ATP depletion is an important factor in DHEA-induced growth inhibition and apoptosis in BV-2 cells

Dehydroepiandrosterone (DHEA), a major steroid secreted by the adrenal gland, is known to have antiproliferative effects but the mechanism is unclear. We recently reported that DHEA induces growth inhibition and apoptosis in BV-2 cells and these effects are inversely associated with glucose concentrations in the medium. Here, we further showed that incubation of BV-2 cells with DHEA under glucose deprivation (G0) led to dose- and time-dependent decrease in cellular ATP levels. The decrease in ATP preceded growth inhibition and apoptosis induced by DHEA and all these effects of DHEA (i.e., loss of ATP, antiproliferation and apoptosis) were prevented by glucose added at 4.5 mg/ml (G4.5) during incubation. In addition, two ATP-depleting agents, iodoacetic acid (IAA) and 2,4-dinitrophenylhydrazine (2,4-DNP), potentiated the antiproliferative and apoptotic effects of DHEA. We also determined whether decrease in nucleic acid synthesis (due to glucose-6-phosphate dehydrogenase inhibition by DHEA) contributes to DHEA-induced antiproliferation and apoptosis. Using a mixture of deoxyribonucleotides (DN) and ribonucleotides (RN), we showed that DNRN had little or no effect on DHEA-induced growth inhibition and apoptosis. We also showed that mevalonic acid (MVA) did not affect DHEA-induced antiproliferation and apoptotic effects, indicating that protein isoprenylation and membrane association are not affected by DHEA in BV-2 cells. Taken together, the present results demonstrate that depletion of ATP by DHEA plays an important role in DHEA-induced antiproliferation and apoptosis of BV-2 cells.     

The magnitude of methylmercury-induced cytotoxicity and cell cycle arrest is p53-dependent

BACKGROUND: Methylmercury (MeHg), a ubiquitous environmental contaminant, is a known potent teratogen selectively affecting the developing central nervous system. While a definitive mechanism for MeHg-induced developmental neurotoxicity remains elusive, in utero exposure has been associated with reduced brain weight and reduction in cell number. This suggests early toxicant interference with critical molecular signaling events controlling cell behavior, i.e., proliferation. METHODS: To examine the role of p53, a major regulator of the G(1)/S and G(2)/M cell cycle checkpoints, in MeHg toxicity, we isolated GD 14 primary embryonal fibroblasts from homozygous wild-type p53 (p53+/+) and homozygous null p53 (p53-/-) mice. Cells were treated at passages 4-7 for 24 or 48 hr with 0, 1.0, or 2.5 microM MeHg and analyzed for effects on viability, cell cycle progression (using BrdU-Hoechst flow cytometric analysis), and apoptosis via annexin V-FITC and propidium iodide (PI) staining. RESULTS: The p53+/+ cells are more sensitive than p53-/- cells to MeHg-induced cytotoxicity, cell cycle inhibition, and induction of apoptosis: at 24 hr, 2.5 microM MeHg reduced p53+/+ cell viability to 72.6% +/- 3.2%, while p53-/- viability was 94.6% +/- 0.4%. The p53-/- cells underwent less necrosis and less apoptosis following MeHg treatment. MeHg (2.5 microM) also halted all cycling in the p53+/+ cells, while 42.6% +/- 7.2% of p53-/- cells were able to reach a new G(0)/G(1) in 48 hr. Time- and dose-dependent accumulation of cells in G(2)/M phase (1.0 and 2.5 microM MeHg) was observed independent of the p53 genotype; however, the magnitude of change was p53-dependent. CONCLUSIONS: These studies suggest that MeHg-induced cell cycle arrest occurs via both p53-dependent and -independent pathways in our model system; however, cell death resulting from MeHg exposure is highly dependent on p53.     

Critical evaluation of techniques to detect and measure cell death--study in a model of UV radiation of the leukaemic cell line HL60.

