Comparison of the structural characterization and biological activity of acidic polysaccharides from <Emphasis Type="Italic">Cordyceps militaris</Emphasis> cultured with different media

Two acidic polysaccharide fractions, CM-jd-CPS2 and CM-jd(Y)-CPS2, were isolated from the fruiting bodies of cultured Cordyceps militaris grown on solid rice medium and silkworm pupa, respectively, by hot-water extraction, ethanol precipitation and fractionation using ion-exchange column (DEAE-cellulose-52) and gel-filtration column (Sephadex G-100) chromatography. Their structural characterizations were performed by gas chromatography and fourier-transform infrared spectroscopy. Some differences existed between their structures, which indicated that culture media could influence the structure of polysaccharides of C. militaris. The antioxidant activities of CM-jd-CPS2 and CM-jd(Y)-CPS2 were evaluated by various methods in vitro. They had strong 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity and ferrous ion-chelating capacity, but moderate reducing power. The antioxidant activities of CM-jd(Y)-CPS2 were slightly higher than those of CM-jd-CPS2. These two acidic fractions were evaluated for proliferation of mouse splenocyte activity in vitro. They both possessed does-dependent mitogenic effects on mouse splenocytes, and could synergistically promote murine T- and B-lymphocytes induced by Con A and LPS. CM-jd(Y)-CPS2 exhibited stronger stimulatory activities upon immunomodulation than CM-jd-CPS2. These results are beneficial for the interpretation of the connection between polysaccharide structures and their biological activities.     

Molecular mechanisms of anti-angiogenic effect of curcumin

Modulation of pathological angiogenesis by curcumin (diferuloylmethane), the active principle of turmeric, seems to be an important possibility meriting mechanistic investigations. In this report, we have studied the effect of curcumin on the growth of Ehrlich ascites tumor cells and endothelial cells in vitro. Further, regulation of tumor angiogenesis by modulation of angiogenic ligands and their receptor gene expression in tumor and endothelial cells, respectively, by curcumin was investigated. Curcumin, when injected intraperitoneally (i.p) into mice, effectively decreased the formation of ascites fluid by 66% in EAT bearing mice in vivo. Reduction in the number of EAT cells and human umbelical vein endothelial cells (HUVECs) in vitro by curcumin, without being cytotoxic to these cells, is attributed to induction of apoptosis by curcumin, as is evident by an increase in cells with fractional DNA content seen in our results on FACS analysis. However, curcumin had no effect on the growth of NIH3T3 cells. Curcumin proved to be a potent angioinhibitory compound, as demonstrated by inhibition of angiogenesis in two in vivo angiogenesis assay systems, viz. peritoneal angiogenesis and chorioallantoic membrane assay. The angioinhibitory effect of curcumin in vivo was corroborated by the results on down-regulation of the expression of proangiogenic genes, in EAT, NIH3T3, and endothelial cells by curcumin. Our results on Northern blot analysis clearly indicated a time-dependent (0-24h) inhibition by curcumin of VEGF, angiopoietin 1 and 2 gene expression in EAT cells, VEGF and angiopoietin 1 gene expression in NIH3T3 cells, and KDR gene expression in HUVECs. Further, decreased VEGF levels in conditioned media from cells treated with various doses of curcumin (1 microM-1mM) for various time periods (0-24h) confirm its angioinhibitory action at the level of gene expression. Because of its non-toxic nature, curcumin could be further developed to treat chronic diseases that are associated with extensive neovascularization.     

Evaluation of the effect of paclitaxel, epirubicin and tamoxifen by cell kinetics parameters in estrogen-receptor-positive ehrlich ascites tumor (eat) cells growing in vitro

