Effects of lysophosphatidic acid (LPA) signaling via LPA receptors on cellular functions associated with ATP reduction in osteosarcoma cells treated with ethidium bromide

Lysophosphatidic acid (LPA) signaling via LPA receptors (LPA₁ to LPA₆) exhibits a variety of malignant properties in cancer cells. Intracellular ATP depletion leads to the development of necrosis and apoptosis. The present study aimed to evaluate the effects of LPA receptor-mediated signaling on the regulation of cancer cell functions associated with ATP reduction. Long-term ethidium bromide (EtBr) treated (MG63-EtBr) cells were established from osteosarcoma MG-63 cells. The intracellular ATP levels of MG63-EtBr cells were significantly lower than that of MG-63 cells. LPAR2, LPAR3, LPAR4 and LPAR6 gene expressions were elevated in MG63-EtBr cells. The cell motile and invasive activities of MG63-EtBr cells were markedly higher than those of MG-63 cells. The cell motile activity of MG-63 cells was increased by LPA₄ and LPA₆ knockdowns. In cell survival assay, cells were treated with cisplatin (CDDP) every 24 h for 3 days. The cell survival to CDDP of MG63-EtBr cells was lower than that of MG-63 cells. LPA₂ knockdown decreased the cell survival to CDDP of MG-63 cells. The cell survival to CDDP of MG-63 cells was inhibited by (2 S)-OMPT (LPA₃ agonist). Moreover, the cell survival to CDDP of MG-63 cells was enhanced by LPA₄ and LPA₆ knockdowns. These results indicate that LPA signaling via LPA receptors is involved in the regulation of cellular functions associated with ATP reduction in MG-63 cells treated with EtBr.

     

Reactivation of herpes simplex virus in a cell line inducible for simian virus 40 synthesis

The reactivation of UV-irradiated herpes simplex virus (HSV) was investigated in irradiated and unirradiated transformed hamster cells in which infectious simian virus 40 (SV40) can be induced. Reactivation was enhanced when the cells were treated with UV light or mitomycin C prior to infection with HSV. The IV dose-response curve of this enhanced reactivation was strikingly similar to that found for induction of SV40 virus synthesis in cells treated under identical condictions. This is the first time that two SOS functions described in bacteria have been demonstrated in a single mammalian cell line.     

Differential proteomic analysis of HeLa cells treated with Honokiol using a quantitative proteomic strategy

Honokiol (HNK) is an active component purified from Magnolia officinalis. HNK exhibits antitumor effects by inducing apoptosis and inhibiting the growth of many cancer cell lines, while proteins involved in antitumor effects in proteomic level are still unclear. In our study, HNK could inhibit HeLa cell proliferation and induce apoptosis in a concentration- and time-dependent manner. We utilized a quantitative proteomic technique termed SILAC (Stable isotope labeling with amino acids in cell culture)-MS (mass spectrometry) to study the differential proteomic profiling of HeLa cells treated by HNK. A total of 85 proteins were changed after HeLa cells were treated with 12 microg/ml HNK for 8 h, and 8 proteins showed up-regulation while 77 proteins down-regulated. The changed proteins were classified into 9 different categories, which covered a broad variety of cellular functions. In conclusion, HNK performs cytotoxicity to HeLa cells through co-operating of many proteins and different pathways.     

A novel function of hepatocyte growth factor in the activation of checkpoint kinase 1 phosphorylation in colon cancer cells

Lysophosphatidic acid (LPA) is a simple biophysical lipid which interacts with at least six subtypes of G protein-coupled LPA receptors (LPA₁–LPA₆). In cancer cells, LPA signaling via LPA receptors is involved in the regulation of malignant properties, such as cell growth, motility, and invasion. The aim of this study was to assess whether LPA receptors regulate cellular functions of fibrosarcoma cells treated with anticancer drug. HT1080 cells were maintained by the stepwise treatment of cisplatin (CDDP) at a range of 0.01 to 1.0 µM for approximately 6 months. The cell motile and invasive activities of long-term CDDP-treated (HT-CDDP) cells were significantly stimulated by LPA treatment, while HT-CDDP cells in the static state showed the low cell motile and invasive activities in comparison with HT1080 cells. Since the expression level of LPAR2 gene was markedly elevated in HT-CDDP cells, LPA₂ knockdown cells were generated from HT-CDDP cells. The cell motile and invasive activities of HT-CDDP cells were reduced by LPA₂ knockdown. In colony assay, large-sized colonies formed by long-term CDDP treatment were suppressed by LPA₂ knockdown. In addition, LPA₂ knockdown cells reduced LPA production by autotaxin (ATX), correlating with ATX expression level. These results suggest that LPA signaling via LPA₂ may play an important role in the regulation of cellular functions in HT1080 cells treated with CDDP.     

