPKs); extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38. LL-Z1640-2, a macrocyclic nonaketide, inhibited HgCl2-induced JNK phosphorylation at 5-100 ng/ml. It also inhibited phosphorylation of ERK and p38 but only at 100 ng/ml. The same doses of radicicol did not suppress MAPKs activation. LL-Z1640-2 (at 100 ng/ml) inhibited HgCl2-induced JNK phosphorylation in NIH 3T3 fibroblasts but not in LLC-PK(1) renal epithelial cells. Thus, LL-Z1640-2 is a potent inhibitor of HgCl2-induced MAPKs activation, especially that of JNK, in CCRF-CEM cells.     

Mechanisms of JP-8 jet fuel cell toxicity. II. Induction of necrosis in skin fibroblasts and keratinocytes and modulation of levels of Bcl-2 family members

JP-8 induces apoptosis in rat lung epithelial cells, primary mouse T lymphocytes, Jurkat T lymphoma cells, and U937 monocytic cells (Stoica et al., 2001). Here, we have observed a different mechanism of cytotoxicity in human keratinocytes grown in culture as well as when grafted onto nude mice. At lower levels of JP-8 (80 microg/ml; 1 x 10(-4) dilution), sufficient to induce apoptosis in other cell types, including lung epithelial cells (Stoica et al., 2001), no apoptosis was observed. At higher levels (>200 microg/ml; 2.5 x 10(-4) dilution), JP-8 is cytotoxic to both primary and immortalized human keratinocytes, as evidenced by the metabolism of calcein, as well as by morphological changes such as cell rounding and cell detachment. There was no evidence of activation of caspases-3, -7, or -8 either by enzyme activity or immunoblot analysis, and the stable expression of a dominant-negative inhibitor of apoptosis (FADD-DN) did not increase the survival of keratinocytes to JP-8. The pattern of poly(ADP-ribose) polymerase (PARP) cleavage was also characteristic of necrosis. PARP has been also been implicated in necrosis via its ability to lower levels of ATP in damaged cells. However, fibroblasts derived from PARP-/- mice underwent necrotic cell death similar to those derived from PARP+/+ mice, indicating that the effects of JP-8 are independent of PARP. Immunoblot analysis further revealed that exposure of keratinocytes to the toxic higher levels of JP-8 markedly downregulates the expression of the prosurvival members of the Bcl-2 family, Bcl-2 and Bcl-x(L), and upregulates the expression of antisurvival members of this family, including Bad and Bak. Bcl-2 and Bcl-x(L) have been shown to preserve mitochondrial integrity and suppress cell death. In contrast, Bak and Bad both promote cell death by alteration of the mitochondrial membrane potential, in part by heterodimerization with and inactivation of Bcl-2 and Bcl-x(L), and either inducing necrosis or activating a downstream caspase program. High intrinsic levels of Bcl-2 and Bcl-x(L) may prevent apoptotic death of keratinocytes at lower levels of JP-8, while perturbation of the balance between pro- and antiapoptotic Bcl-2 family members at higher levels may ultimately play a role in necrotic cell death in human keratinocytes. Finally, when human keratinocytes were grafted to form a human epidermis on nude mice, treatment of these grafts with JP-8 revealed cytotoxicity and altered histology in vivo.     

Effects of glycoprotein isolated from Rhus verniciflua stokes on TPA-induced apoptosis and production of cytokines in cultured mouse primary splenocytes

Glycoprotein of Rhus verniciflua Stokes (RVS glycoprotein) was isolated and identified using SDS-PAGE. To study the anti-apoptotic effects of RVS glycoprotein on mouse splenocytes, splenocytes were exposed to 100 nM TPA (61.68 ng/ml) for 3 h with or without RVS glycoprotein (100 microg/ml). Results from our experiment showed that RVS glycoprotein protects from splenocyte apoptosis induced by 12-O-tetradecanoylphorbol 13-acetate (TPA). We also studied the effects of RVS glycoprotein on the proliferation of T/B cells and the production of cytokines. Our results showed that Concanavalin A (Con A)-induced T cell proliferation and the production of interleukin-2 (IL-2)/interleukin-4 (IL-4) were reduced, and that Lipopolysaccharide (LPS)-induced B cell proliferation and the Tumor necrosis factor alpha (TNF-alpha) were reduced significantly by the addition of 50 microg/ml RVS glycoprotein (P<0.01), compared to the control. These results indicate that RVS glycoprotein has the capacity to modulate apoptosis, cytokine production and T/B cell proliferation in splenocytes.     