In this study the antiproliferative effects of Paclitaxel (PAC), Epirubicin (EPI) and Tamoxifen (TAM) on growth kinetics of Ehrlich Ascites Tumor (EAT) cells were examined in culture. An estrogen-receptor-positive ER (+) hyperdiploid EAT cell line growing in vitro was also analysed in the present study. IC50 doses of PAC, EPI and TAM (12 microg/ml, 12 microg/ml and 2 microg/ml, respectively) were used. Cells were treated with the above doses for 0, 4, 8, 16, 24 and 32 hrs. At the end of these periods, living cell numbers were determined by collecting EAT cells in every group for growth study rate and for MTT assay. Therefore, the mitotic index was determined in the same experimental groups. The proliferation of EAT cells, inhibited by PAC, EPI and TAM concentrations was compared to control with increasing treatment time (4-32 hrs). Treatment of PAC, EPI and TAM alone for 24 hrs decreased the proliferation rate of EAT cells by 50% with respect to control. The inhibition of proliferation rate was higher in double drug treatment than that in single drug treatment with increased treatment time. In the treatment of three drugs applied for 32 hrs, this effect reached a maximum and proliferation rate decreased by 12% as compared to the (100%) control. In our studies, when the mitotic index parameter data were evaluated to determine which phase of the cell cycle was affected by PAC to cause the repression of cell reproduction, it was found that PAC exerted of its cytotoxic effect by causing cell accumulation at mitosis. The accumulation of the cells resulted in an increase in mitotic index values, which was an expected consequence of PAC treatment. It was observed that depending on the drug treatments, inhibition of proliferation rate and mitotic index in EAT cells were increased with respect to control, being with statistically significant occurrence (p < 0.01 - p < 0.001). As a result, concomitant treatment combined with hormonal therapy has given improved results compared with single treatment and PAC + EPI + TAM treatments had a maximum synergistic effect for 32 hrs (p < 0.001).     

Chalone-like inhibition of Ehrlich ascites cell proliferation in vitro by an ultrafiltrate obtained from the ascitic fluid.

An aqueous ultrafiltrate (10 000-50 000 dalton) prepared from the cell-free ascitic fluid of mice bearing Ehrlich ascites tumour (EAT) in the plateau phase of growth (12-16 days after transplantation) was investigated with regard to its inhibitory effects on the proliferation of EAT cells in a 24-hr suspension culture. The following results were obtained: (1) The in vitro proliferation of cells obtained from the plateau phase of in vivo growth was reversibly inhibited. (2) The dose-response curves show a plateau with a maximum inhibition of about 50%, which suggests that not all cells can be affected. (3) Young cells (4-6 days after transplantation) were not inhibited. (4) Preincubation of plateau phase cells in the culture medium before treatment abolishes the inhibitory effect of the ultrafiltrate. This effect of preincubation is dependent on time and serum concentration. It provides the possibility to differentiate between true "chalone-like" and cytotoxic effects. (5) the inhibitory properties of the ultrafiltrate are destroyed by heating or trypsin treatment. (6) Extracts prepared in the same way from ascitic fluid of mice bearing lymphocytic leukemia L1210 do not inhibit the proliferation of EAT cells. Corresponding extracts from ascitic fluid of mice bearing myelocytic leukemia YM were found to be inhibitory; however, the inhibitory effect was also found on preincubated cells and is therefore considered to be due to an unspecific cytotoxicity. In conclusion, evidence was obtained for a factor from the ascitic fluid of mice bearing EAT, which prevents EAT cells from entering the proliferating state.     

Butyrate-induced proapoptotic and antiangiogenic pathways in EAT cells require activation of CAD and downregulation of VEGF

Butyrate, a short-chain fatty acid produced in the colon, induces cell cycle arrest, differentiation, and apoptosis in transformed cell lines. In this report, we study the effects of butyrate (BuA) on the growth of Ehrlich ascites tumor (EAT) cells in vivo. BuA, when injected intraperitoneally (i.p) into mice, inhibited proliferation of EAT cells. Further, induction of apoptosis in EAT cells was monitored by nuclear condensation, annexin-V staining, DNA fragmentation, and translocation of caspase-activated DNase into nucleus upon BuA-treatment. Ac-DEVD-CHO, a caspase-3 inhibitor, completely inhibited BuA-induced apoptosis, indicating that activation of caspase-3 mediates the apoptotic pathway in EAT cells. The proapoptotic effect of BuA also reflects on the antiangiogenic pathway in EAT cells. The antiangiogenic effect of BuA in vivo was demonstrated by the downregulation of the secretion of VEGF in EAT cells. CD31 immunohistochemical staining of peritoneum sections clearly indicated a potential angioinhibitory effect of BuA in EAT cells. These results suggest that BuA, besides regulating other fundamental cellular processes, is able to modulate the expression/secretion of the key angiogenic growth factor VEGF in EAT cells.     