pH-dependent influence of a quaternary ammonium salt and an aminoester on the yeast Saccharomyces cerevisiae ultrastructure

Quaternary ammonium salts inhibited the growth of yeast especially at pH higher (pH 8) than optimal. It was postulated that compounds integrate with the cell membrane and interfere with its functions. The yeast cell ultrastructure investigated under an electron microscope confirms this hypothesis. A relatively high percentage of cells treated at pH 6 with the quaternary ammonium salt of alanine derivative (DMALM-12) at the minimal inhibitory concentration showed an irregularity in the cell shape. No such irregularity was observed in the control. Besides, in the cells treated with the drug, practically no lipid droplets were seen at all. Inside the control cells, electron-dense round bodies were clearly seen and interpreted as vacuoles. These bodies were absent in the cells treated with DMALM-12. Although the yeast cells growing at pH 8 showed a more or less normal shape, they seemed to have difficulty in budding - no fully developed buds were found in the preparations. Only some convexities of the cell wall were seen that could be the beginning of budding which stopped early after the start. Some changes in the round bodies interpreted as vacuoles were visible: they were less dense and full of granules.     

Human Decidua Basalis mesenchymal stem/stromal cells reverse the damaging effects of high level of glucose on endothelial cells in vitro

Recently, we reported the therapeutic potential of mesenchymal stem/stromal cells (MSCs) from the maternal decidua basalis tissue of human term placenta (DBMSCs) to treat inflammatory diseases, such as atherosclerosis and cancer. DMSCs protect endothelial cell functions from the negative effects of oxidative stress mediators including hydrogen peroxide (H₂O₂) and monocytes. In addition, DBMSCs induce the generation of anti-cancer immune cells known as M1 macrophages. Diabetes is another inflammatory disease where endothelial cells are injured by H₂O₂ produced by high level of glucose (hyperglycaemia), which is associated with development of thrombosis. Here, we investigated the ability of DBMSCs to reverse the damaging effects of high levels of glucose on endothelial cells. DBMSCs and endothelial cells were isolated from human placental and umbilical cord tissues, respectively. Endothelial cells were incubated with glucose in presence of DBMSCs, and their functions were evaluated. The effect of DBMSCs on glucose- treated endothelial cell expression of genes was also determined. DBMSCs reversed the effects of glucose on endothelial cell functions including proliferation, migration, angiogenesis and permeability. In addition, DBMSCs modified the expression of several genes mediating essential endothelial cell functions including survival, apoptosis, permeability and angiogenesis. We report the first evidence that DBMSCs protect the functions of endothelial cells from the damaging effects of glucose. Based on these results, we establish that DBMSCs are promising therapeutic agents to repair glucose-induced endothelial cell injury in diabetes. However, these finding must be investigated further to determine the pathways underlying the protective role of DBMSCs on glucose-stimulated endothelial cell Injury.     

Immunologic suppression of carcinogenesis in spontaneously hypertensive rats (SHR) with T cell depression

A strain of spontaneously hypertensive rats (SHR) showed a selective depression of T cell functions brought about by aging. Conversely, this strain had a high NK cell activity as compared to other normal rat strains. This SHR strain was found to be much more sensitive to the carcinogenic activity of low doses of MCA than were WKA rats with normal T cell functions. Allogeneic thymus grafts almost completely restored the T cell functions of SHR, whereas injection of an immunopotentiator, NSP, enhanced NK cell activity and also caused a partial recovering of T cell functions. When immunologic restoration was achieved, generation of killer T cells to syngeneic SMT-5 tumor cells was induced and the cytotoxic activity of NK cells to K-562 cells was also enhanced. But the cytotoxic activity to the SMT-5 cells of NK cells and macrophages from the treated or untreated SHR was not detected. Allogeneic thymus grafts induced a significant transplantation resistance against a syngeneic SMT-5 tumor and injection of NSP enhanced only the survival days of the rats. Allogeneic thymus grafts also significantly suppressed the incidence of tumors induced by MCA, whereas the injection of NSP was not effective in the prevention of tumor development but was effective in prolongation of latency periods. These results support the hypothesis that immune surveillance mediated by T cells is an important mechanism for the control of tumor development.     