雷帕霉素对肾小管上皮细胞增殖和凋亡的影响

目的 探讨雷帕霉素对肾小管上皮细胞增殖和凋亡的影响。方法 体外培养肾小管上皮细胞(HK-2细胞),选择100, 200, 400, 800 ng/ml雷帕霉素作用于HK-2细胞24、48、72h。利用 MTT实验分析雷帕霉素对HK-2细胞增殖的影响,并计算各浓度及不同时间的增殖抑制率优化雷帕霉素作用的浓度和时间;选择最佳的作用浓度和作用时间处理HK-2细胞,通过流式细胞分析技术,分析雷帕霉素对HK-2细胞的凋亡的影响。结果 MTT实验显示不同浓度的雷帕霉素,对增殖有抑制作用,且表现浓度依赖性和时间依赖性;RAPA可以促进HK-2细胞的凋亡。结论 通过HK-2细胞模型研究,发现雷帕霉素可抑制肾小管上皮细胞增殖、促进细胞凋亡。     

Apoptosis, necrosis, and cell proliferation induced by S-(1,2-dichlorovinyl)-L-cysteine in primary cultures of human proximal tubular cells.

Apoptosis, necrosis, and cell proliferation induced by S-(1,2-dichlorovinyl)-L-cysteine (DCVC), the cysteine conjugate of the environmental and occupational contaminant trichloroethylene, were studied in primary cultures of human proximal tubular (hPT) cells. Cells from male and female donors were incubated with a range of concentrations of DCVC (10 to 1000 microM) for up to 48 h, and assessments of cellular morphology (phase-contrast microscopy), necrosis (lactate dehydrogenase (LDH) release), apoptosis(cell cycle analysis, annexin V staining, and caspase activation), and proliferation (cell cycle analysis and DNA synthesis) were made. Time- and concentration-dependent changes in cellular morphology, including elongation of cell shape, formation of intracellular vesicles, and formation of apoptotic bodies, were observed. Significant increases in LDH release occurred in hPT cells incubated with < or =100 microM DCVC for at least 24 h. hPT cells from males were modestly more sensitive to DCVC than those from females, with maximal LDH release of 78 and 65% in cells from males and females, respectively. Flow cytometry analysis of propidium iodide-stained and DCVC-treated hPT cells showed that apoptosis occurred at markedly lower concentrations (10 microM) and at much earlier incubation times (2 h) than necrosis. A small increase was also noted in the percentage of cells in S-phase after a 4-h treatment with as little as 10 microM DCVC, suggesting that cell proliferation was stimulated. This was supported further by increased DNA synthesis. These results show that DCVC causes apoptosis and enhances cell proliferation in hPT cells at environmentally relevant doses and at earlier time points and lower concentrations than necrosis.     

Effects of ginseng saponins isolated from red ginseng on ultraviolet B-induced skin aging in hairless mice

It is well-known that chronic ultraviolet B (UVB) exposure at low-dose causes skin photoaging including increases in skin thickness and wrinkle formation and reduction in skin elasticity. This study examined the effects of total saponins and ginsenoside Rb₁ isolated from Red Ginseng roots on skin thickness, elasticity, and wrinkle formation caused by long-term, low-dose UVB irradiation in hairless mice. The topical application of total ginseng saponins (10 pg or 100 ng/mouse) and ginsenoside Rb₁ (100 fg, 10 pg, or 1 ng/mouse) significantly inhibited increases in skin thickness and wrinkle formation and the reduction in skin elasticity induced by long-term UVB irradiation. Furthermore, we examined the histological effects of total saponins and ginsenoside Rb₁ in the skin of UVB-irradiated hairless mice. The increases in apoptotic, Ki-67-, and 8-hydroxy-2′-deoxyguanosine-positive cells induced by UVB exposure were prevented by the topical application of total saponins and ginsenoside Rb₁. Furthermore, total saponins and ginsenoside Rb₁ prevented the disruption of collagen fibers induced by the long-term UVB irradiation. Ginsenoside Rb₁ (100 fg, 10 pg, and 1 ng/ml) increased the Bcl-2 expression level in UVB-treated human keratinocytes. The protective effect of ginsenoside Rb₁ on UVB-mediated apoptosis may be due to the up-regulation of Bcl-2 expression. These results suggest that the protective effect of ginsenoside Rb₁ on skin photoaging induced by chronic UVB exposure may be due to the increase in collagen synthesis and/or the inhibition of matrix metalloproteinase expression in dermal fibroblasts.     