Antitumor effect of Bothrops jararaca venom

Many experimental studies have been carried out using snake venoms for the treatment of animal tumors, with controversial results. While some authors have reported an antitumor effect of treatment with specific snake venom fractions, others have reported no effects after this treatment. The aim of this study was to evaluate the effect of Bothrops jararaca venom (BjV) on Ehrlich ascites tumor (EAT) cells in vivo and in vitro. In the in vivo study, Swiss mice were inoculated with EAT cells by the intraperitoneal (i.p.) route and treated with BjV venom (0.4 mg/kg, i.p.), on the 1st, 4th, 7th, 10th, and 13th days. Mice were evaluated for total and differential cells number on the 2nd, 5th, 8th, 11th and 14th days. The survival time was also evaluated after 60 days of tumor growth. In the in vitro study, EAT and normal peritoneal cells were cultivated in the presence of different BjV concentrations (2.5, 5.0, 10.0, 20.0, 40.0, and 80 microg) and viability was verified after 3, 6, 12 and 24 h of cultivation. Results were analyzed statistically by the Kruskal-Wallis and Tukey tests at the 5% level of significance. It was observed that in vivo treatment with BjV induced tumor growth inhibition, increased animal survival time, decreased mortality, increased the influx of polymorphonuclear leukocytes on the early stages of tumor growth, and did not affect the mononuclear cells number. In vitro treatment with BjV produced a dose-dependent toxic effect on EAT and peritoneal cells, with higher effects against peritoneal cells. Taken together, our results demonstrate that BjV has an important antitumor effect. This is the first report showing this in vivo effect for this venom.     

软骨多糖诱导小鼠S180细胞凋亡的作用机制

目的研究软骨多糖对S180荷瘤小鼠的作用,并探讨其抑瘤作用机制。方法采用小鼠肉瘤S180细胞建立动物腹水瘤模型,通过腹腔注射软骨多糖进行治疗,治疗期间抽取腹水瘤细胞进行细胞生物学分析。通过HE染色,流式细胞术、TUNEL法检测细胞形态学方面、细胞周期及凋亡率的变化情况;通过免疫荧光方法检测Fas、增殖细胞核抗原(PCNA)的表达情况。结果软骨多糖可以明显提高S180荷瘤小鼠的生存率,细胞形态学观察可见细胞出现细胞质浓缩、核固缩及凋亡小体等现象。软骨多糖作用后的S180细胞,其细胞周期被阻遏于G2/M期,Fas蛋白的表达水平于给药24 h后升高,增殖细胞核抗原PCNA表达下降。结论软骨多糖可能通过影响肿瘤细胞周期和Fas、PCNA蛋白的表达来诱导S180细胞凋亡,并显著抑制肿瘤细胞的生长,延长S180荷瘤小鼠的生存时间,研究证实动物软骨多糖具有潜在的药用价值。     

Involvement of EAT/mcl-1, an anti-apoptotic bcl-2-related gene, in murine embryogenesis and human development

Apoptosis plays an important regulatory role in mammalian embryogenesis and development. EAT/mcl-1 (EAT), an anti-apoptotic bcl-2-related gene, was isolated during the early differentiation of a human embryonal carcinoma cell line, an event which serves as a model of early embryogenesis. EAT is involved in apoptotic regulation and is believed to also function as an immediate-early gene. Thus it was hypothesized that EAT would be expressed during early embryogenesis and would be involved in the regulation of apoptosis during this critical period. To clarify this early expression, two antibodies to EAT were generated by use of immunizing oligopeptide (aa 37-55) and recombinant protein (aa 31-229) for use in immunohistochemistry and immunoblotting, respectively. With these antibodies, we then determined EAT expression during murine embryogenesis and in human development, using human fetal tissue of 6 to 23 gestational weeks. During murine embryogenesis, the EAT protein was found to be rapidly induced after fertilization, to peak at the 2-cell stage, to remain constant until the 8-cell stage, and then to decrease to below unfertilized egg levels in blastocysts. EAT expression patterns in early human development were found to essentially overlap those observed in adult tissues which suggest that EAT expression continues until adulthood in terminally differentiated tissues. Among tissues distinct to fetal development, EAT was detected in the mesonephric (Wolffian) duct and paramesonephric (Müllerian) duct. It is also noteworthy that prominent EAT immunoreactivity was also observed in large primary oocytes in 21-week fetal ovary, but was not detected in primordial germ cells in 23-week fetal testis. In summary, EAT expression was detected in hematopoietic, epithelial, neural, endocrine, and urogenital cells; this provides evidence that EAT, as an anti-apoptotic molecule, possibly functions to regulate apoptosis during development in these systems.     