Expression of macrophage functions in hybrids of a myeloma cell line with inflammatory macrophages: Evidence for negative control mechanisms in the expression of macrophage functions

Mouse inflammatory macrophages from C57BL/6N mice were fused with BALB/c mouse-derived myeloma cells (the CANS series). The hybrids in the early period after cell fusion (8 weeks) showed no macrophage functions (chemotaxis, EA and EAC rosette-forming abilities, phagocytosis or lysozyme production). EA rosette-forming ability was observed when these hybrids were treated with trypsin, whereas other macrophage functions were not. After prolonged culture, the hybrids (12 clones of 13 randomly selected) showed all the macrophage functions along with chromosome loss. Myeloma cell functions ( light chain production) were found in the young hybrids soon after cell fusion but were absent in the aged hybrids. These results indicated that reexpression of macrophage properties, except for EA rosette-forming abilities, takes place after the loss of chromosomes or genes repressing the expression of macrophage functions.     

Protective role of vitamin E on mefenamic acid-induced alterations in erythrocytes

Erythrocyte osmotic fragility (O.F.), acetylcholinesterase (AChE) activity,and the level of malonyl dialdehyde (MDA) of control, mefenamic acid treated, and mefenamic acid with vitamin E treated rats were investigated. Administration of mefenamic acid to albino rats brought about a significant increase in the osmotic fragility of red cells and a significant (p<0.01) decrease in the activity of AChE. We have also observed increased red cell level of MDA and decreased cholesterol (Chl), hemoglobin (Hb), and reduced glutathione (GSH) content. Supplementation of vitamin E to the mefenamic acid treated rats restored the O.F., AChE activity, level of MDA, and Chl, Hb, and GSH content almost to normal. These observations suggest that mefenamic acid causes functional impairment of red cell membrane, while vitamin E shows its protective role in maintaining normal red cell functions.     

Effect of millimeter waves on cyclophosphamide induced suppression of T cell functions

The effects of low power electromagnetic millimeter waves (MWs) on T cell activation, proliferation, and effector functions were studied in BALB/c mice. These functions are important in T-lymphocyte mediated immune responses. The MW exposure characteristics were: frequency = 42.2 GHz; peak incident power density = 31 +/- 5 mW/cm(2), peak specific absorption rate (SAR) at the skin surface = 622 +/- 100 W/kg; duration 30 min daily for 3 days. MW treatment was applied to the nasal area. The mice were additionally treated with cyclophosphamide (CPA), 100 mg/kg, a commonly used immunosuppressant and anticancer drug. Four groups of animals were used in each experiment: naive control (Naive), CPA treated (CPA), CPA treated and sham exposed (CPA + Sham), and CPA treated and MW exposed (CPA + MW). MW irradiation of CPA treated mice significantly augmented the proliferation recovery process of T cells (splenocytes). A statistically significant difference (P <.05) between CPA and CPA + MW groups was observed when cells were stimulated with an antigen. On the other hand, no statistically significant difference between CPA and CPA-Sham groups was observed. Based on flow cytometry of CD4(+) and CD8(+) T cells, two major classes of T cells, we show that CD4(+) T cells play an important role in the proliferation recovery process. MW exposure restored the CD25 surface activation marker expression in CD4(+) T cells. We next examined the effector function of purified CD4(+) T cells by measuring their cytokine profile. No changes were observed after MW irradiation in interleukin-10 (IL-10) level, a Th2 type cytokine, while the level of interferon-gamma (IFN-gamma), a Th1 type cytokine was increased twofold. Our results indicate that MWs enhance the effector function of CD4(+) T cells preferentially, through initiating a Th1 type of immune response. This was further supported by our observation of a significant enhancement of tumor necrosis factor-alpha (TNF-alpha) production by peritoneal macrophage's in CPA treated mice. The present study shows MWs ameliorate the immunosuppressive effects of CPA by augmenting the proliferation of splenocytes, and altering the activation and effector functions of CD4(+) T cells.     