The reliability of eight distinct methods (Giemsa staining, trypan blue exclusion, acridine orange/ethidium bromide (AO/EB) double staining for fluorescence microscopy and flow cytometry, propidium iodide (PI) staining, annexin V assay, TUNEL assay and DNA ladder) for detection and quantification of cell death (apoptosis and necrosis) was evaluated and compared. Each of these methods detects different morphological or biochemical features of these two processes. The comparative analysis of the 8 techniques revealed that AO/EB (read in fluorescence microscopy) provides a reliable method to measure cells in different compartments (or pathways) of cell death though it is very time consuming. PI staining and TUNEL assay were also sensitive in detecting very early signs of apoptosis, but do not allow precise quantification of apoptotic cells. These three methods were concordant in relation to induction of apoptosis and necrosis in HL60 cells with the various UV irradiation time periods tested. Both AO/EB (read by flow cytometry) and annexin V-FITC/PI failed to detect the same number of early apoptotic cells as the other three methods. Trypan blue is valueless for this purpose. Giemsa and DNA ladder might be useful as confirmatory tests in some situations.     

Ellipticine induces apoptosis through p53-dependent pathway in human hepatocellular carcinoma HepG2 cells

Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole), one of the simplest naturally occurring alkaloids, was isolated from the leaves of the evergreen tree Ochrosia elliptica Labill (Apocynaceae). Here, we reported that ellipticine inhibited the cell growth of human hepatocellular carcinoma cell line HepG2 and provided molecular understanding of this effect. The XTT assay results showed that ellipticine decreased the cell viability of HepG2 cells in a dose- and time-dependent manner, and the IC50 value was 4.1 microM. Furthermore, apoptosis induction by ellipticine in HepG2 cells was verified by the appearance of DNA fragmentation and annexin V-FITC/propidium iodide (PI) staining assay. Ellipticine treatment was found to result in the upregulation of p53, Fas/APO-1 receptor and Fas ligand. Besides, ellipticine also initiated mitochondrial apoptotic pathway through regulation of Bcl-2 family proteins expression, alteration of mitochondrial membrane potential (DeltaPsim), and activation of caspase-9 and caspase-3. Taken together, ellipticine decreased the cell growth and induced apoptosis in HepG2 cell.     

CRH functions as a growth factor/cytokine in the skin

We tested the effect of CRH and related peptides in a large panel of human skin cells for growth factor/cytokine activities. In skin cells CRH action is mediated by CRH-R1, a subject to posttranslational modification with expression of alternatively spliced isoforms. Activation of CRH-R1 induced generation of both cAMP and IP3 in the majority of epidermal and dermal cells (except for normal keratinocytes and one melanoma line), indicating cell type-dependent coupling to signal transduction pathways. Phenotypic effects on cell proliferation were however dependent on both cell type and nutrition conditions. Specifically, CRH stimulated dermal fibroblasts proliferation, by increasing transition from G1/0 to the S phase, while in keratinocytes CRH inhibited cell proliferation. In normal and immortalized melanocytes CRH effect showed dichotomy and thus, it inhibited melanocyte proliferation in serum-containing medium CRH through G2 arrest, while serum free media led instead to CRH enhanced DNA synthesis (through increased transition from G1/G0 to S phase and decreased subG1 signal, indicating DNA degradation). CRH also induced inhibition of early and late apoptosis in the same cells, demonstrated by analysis with the annexin V stains. Thus, CRH acts on epidermal melanocytes as a survival factor under the stress of starvation (anti-apoptotic) as well as inhibitor of growth factors induced cell proliferation. In conclusion, CRH and related peptides can couple CRH-R1 to any of diverse signal transduction pathways; they also regulate cell viability and proliferation in cell type and growth condition-dependent manners.     