软骨多糖对L1210白血病小鼠生长抑制作用的研究

目的:研究软骨多糖对L1210白血病小鼠的生长抑制作用,并探讨其抑瘤作用机理。方法:建立DBA/2小鼠L1210腹水瘤模型,将小鼠分为对照组、低剂量组、中剂量组和高剂量组进行实验,通过腹腔注射软骨多糖治疗,每天称重并记录小鼠的生存时间,计算生命延长率。于0h、24h、48h、72h抽取治疗组小鼠的腹水瘤细胞进行细胞周期的分析;采用常规HE染色观察细胞形态学变化;应用免疫荧光方法检测BCL-2和BAD蛋白表达变化,以进一步探讨软骨多糖的抑瘤机制。结果:软骨多糖可以明显提高DBA/2小鼠的生存率;细胞形态学观察可见细胞出现细胞浆浓缩、核固缩及凋亡小体等现象;软骨多糖作用后的L1210细胞,其细胞周期被阻遏于Go/G1期,24h-48h凋亡率迅速上升;Bad蛋白的表达水平于给药24h-72h后升高,抗凋亡基因Bcl-2表达下降。结论:软骨多糖可能通过影响肿瘤细胞周期和Bad、Bcl-2蛋白的表达来诱导L1210细胞凋亡,并显著抑制肿瘤细胞的生长,延长DBA/2小鼠的生存时间,是一种新型的抑癌活性物质。     

Reduced growth of Ehrlich ascites tumor cells in creatine depleted mice fed beta-guanidinopropionic acid

The effect of implantation of Ehrlich ascites tumor (EAT) cells on creatine distribution was investigated. It was also studied how depletion of creatine by feeding creatine-analogue beta-guanidinopropionic acid (beta-GPA) affects the growth of EAT cells in mice. Enhanced mobilization of creatine from host tissues to EAT cells against a greater concentration gradient was observed. The creatine (but not creatinine) level in blood plasma was lowered to 22% of the normal value by beta-GPA feeding alone and assimilation of 14C-creatine into EAT cells was inhibited. The growth of EAT cells was significantly reduced and the duration of survival of mice after implantation of EAT cells was extended when the creatine concentration was decreased. A decrease in daily food consumption and the degree of muscle atrophy after implantation of EAT cells was less in beta-GPA than control groups. In the creatine-depleted mice, the rate of increase in total EAT cell number and the volume of abdominal ascites were approximately half of the control values, and more dead EAT cells were observed. These results suggest that supplementation of beta-GPA inhibits creatine transfer to EAT cells and reduces the growth of cancer cells.     

Progression of Chronic Liver Inflammation and Fibrosis Driven by Activation of c-JUN Signaling in Sirt6 Mutant Mice

The human body has a remarkable ability to regulate inflammation, a biophysical response triggered by virus infection and tissue damage. Sirt6 is critical for metabolism and lifespan; however, its role in inflammation is unknown. Here we show that Sirt6-null (Sirt6^−/−) mice developed chronic liver inflammation starting at ∼2 months of age, and all animals were affected by 7–8 months of age. Deletion of Sirt6 in T cells or myeloid-derived cells was sufficient to induce liver inflammation and fibrosis, albeit to a lesser degree than that in the global Sirt6^−/− mice, suggesting that Sirt6 deficiency in the immune cells is the cause. Consistently, macrophages derived from the bone marrow of Sirt6^−/− mice showed increased MCP-1, IL-6, and TNFα expression levels and were hypersensitive to LPS stimulation. Mechanistically, SIRT6 interacts with c-JUN and deacetylates histone H3 lysine 9 (H3K9) at the promoter of proinflammatory genes whose expression involves the c-JUN signaling pathway. Sirt6-deficient macrophages displayed hyperacetylation of H3K9 and increased occupancy of c-JUN in the promoter of these genes, leading to their elevated expression. These data suggest that Sirt6 plays an anti-inflammatory role in mice by inhibiting c-JUN-dependent expression of proinflammatory genes.     