白介素10对肿瘤坏死因子-α诱导心肌细胞凋亡的影响及其作用机制

目的 探讨凋亡诱导因子（AIF）在肿瘤坏死因子-α（TNF-α）诱导心肌细胞凋亡途径中的作用,以及白介素-10（IL-10）对TNF-α诱导心肌细胞凋亡的影响及其可能的作用机制.方法 原代培养的乳鼠心肌细胞,分为六组（对照组、TNF-α 100 ng/ml 12 h、18 h和24 h组,IL-10 50ng/ml 19 h、IL-10 50.g/ml＋TNF-α 100 ng/ml 18 h组）.用流式细胞仪和Hoeehest 33258染色检测心肌细胞凋亡;weatertrl blot检测AIF蛋白的表达.结果 TNF-α 100ng/ml 12 h、18 h、24 h组心肌细胞的凋亡率较对照组均明显升高：12 h组流式细胞仪法（5.08±0.79）%与（2.2±0.77）%;18 h（14.39±2.31）%与（2.2±0.77）%;24（4.61±0.84）%与（2.2±0.77）%.18 h组Hoechst 33258法（18.936±2.79t）%与（2.890±1.326）%,P〈0.01;18 h凋亡率达高峰,明显高于12 h、24 h组[分别为（14.39±2.31）%与（5.08±0.79）%;（14.39±2.31）%与（4.61±0.84）%]加入TNF-α后12 h、18 h、24 h,较对照组AIF蛋白的表达明显增加,18h的AIF蛋白的表达增加更明显.结论AIF可能是TNF-α诱导心肌细胞凋亡的途径之一;IL-10对TNF-α诱导的心肌细胞凋亡有保护作用.     

Effect of environmental pollutants on the production of pro-inflammatory cytokines by normal human dermal keratinocytes

The effect of the environmental pollutants, diesel exhaust particles (DEP) and formaldehyde (FA), on the production of pro-inflammatory cytokines (interleukin (IL)-1alpha, IL-1beta, tumor necrosis factor (TNF)-alpha and IL-8) by normal human dermal keratinocytes (hKCs) was investigated. Normal hKCs were incubated with various concentrations of DEP (0.4, 0.8, 4, or 20 microg/ml) or FA (0.25, 0.5, 1, or 5 microg/ml), and cytokine production was then determined by enzyme-linked immunosorbent assay (ELISA). DEP (20 microg/ml) induced IL-1beta production without altering cell growth. The increased production of IL-1beta induced by this concentration of DEP was further enhanced by the presence of phorbol 12-myristate 13-acetate (PMA), although PMA alone did not affect the levels of IL-1beta. IL-8 production was also increased by DEP (0.4 and 0.8 microg/ml), which is consistent with the results that these concentrations of DEP increased the number of cells significantly after 72 h incubation. Although FA alone did not stimulate the production of IL-1beta or IL-8 by keratinocytes, FA (0.5 microg/ml and 5 microg/ml) significantly increased IL-8 and IL-1beta production, respectively, in cells stimulated with PMA. IL-1alpha production was not modulated by FA or DEP even in the presence of PMA. TNF-alpha was produced by unstimulated keratinocytes at barely detectable levels after 48 h incubation. Although basal levels of TNF-alpha in the culture supernatants were increased after stimulation with PMA, neither pollutant alone nor combination with PMA affected the levels of TNF-alpha. These in vitro findings suggest that environmental pollutants may act as modulating factors of cutaneous inflammation by affecting the ability of keratinocytes to release pro-inflammatory cytokines.     