脱氢表雄酮调节HepG2/GFP-HBx细胞p16基因甲基化及其与细胞周期和凋亡的相关性

目的探讨HBx与p16基因甲基化的关系,研究脱氢表雄酮（DHEA）对p16基因甲基化以及细胞周期和细胞凋亡的调节作用。方法以HepG2、HepG2/GFP和HepG2/GFP-HBx三种细胞为材料,采用MTT法检测细胞生长;流式细胞术检测细胞周期和凋亡率;MSP-PCR检测p16基因甲基化水平。比较分析HBx与p16基因启动子甲基化、DHEA与各检测指标的关系。结果HepG2/GFP-HBx细胞与HepG2细胞和HepG2/GFP细胞相比,细胞的增殖速度提高（P〈0.05）,G0/G1期细胞减少,S期细胞增多（P〈0.05）,凋亡率降低（P〈0.05）;HepG2/GFP-HBx细胞的p16基因启动子呈现高水平甲基化,HepG2和HepG2/GFP细胞呈现高水平非甲基化。100μmol/L的DHEA使三种细胞的增殖速度降低（P〈0.05）,G0/G1期细胞增加,S期细胞减少（P〈0.05）,凋亡率提高（P〈0.05）。DHEA可下调HepG2/GFP-HBx细胞的p16基因启动子甲基化水平,但对HepG2和HepG2/GFP细胞的p16基因启动子甲基化水平不产生影响。结论HBx引起肝癌细胞p16基因甲基化,并缩短细胞周期抑制细胞凋亡;DHEA可明显下调HBx引起的p16基因甲基化水平,延长细胞周期促进细胞凋亡,在无HBx基因整合的情况下,DHEA对肝癌细胞生长、细胞周期和凋亡的影响不通过p16基因甲基化的途径实现。     

Apoptosis induced by selenium in human glioma cell lines

Several studies have shown that selenium can inhibit tumorigenesis in tissues. However, little is known about the mechanism and the effect of selenium on DNA, especially in brain tumor cells. In this study we examined the biological effect of selenium on human glioma cell lines (A172 and T98G). Selenium exhibited an antiproliferative effect on these cell lines (and induced the typical ladder pattern of DNA fragmentation commonly found in apoptosis), which were prevented by catalase. Few effects of selenium on NTI4 fibroblasts were found. These findings demonstrate that selenium may induce, by apoptosis, cell death of human glioma cell lines, which are resulting from free radical oxygen forming.     

Characterization of Vero cell growth and death in bioreactor with serum-containing and serum-free media

The density of viable cells in a culture results from a balance between cell proliferation and cell death. The aim of this study was to characterize and compare these two phenomena in Vero cell cultures in one serum containing medium (ScA) and one serum free medium (SfB) in bioreactors. Cell growth was evaluated by cell counting(after crystal violet staining) and cell cycle analysis. Necrosis and apoptosis were characterized and quantified by measuring the release of LDH, trypan blue exclusion,annex in V-FITC/PI staining and TUNEL assay. ScA supported a higher maximal viable-cell density(2.3 × 10⁶ vs. 1.8 × 10⁶ cells ml^-1). However, cell cycle analysis showed that cell division was more active in SfB than in ScA. LDH release in the supernatant increased much earlier in SfB than in ScA (one vs. five days), but trypan blue counts showed no apparent difference in the viability of the cultures. Apoptosis, evidenced by annexin V-FITC/PI staining, could be detected in the population of suspension cells detached from microcarriers, but not among adherent cells; positivity of the TUNEL assay occurred later than that of the annexin V-FITC/PI staining. Our data indicate that the lower cell yield in SfB,compared with that in ScA, results from a higher cell death rate. Apparently, cells die from apoptosis followed by secondary necrosis. This revised version was published online in July 2006 with corrections to the Cover Date.     