IFN-beta inhibits T cell activation capacity of central nervous system APCs

We have previously investigated the physiological effects of IFN-beta on chronic CNS inflammation and shown that IFN-beta(-/-) mice develop a more severe experimental autoimmune encephalomyelitis than their IFN-beta(+/-) littermates. This result was shown to be associated with a higher activation state of the glial cells and a higher T cell cytokine production in the CNS. Because this state suggested a down-regulatory effect of IFN-beta on CNS-specific APCs, these results were investigated further. We report that IFN-beta pretreatment of astrocytes and microglia (glial cells) indeed down-modulate their capacity to activate autoreactive Th1 cells. First, we investigated the intrinsic ability of glial cells as APCs and report that glial cells prevent autoreactive Th1 cells expansion while maintaining Ag-specific T cell effector functions. However, when the glial cells are treated with IFN-beta before coculture with T cells, the effector functions of T cells are impaired as IFN-gamma, TNF-alpha, and NO productions are decreased. Induction of the T cell activation marker, CD25 is also reduced. This suppression of T cell response is cell-cell dependent, but it is not dependent on a decrease in glial expression of MHC class II or costimulatory molecules. We propose that IFN-beta might exert its beneficial effects mainly by reducing the Ag-presenting capacity of CNS-specific APCs, which in turn inhibits the effector functions of encephalitogenic T cells. This affect is of importance because activation of encephalitogenic T cells within the CNS is a prerequisite for the development of a chronic progressive CNS inflammation.     

Somatostatin Derivative (smsDX) Targets Cellular Metabolism in Prostate Cancer Cells after Androgen Deprivation Therapy

Cancer cell metabolism responsive to androgen deprivation therapy (ADT) may be involved in the development and progression of prostate cancer and the ultimate failure of androgen-deprivation therapy. To investigate the metabolism regulation effects on androgen-independent growth of prostate cancer, an established LNCaP-s cell model that resembles the clinical scenario of castration-resistant prostate cancer (CRPC), was used in this current study. This cell line was cultured from androgen-sensitive LNCaP parental cells, in an androgen-reduced condition, resembling clinical androgen deprivation therapy. To assess the effects of smsDX on the invasiveness of prostate cancer cells we used wound healing assay and Matrigel™ invasion assay. We evaluated differentially expressed proteins of the parental LNCaP cells and LNCaP-s cells after ADT by means of two-dimensional gel electrophoresis (2-DE) followed by MALDI-TOF mass spectrometric analysis. The covered area in the wound and the number of cells invading through a Matrigel chamber were significantly smaller for cells treated with smsDX than they were for control cells treated with vehicle. 56 proteins were found differentially expressed in LNCaP-s cells compared to LNCaP cells, majority of them were down-regulated after ADT treatment. 104 proteins of LNCaP cells and 86 in LNCaP-s cells, separately, were found differentially expressed after treatment with smsDX, When we explored these protein functions within the website UniProtKB/Swiss-Prot, surprisingly, most of the proteins were found to be involved in the cellular metabolism and mitochondrial function regulation. LNCaP-s as potential metastatic androgen-independent cancer cells, its metabolism and mitochondrial functions could be altered by a new somatostatin derivative smsDX, the smsDX regulatory effects on metabolism in LNCaP-s deliver more therapeutic information with the treatment of CRPC.     

Regulation of focal adhesion targeting and inhibitory functions of the FAK related protein FRNK using a novel estrogen receptor "switch"

Focal adhesion kinase (FAK) is a regulator of numerous adhesion-dependent processes including cell migration, cell proliferation, and cell survival. The C-terminal domain of FAK, FAK-related nonkinase (FRNK), is autonomously expressed and functions as an inhibitor of FAK signaling. Previous attempts to use FRNK as a tool to dissect FAK signaling have been limited because of an inability to temporally regulate the inhibitory functions of FRNK. In this report, we describe and characterize a conditionally targeted form of FRNK that was created by fusing the hormone-binding domain of the estrogen receptor (ER*) to the C-terminus of FRNK. In the absence of added hormone, FRNK-ER* was diffusely distributed throughout the cytoplasm of cells. Upon addition of hormone, the cytoplasmic pool of FRNK-ER* was rapidly redistributed to focal adhesions. We demonstrate that cells expressing FRNK-ER* show a hormone-dependent decrease in FAK tyrosine phosphorylation and cell migration. Furthermore, when cells expressing of FRNK-ER* were treated with hormone, the cells responded with a dramatic change in cell morphology, suggesting a role for FAK in the regulation of the adhesive properties of focal adhesions.     