Camelliin B induced apoptosis in HeLa cell line.

Gordonia axillaris (Roxb.) Dietrich (Theaceae) is a native to Taiwan and the leaves have been used as an astringent folk medicine. Camelliin B (CB), a macrocyclic hydrolyzable tannin, was isolated from G. axillaris and showed cytotoxic effects in human carcinoma cells. Among the target cells (SKHep-1, Ha-22T, DU-145, AGS, and HeLa), the cervical carcinoma cell line, HeLa, was more sensitive to CB than were Chang normal liver cells and primary-cultured normal gingival and cervical fibroblasts. Furthermore, the cytotoxic effects of CB showed dose-dependency at 3.2-100.0 microg/ml in HeLa for 1,24,48, and 72 h and with an IC(50) value of 46.3 microg/ml for 48 h. However, the IC(50) value of CB in primary-cultured normal cervical fibroblasts was 108.0 microg/ml. Therefore, the selectivity shown by CB was ascribed to the difference in growth speed between normal and tumor cells. HeLa cells and primary-cultured normal cervical fibroblasts were treated with 50.0 and 100.0 microg/ml CB for 48 h, respectively, and exhibited chromatin condensation, indicating the occurrence of apoptosis. Flow cytometric analysis demonstrated the presence of apoptotic cells with low DNA content, a decrease of cell population at the G(1) phase, and a concomitant increase of cell population at the G(2)/M phase. CB also caused DNA fragmentation and inhibited PARP degradation in HeLa cells. However, CB did not significantly inhibit Bcl-2 expression in HeLa cells at 50.0 microg/ml, only at 100.0 microg/ml for 48 h. These results suggest that CB induced apoptosis, without direct inhibition of Bcl-2 expression in HeLa cells.     

Basic fibroblast growth factor sensitizes NIH 3T3 cells to apoptosis induced by cisplatin

One mechanism by which chemotherapeutic agents kill tumor cells is by induction of apoptosis. Basic fibroblast growth factor (bFGF/FGF-2) has been reported to inhibit apoptosis in NIH 3T3 cells treated with chemotherapy drugs. We have investigated how bFGF modulates apoptosis induced by cisplatin in NIH 3T3 cells. Treatment with 10 microgram/ml cisplatin for 12 h induced apoptosis in 2 to 13% of the cells at 24 h post-treatment. Preincubation with 10 ng/ml bFGF for 24 h led to cisplatin-induced apoptosis in 20% to 50% of the cells. Preincubation with lower concentrations of bFGF (0.1-1 ng/ml) or simultaneous addition of bFGF and cisplatin had no effect on the amount of apoptosis. Pretreatment with bFGF also significantly decreased the dose-dependent survival of NIH 3T3 cells exposed to cisplatin, as determined by colony formation. Cells treated with 10 ng/ml bFGF showed a distinct morphology, appearing smaller and more refractile, before cisplatin exposure. The enhancement of cisplatin-induced apoptosis and the morphology shift demonstrated the same dose response to bFGF, and both effects were reversible if bFGF was removed from the medium for 24 h before cisplatin treatment. Mitogenic response to bFGF by NIH 3T3 cells saturated at 0.5 ng/ml, as measured by (3)H-thymidine uptake, and this response was blocked by coaddition of suramin, an inhibitor of FGF ligand-receptor interactions. Suramin did not reverse the enhancement of cisplatin-induced apoptosis by bFGF. Therefore, bFGF sensitized NIH 3T3 cells to cisplatin, and this effect might be mediated through a pathway separate from that used for mitogenic signaling.     

Effect of glutathione depletion on inhibition of cell cycle progression and induction of apoptosis by melphalan (L-phenylalanine mustard) in human colorectal cancer cells.