The cytotoxic nature of Acanthopanax sessiliflorus stem bark extracts in human breast cancer cells

Acanthopanax sessiliflorus, a small woody shrub has traditionally been referred to have anticancer activity, but it has not been scientifically explored so far. Therefore, to investigate the anticancer effects of A. sessiliflorus stem bark extracts (ASSBE), MDA-MB-231 and MCF-7 human breast cancer cells were treated with one of its bioactive fractions, n-hexane (ASSBE-nHF). Cytotoxicity (24 h) was determined by MTT assay and antiproliferative effect was assessed by counting cell numbers after 72 h treatment using hemocytometer. The role of ASSBE-nHF on apoptosis was analysed by annexin V-FITC/PI, Hoechst 33342 staining, DNA fragmentation pattern and immunoblotting of apoptosis markers. For the assay of enhanced production of ROS and mitochondrial membrane depolarization, specific stains such as DCFH-DA and JC-1 were used, respectively. To understand the mode of action of ASSBE-nHF on MCF-7 cells, cells were pre-treated with antioxidant, n-acetylcysteine. The hexane fraction of ASSBE showed maximum activity towards human breast cancer cells compared to other two fractions at a minimal concentration of 50 μg/ml. The annexin V-FITC/PI, Hoechst 33342 staining, DNA fragmentation and immunoblotting assays showed that ASSBE-nHF induces non-apoptotic cell death in MCF-7 and MDA-MB-231 cells. ASSBE-nHF significantly increased the production of ROS and decreased the mitochondrial membrane potential (MMP) in MCF-7 cells. Similarly, it decreased the MMP in MDA-MB-231 cells, but had no effect on ROS production. Further, the cytotoxic effect of ASSBE-nHF in MCF-7 cells was not significantly reversed even in the presence of n-acetylcysteine, an antioxidant. These findings revealed that ASSBE-nHF induces non-apoptotic cell death via mitochondria associated with both ROS dependent and independent pathways in human breast cancer cells.     

香烟烟雾提取物抑制肺泡上皮细胞的增殖并诱导其凋亡

香烟烟雾提取物（cigarette smoke extract,CSE）中含有丰富的氧化剂和自由基,由它所引起的氧化应激可导致肺泡壁的损伤进而发展为肺气肿.近年来,围绕CSE损伤肺泡壁作用机制的研究较为活跃,但其结果却一直存在着分歧.本实验的目的是观察CSE对肺泡Ⅱ型上皮细胞的损伤作用并探讨与其相关的分子机制.MTT比色法的结果显示,CSE以时间和剂量依赖性的方式降低细胞的增殖活力,流式细胞术的分析结果表明细胞增殖周期被阻滞在G1/S期.Hoechst 33258染色以及透射电镜观察从形态上确认CSE诱导细胞凋亡的发生,DNA梯的出现和Annexin V-FITC/碘化丙啶双染色的结果从分子水平得到进一步的证实.同时,运用流式细胞术检测到CSE诱导的凋亡伴随着Fas受体的高表达和caspase-3的显著活化.另外,使用H2DCFDA染色,经激光共聚焦显微镜术测得细胞内氧自由基在细胞受到CSE刺激以后大量快速积累.结果表明CSE能够抑制肺泡Ⅱ型上皮细胞来源的A549细胞的生长和增殖,并诱导细胞凋亡,由Fas受体所介导的死亡受体途径参与此凋亡过程,而CSE所引起的氧化应激则可能是阻止肺泡上皮细胞生长增殖并诱导其凋亡的始动因素.     

Adrenomedullin is anti-apoptotic in osteoblasts through CGRP1 receptors and MEK-ERK pathway