Heat-induced K+ loss, trypan blue uptake, and cell lysis in different cell lines: effect of serum

Experiments were performed with three different cell lines, mouse fibroblast LM cells, HeLa S3 cells, and Ehrlich Ascites Tumor (EAT) cells, to establish the possible importance of hyperthermic-induced alterations in cellular K+ content in the mechanism of cell killing by heat. At different time points after the hyperthermic treatment, the K+ content in the cells, the uptake of the dye Trypan Blue (TB), and cell lysis were assayed. Heat-induced K+ loss preceded TB uptake which was followed by the heat-induced cell lysis. Lysis was assayed as disappearance of cells by counting the cells at different time points in a hematocytometer. The presence of serum during and after the heat treatment was of considerable importance with respect to K+ loss and TB uptake. K+ loss and TB uptake after the heat treatment were less when serum was present during and after hyperthermia. To protect against cell lysis, however, the serum had to be present during a preincubation period of 24 h. Clonogenic ability was not affected by the presence of serum. It is concluded that the intracellular K+ level of hyperthermic-treated cells is not a direct cause for cell killing and that heat-induced alterations in the cell leading to cell lysis are different from the processes decreasing cellular K+ content and permeabilizing the plasma membrane for trypan blue.     

Subpopulations of mononuclear leukocytes associated with inhibition of Ehrlich ascites tumor growth by treatment with Bothrops jararaca venom

Snake venoms have been used as antineoplastic substances in several experimental models. We demonstrated in previous studies that Bothrops jararaca venom (BjV) induces inhibition of Ehrlich ascites tumor (EAT) growth accompanied by an increase of mononuclear (MN) leukocytes in all groups inoculated with EAT and/or venom. The objective of the present study was to characterize the subpopulations of MN leukocytes involved in the inhibition of EAT growth by treatment with BjV. Swiss mice were inoculated with 1.0x10(3) EAT cells by the intraperitoneal route and treated with 0.4 mg/kg of BjV by the same route (Group TV). Treatment was started 24 h after tumor cell inoculation and consisted of five intraperitoneal injections performed at 72 h intervals. After 2, 8 and 14 days, groups of animals were sacrificed and the number of B, TCD4 and TCD8 lymphocytes, macrophages and natural killer cells present in the peritoneal cavity was determined by flow cytometry. The control group consisted of animals inoculated with EAT and treated with 0.1 ml of saline under the same conditions as the experimental group (Group T). Two additional control groups consisted of animals not inoculated with EAT and treated with saline or venom. Data were analyzed statistically by the Kruskal-Wallis non-parametric test for independent samples. On the 2nd and 8th day we observed a difference between groups T and TV (group T > group TV) for all cell types, except natural killer cells, that only differed on the 2nd day. However, on the 14th day there was no difference in MN cells among groups. These data suggest that the inhibition of EAT is related to the toxic action of BjV on tumor cells and/or to the proteolytic effect of the venom on the mediators produced by the cells for growth modulation.     

Effects of trypsin on X-ray-induced cell killing, chromosome abnormalities and kinetics of DNA repair in mammalian cells

When cells are trypsinized before irradiation a potentiation of X-ray damage may occur. This is known as the 'trypsin effect'. Potentiation of X-ray damage on cell killing was seen in V79 Chinese hamster cells but was marginal in Chinese hamster ovary (CHO K1) cells and not evident in murine Ehrlich ascites tumour (EAT) cells. Trypsinization did however increase the number of X-ray-induced chromosomal abnormalities in all 3 lines. To investigate the possibility that trypsin acts by digestion of proteins in chromatin, further experiments were performed to monitor DNA damage and repair. Induction of DNA breaks by X-rays was unaffected by trypsin but trypsinized EAT (suspension) cells repaired single-strand breaks (ssb) less rapidly than controls indicating an inhibitory effect of trypsin on ssb repair. However double-strand break (dsb) repair was unaffected by trypsin. It was also found that the EDTA solution in which the trypsin was dissolved also contributes to the inhibition of dsb repair. The results show that trypsinization can enhance X-ray-induced cell killing, chromosomal damage and DNA repair, the effect varying between cell lines.