Intracellular levels of glutathione have been shown to affect the sensitivity of cells to cell death-inducing stimuli, as well as the mode of cell death. We found in five human colorectal cancer cell lines (HT-29, LS-180, LOVO, SW837, and SW1116) that GSH depletion by L-buthionine-[S,R]-sulfoximine (BSO) below 20% of control values increased L-phenylalanine mustard (L-PAM; Melphalan) cytotoxicity 2- to 3-fold. Effects on kinetics of both cell cycle progression and cell death were further investigated in the HT-29 cell line. BSO treatment alone had no effect on cell cycle kinetics, but did enhance the inhibition of S phase progression as induced by L-PAM; at high concentration of of L-PAM, BSO pretreatment resulted in blockage in all phases of the cell cycle. Yet, BSO pretreatment did not affect the intracellular L-PAM content. L-PAM induced apoptosis in both normal and GSH-depleted cells. A combination of annexin V labeling and propidium iodide staining revealed that even the higher concentration of L-PAM (420 microM) did not induce apoptosis until 48 hr after treatment, but that induction of cell death was markedly accelerated as a result of GSH depletion: 48 hours after L-PAM (420 microM) treatment, GSH-depleted cells showed a 4-fold increase in DNA fragmentation and a 7-fold increase in the fraction of apoptotic (annexin V-positive) cells as compared to cells with normal GSH levels. Various antioxidant treatment modalities could not prevent this potentiating effect of GSH depletion on L-PAM cytotoxicity, suggesting that reactive oxygen species do not play a role. These data show that after BSO treatment the mode of L-PAM-induced cell death does not necessarily switch from apoptosis to necrosis.     

Cytotoxic and apoptotic activity of essential oil from Ocimumviride towards COLO 205 cells

We investigated the apoptosis inducing effect of essential oil (EO) from aerial parts of Ocimumviride in human colorectal adenocarcinoma cells (COLO 205 cell line). The COLO 205 cells were exposed to 0.0125–0.1 μl/ml of EO for 24, 48 and 72 h. Growth inhibition was determined by sulphorhodamine B (SRB) assay. Double staining with acridine orange and ethidium bromide for nuclear changes was performed. Cell cycle analysis and change in mitochondrial membrane potential was quantified by flow cytometry. Subsequently, using annexin V/PI assay, the proportion of cells actively undergoing apoptosis was determined. Changes in DNA were observed by DNA ladder assay. Eventually the surface morphology of apoptotic cells was studied by scanning electron microscopy. EO is cytotoxic to COLO 205 cells in dose and time-dependent manner, as is evident by SRB assay. This observed cell death was due to apoptosis, as established by annexin V/PI assay, DNA ladder formation and scanning electron microscopy. Our results reveal that EO has apoptosis inducing effect against COLO 205 cells in vitro and is a promising candidate for further anti-cancer study.     

Effects of octenidine mouth rinse on apoptosis and necrosis of human fibroblasts and epithelial cells – an in vitro study

This study aimed at comparing the cytotoxicity of a new octenidine mouth rinse (MR) on gingival fibroblasts and epithelial cells using different established MRs. Octenidol (OCT), Chlorhexidine 0.2% (CHX), Meridol (MER), Oral B (OB), and control (PBS only) were used. Human primary gingival fibroblasts (HGFIBs) and human primary nasal epithelial cells (HNEPCs) were cultivated in cell-specific media (2?×?10⁵ cells/well) and treated with a MR or PBS for 1, 5, and 15?min. All tests were performed in duplicate and repeated 12 times. The apoptosis and necrosis were determined using a Caspase-3/7 assay and LDH assay, respectively. The data were analyzed using two-way analysis of variance with subsequent Mann–Whitney U-test. No significant differences could be found between the incubation times of the MR, neither for apoptosis nor necrosis (p?>?0.05). Regarding apoptosis of HGFIBs, MRs had no influence at all. In HNEPCs, OCT induced relevantly lower apoptosis than CHX (p?=?0.01). Considering necrosis, MER showed the lowest numbers of necrotic HGFIBs and HNEPCs, whereas OB induced the highest number of necrotic cells. The differences between both MR were statistically relevant (p?相似文献   

Suppressive activity of fexofenadine hydrochloride on the production of eosinophil chemoattractants from human nasal fibroblasts in vitro