Inhibition of growth and induction of enzyme activities in a clonal human hepatoma cell line (Li-7A): comparison of the effects of epidermal growth factor and an anti-epidermal growth factor receptor antibody

A cloned human hepatoma cell line (Li-7A), possessing epidermal growth factor (EGF) receptors numbering in the range of 10-20 pmol/10(6) cells, was inhibited in its growth by EGF as well as an antagonist monoclonal antibody (MoAb) to the EGF receptor. The mode of action of the two ligands of EGF receptors appeared to be different as indicated by the following results: 1) EGF induced marked alteration in cell morphology, whereas the antibody did not; 2) cellular protein accumulated in the EGF-treated cells but not in the antibody treated cells; and 3) ectoATPase activities were greatly enhanced in Li-7A cells treated with EGF and cholera toxin but were unaffected in cells treated with antibody and cholera toxin. The last result also suggests that expression of ectoATPase activities is under the regulation of both EGF and cholera toxin. Li-7A cells provide an additional valuable experimental system for the study of EGF action, as well as the interactive effects of EGF and cholera toxin. The enrichment of the ATPase activities in the EGF-cholera toxin-treated cells can be exploited for the detailed study and isolation of these enzymes and elucidation of their physiological functions.     

Human amniotic epithelial cells induce apoptosis of cancer cells: a new anti-tumor therapeutic strategy

Background aimsAmniotic membrane (AM), the innermost layer of human placenta, is composed of a single layer of epithelial cells, a basement membrane and an avascular stroma. The AM has many functions and properties, among which angiogenic modulatory and immunoregulatory effects are applicable in cancer therapy. Because these functions belong to amniotic epithelial cells, in this study we compared the anti-cancer effect of amniotic epithelial cells and the whole AM.MethodsThe effect of the AM and the amniotic epithelial cells on cancer cell apoptosis was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay, terminal deoxynucleotidyl transferase dUTP nick end labeling assay and immunocytochemistry. The effect of the AM on angiogenesis in conditions both with and without epithelial cells was also evaluated using rat aortic ring assay.ResultsThere was a decrease in cancer cell viability after adding either AM or amniotic epithelial cell supernatant to cancer cells. A significant increase in caspase-3 and caspase-8 expression in cancer cells treated with amniotic epithelial cell supernatant was observed. The recorded media also demonstrated the possible induction of apoptosis in cancer cells treated with the amniotic epithelial cell supernatant. In the aorta ring assay, the AM showed an anti-angiogenic effect in the presence of its epithelial cells; however, this effect was altered to initiate angiogenesis when amniotic epithelial cells were removed from the AM.ConclusionsThese results suggest that amniotic epithelial cells, with their anti-angiogenic effect and induction of apoptosis, are candidates for cancer therapeutic agents in the near future.     

羟基喜树碱诱导肝癌细胞凋亡前后线粒体疏水蛋白质组的定量分析

抗癌剂羟基喜树碱可以通过线粒体途径诱导肝癌细胞凋亡.应用定量蛋白质组学技术分析羟 基喜树碱诱导肝癌细胞凋亡前后的线粒体疏水蛋白质差异表达,探讨癌细胞凋亡机制及羟基 喜树碱的抗癌机理.分离提取羟基喜树碱诱导肝癌细胞凋亡前后的线粒体,并采用顺序抽提法提取疏水蛋白质;用含稳定同位素亲和标签的c-ICAT试剂标记蛋白,利用基于多维色谱线性离子阱/静电场轨道阱质谱联用技术的鸟枪(shotgun)法策略分析鉴定了在肝癌细胞凋亡前后的线粒体中表达量差异有显著统计学意义(P<0.05)的疏水蛋白144种,其中, 12种蛋白的表达量在凋亡细胞中下调,而表达量在羟基喜树碱诱导细胞凋亡后上调10倍以上的蛋白43种.这些蛋白主要与细胞分裂增殖、分化凋亡、能量代谢、核酸代谢以及信号转导相关.该研究结果为在亚细胞定量蛋白质组水平上深入探讨羟基喜树碱的作用机理提供了新的实验依据,亦为研究肿瘤细胞凋亡机制提供了新的思路.     