The influence of fexofenadine hydrochloride (FEX; CAS 138452-21-8) on the production of eosinophil chemoattractants, RANTES and eotaxin, from nasal polyp fibroblasts (NPFs) was examined in vitro. Seventh to tenth generation NPFs were cultured with or without 1 microg/ml lipopolysaccharide (LPS) in the presence of various concentrations of FEX. After 24 h, the culture supernatants were obtained and assayed for eosinophil chemoattractants by enzyme-linked immunosorbent assay (ELISA). FEX at a dose of more than 250 ng/ml (but not 100 ng/ml) inhibited RANTES and eotaxin production in response to LPS stimulation, when the agent was added at starting of cell cultures. FEX also showed suppressive effect on RANTES and eotaxin production from NPFs after stimulation with tumor necrosis factor alpha (TNF-alpha). However, the addition of FEX at 12 h after culture could not inhibit factor production. The influence of FEX on messenger RNA (mRNA) expression in NPFs was then examined. The addition of FEX at 100 ng/ml scarcely affected the expression of RANTES and eotaxin mRNA. On the other hand, 250 ng/ ml of FEX significantly inhibited these mRNA expressions that were enhanced by LPS stimulation.     

Apoptosis induction by 4beta-acetoxyscirpendiol from Paecilomyces tenuipes in human leukaemia cell lines

The carpophores of Paecilomyces tenuipes are known in the Orient for their strong antitumor activity. In continuation of our study on acetoxyscirpendiol (ASD, 4beta-acetoxyscirpene-3alpha,15-diol) as a cytotoxic component from this fungus, we report particularly on the mode of action of ASD in inducing apoptosis in human MOLT-4, THP-1 and Jurkat T cell leukaemia in vitro. The antiproliferative effects of ASD seem attributable to its induction of apoptosis in the cells, as it blocked the cell cycle, induced hypodiploidity and bound annexin V and also cleaved poly-(ADP-ribose) polymerase (PARP) in these cell lines. The 50% inhibitory concentrations (IC50) of ASD on MOLT-4, THP-1 and Jurkat T cells were found to be 60, 85 and 60 ng/ml, respectively. ASD arrested the cell cycle at the G1/S transition and showed hypodiploidity due to the accumulation of sub-G0 population. Annexin V binding was increased in the presence of ASD in the MOLT-4 cell line in a time-dependent manner. ASD and three of its derivatives also induced cleavage of PARP in both MOLT-4 and Jurkat T cell lines. From these data, it is suggested that ASD exerts its cytotoxic activity by inducing apoptosis in leukaemia cell lines in vitro.     

Transfection of annexin 1 in monocytic cells produces a high degree of spontaneous and stimulated apoptosis associated with caspase-3 activation

Transfection of the pre-monomyelocytic U937 cell line with a plasmid coding for full-length annexin 1 (ANX1, 347 amino acid) leads to cell death by promoting apoptosis. In addition, over-expression of the N-terminal and the first domain of the protein (144 amino acids, clone ANX1-S), which does not contain the Ca2+ binding sites, gives susceptibility to cell apoptosis following activation by either 5 ng ml(-1) tumour necrosis factor (TNF)-alpha or 1 - 40 microg ml-1 etoposide. This was demonstrated by using the fluorescent labelled annexin V, cell cycle and nuclear staining analyses. Transfection with an empty plasmid (clone CMV) or with a plasmid carrying the cDNA antisense for ANX1 (clone ANX1-AS) did not alter U937 cells to the degree of apoptosis promoted by either stimulant. Treatment of CMV U937 cells with TNF-alpha increased ANX1 mRNA and protein expression in a time-dependent manner, with maximal increases at 3 and 6 h, respectively. Clone ANX1-S showed higher constitutive (more than 2 fold) and activated caspase-3 activity, associated with higher phospholipase A2 (PLA2) activity (in the region of +50 - 100%), whereas expression of cytosolic PLA2 Bax and Bcl-2 were similar in all cell clones, as determined by Western blotting. In conclusion, this study demonstrates a complex regulatory role of cell apoptosis for ANX1, at least with regards to cells of the myelo-monocytic lineage.     

丹酚酸B对转化生长因子β_1诱导心肌成纤维细胞增殖的影响

目的:探讨丹酚酸B对转化生长因子β1(TGF-β1)诱导心肌成纤维细胞增殖的影响。方法:培养原代心肌成纤维细胞;采用波形蛋白、肌动蛋白及纤维连接蛋白免疫组化染色鉴定心肌成纤维细胞,计算阳性细胞率;加入TGF-β1,使其终质量浓度为5、10、20、40 ng/ml,以MTT法测定细胞增殖活性;加入丹酚酸B 100μl,使其终浓度为10、30、100μmol/L,1 h后加100μl TGF-β1,使其终质量浓度为20 ng/ml,以MTT法测定细胞增殖活性。结果:倒置显微镜下观察细胞呈梭形,排列较紧密,有的重叠交叉生长;免疫组化染色鉴定该细胞为心肌成纤维细胞,纯度>99%;10⁴0 ng/ml的TGF-β1均可显著诱导心肌成纤维细胞增殖,1040 ng/ml的TGF-β1均可显著诱导心肌成纤维细胞增殖,10¹00μmol/L的丹酚酸B均可显著抑制TGF-β1诱导的心肌成纤维细胞增殖。结论:丹酚酸B对TGF-β1诱导心肌成纤维细胞增殖具有抑制作用。     

Aristolochic acid I-induced DNA damage and cell cycle arrest in renal tubular epithelial cells in vitro

DNA damage is a critical event preceding cellular apoptosis or necrosis. This study was carried out to investigate the effect of aristolochic acid I (AAI) on DNA damage and cell cycle in porcine proximal tubular epithelial cell lines (LLC-PK1 cells). LLC-PK1 cells were stimulated with AAI at the concentrations of 80, 320, and 1,280 ng/ml for 24 h. DNA damage was examined by comet assay and the cell cycle was assayed by flow cytometry (FCM), cellular apoptosis and lysis were examined simultaneously. Cellular nuclear changes were observed by electron microscopy and the expression of wild-type p53 protein and mRNA were measured by FCM and RT-PCR. We found that AAI-induced DNA damage prior to apoptosis and lysis in LLC-PK1 cells in a dose-dependent manner (P<0.01). The percentage of cells in the G2/M phase that were treated with AAI (320 and 1,280 ng/ml) for 24 h increased significantly (P<0.01). Electron micrographs showed the nuclear abnormalities in AAI-treated cells. The expression of p53 protein and mRNA did not change in the AAI-treated cells. AAI may cause DNA damage and cell cycle arrest in LLC-PK1 cells through a wild-type p53-independent pathway, prior to apoptosis or necrosis. This study on the molecular mechanism of AAI-induced toxicity may explain why tubular epithelial cells present limited proliferation and regeneration abilities in the clinical presentation of AAI-associated nephrotoxicity.     

Hydrogen peroxide-induced apoptosis and necrosis in human lung fibroblasts: protective roles of glutathione

Although reactive oxygen species (ROS)-related cell damage has been implicated in pathogenesis of fibrogenetic pulmonary disorders, features of ROS-mediated cell death in human lung fibroblasts are not completely understood. We therefore examined the effects of hydrogen peroxide (H2O2) on cell growth kinetics in human lung fibroblasts (HFL-1 cells) and tested the roles of antioxidants on the H2O2-induced cell death (i.e., necrosis and apoptosis) in HFL-1 cells. We found that the relatively low concentrations of H2O2 ranging from 10 microM to 100 microM induced predominantly apoptosis, whereas higher concentration of H2O2 ranging 1 mM-10 mM induced predominantly necrosis in HFL-1 cells. Extracellular supplementation of glutathione (GSH) in culture media significantly abolished the H2O2-induced cell death, whereas GSH-depleted cells by pretreatment with buthionine sulfoxime (BSO) were likely to undergo cell death caused by a lower concentration of H2O2 than normal HFL-1 cells without BSO treatment. These results indicate that H2O2 induces both necrosis and apoptosis of human lung fibroblasts at least in part through the action of ROS and that modulation of the ROS production inside and outside of cells may influence the cell survival during oxidative insults.