Transformation of ultraviolet-irradiated human fibroblasts by simian virus 40 is enhanced by cellular DNA repair functions

Human fibroblasts irradiated with ultraviolet light were either tested for survival (colony formation) or infected with simian virus 40 and examined for transformation (foci formation). For normal cell cultures, the fractions of surviving colonies which were also transformed increased with increasing irradiation dose. In contrast, little increase in the transformation of ultraviolet-irradiated repair-deficient (xeroderma pigmentosum and xeroderma pigmentosum variant) cells was observed. Similar experiments with xeroderma pigmentosum variant cells treated with caffeine following irradiation indicated that, under these conditions, the deficient cells produced more transformants among the survivors of ultraviolet irradiation than did unirradiated cells. These results suggest (1) that DNA repair functions, not DNA damage per se, are required for enhanced viral transformation in normal cells; (2) that functions involved in excision repair and functions needed for replication of ultraviolet-damaged DNA appear necessary for this stimulation; and (3) that blocking DNA replication in ultraviolet-irradiated xeroderma pigmentosum variant cells by caffeine enhances viral transformation.     

麦芽酚对活性氧损伤人神经瘤细胞的保护作用

以人神经瘤细胞株 (SH SY5Y)为材料 ,使用过氧化氢 (H2 O2 )产生过量活性氧诱导SH SY5Y细胞株进入氧化应激状态 .研究麦芽酚对过量活性氧造成的SH SY5Y细胞株氧化损伤的保护作用 .分析活性氧对细胞膜蛋白和DNA的损伤 ,细胞线粒体功能变化 ,白介素 6 (IL 6 )的表达变化以及细胞核因子κB(NF κB)的激活 .结果显示 ,2mmol L麦芽酚保护细胞 2h后 ,对细胞膜蛋白和DNA的损伤均有明显的保护作用 ,减少了膜蛋白的氧化和细胞DNA片段化的形成 ,细胞线粒体功能损伤减小 ,细胞表达的IL 6减少 ,被激活的NF κB水平同时降低 .结果证明 ,麦芽酚可以有效保护活性氧对神经细胞的氧化损伤 ,维持细胞的正常生理功能     

Implications of altered glutathione metabolism in aspirin-induced oxidative stress and mitochondrial dysfunction in HepG2 cells

We have previously reported that acetylsalicylic acid (aspirin, ASA) induces cell cycle arrest, oxidative stress and mitochondrial dysfunction in HepG2 cells. In the present study, we have further elucidated that altered glutathione (GSH)-redox metabolism in HepG2 cells play a critical role in ASA-induced cytotoxicity. Using selected doses and time point for ASA toxicity, we have demonstrated that when GSH synthesis is inhibited in HepG2 cells by buthionine sulfoximine (BSO), prior to ASA treatment, cytotoxicity of the drug is augmented. On the other hand, when GSH-depleted cells were treated with N-acetyl cysteine (NAC), cytotoxicity/apoptosis caused by ASA was attenuated with a significant recovery in oxidative stress, GSH homeostasis, DNA fragmentation and some of the mitochondrial functions. NAC treatment, however, had no significant effects on the drug-induced inhibition of mitochondrial aconitase activity and ATP synthesis in GSH-depleted cells. Our results have confirmed that aspirin increases apoptosis by increased reactive oxygen species production, loss of mitochondrial membrane potential and inhibition of mitochondrial respiratory functions. These effects were further amplified when GSH-depleted cells were treated with ASA. We have also shown that some of the effects of aspirin might be associated with reduced GSH homeostasis, as treatment of cells with NAC attenuated the effects of BSO and aspirin. Our results strongly suggest that GSH dependent redox homeostasis in HepG2 cells is critical in preserving mitochondrial functions and preventing oxidative stress associated complications caused by aspirin treatment.