Adrenomedullin (ADM) has been shown to mediate multifunctional responses in cell culture and animal system such as regulation of growth and apoptosis. ADM stimulates the proliferation of osteoblasts in vitro and promotes bone growth in vivo. The ability of ADM to influence osteoblastic cell number through inhibition of apoptosis has not yet been studied. To address this question we have investigated its effect on the apoptosis of serum-deprived osteoblastic cells using mouse MC3T3-E1 cells which express both ADM and ADM receptors. Treatment with ADM significantly blunted apoptosis, evaluated by caspase-3 activity, DNA fragmentation quantification and annexin V-FITC labeling. This effect was abolished by the subtype-1 CGRP receptor antagonist, CGRP(8-37). Both ADM and its specific receptor antagonist, the (22-52) ADM fragment exhibited a similar anti-apoptotic effect. Thus, our data suggest that ADM exerts anti-apoptotic effects through CGRP1 receptors. This was substantiated by a similar protective effect of CGRP on MC3T3-E1 cells apoptosis. Accordingly, neutralization of endogenous ADM by a specific antibody enhanced apoptosis. Finally, the selective inhibitor of MAPK kinase (MEK), PD98059, abolished the apoptosis protective effect of ADM and prevented ADM activation of ERK1/2. These data show that ADM acts as a survival factor in osteoblastic cells via a CGRP1 receptor-MEK-ERK pathway, which provides further understanding on the physiological function of ADM in osteoblasts.     

Effect of non-esterified fatty acids on bovine granulosa cell steroidogenesis and proliferation in vitro

In high-yielding dairy cows, the negative energy balance (NEB) during the first weeks post partum may influence dominant follicle growth and steroidogenesis. Since non-esterified fatty acid (NEFA) concentrations are elevated during NEB and are shown to be toxic for several cell types, we investigated the individual and combined effects of the three main NEFA's on granulosa cell proliferation and steroidogenesis in vitro. Granulosa cells from large follicles were cultured for two days in serum free medium in the presence of palmitic (C16:0) (PA), stearic (C18:0) (SA) and/or oleic acid (C18:1) (OA). Addition of 150, 300 or 500 microM of PA and SA inhibited cell proliferation (P<0.05) while OA only elicited such an effect at 500 microM (P<0.01). In the combination treatment (150 microM of each fatty acid), cell numbers were also reduced (P<0.01). These inhibitory effects on cell number are partly due to the induction of apoptosis by these NEFA's, as was demonstrated by annexin V-FITC/propidium iodide staining of the granulosa cells. Oestradiol-17beta production was stimulated by all doses of PA, by 300 and 500 microM of SA and by 500 microM of OA (P<0.05). Combined treatment with 150 microM of each fatty acid also stimulated oestradiol-17beta production per 10(4) cells (P<0.05). We can conclude that PA, SA and to a lesser degree OA modulate granulosa cell proliferation and steroidogenesis in vitro. These effects may be involved in the occurrence of ovarian dysfunction during the postpartum period in high-yielding dairy cows.     

Molecular mechanisms involved in the antiproliferative action of protein tyrosine phosphatase inhibitor potassium bisperoxo(1,10-phenanthroline)oxovanadate

Potassium bisperoxo(1,10-phenanthroline)oxovanadate, bpV(phen), a powerful protein phosphotyrosine phosphatase inhibitor and a potent insulinomimetic, influenced three fundamental cellular processes in HL-60 human leukemic cells: 1) inhibition of proliferation, 2) induction of differentiation and 3) apoptotic cell death. In the presence of micromolar concentrations of bpV(phen) cell number and DNA synthesis decreased progressively with time of incubation. A single treatment with bpV(phen) (3 microM) activated a differentiation program; after 6 days of incubation 82% of cells were differentiated, but differentiation started already within the first 24 h. Concentrations of 5-10 microM bpV(phen) caused the characteristic DNA ladder pattern, starting after 4.5 h. Differentiation in HL-60 cells appear to be associated with activation of extracellular signal-regulated kinase while apoptosis is connected with phosphorylation and activation of both extracellular signal-regulated kinase and c-Jun N-terminal kinase in a concentration and time-dependent manner. The antiproliferative and apoptotic action of bpV(phen) could be exploited in combination chemotherapy in leukemia.     

2-Methoxyestradiol Induces Mitotic Arrest,Apoptosis, and Synergistic Cytotoxicity with Arsenic Trioxide in Human Urothelial Carcinoma Cells

2-Methoxyestradiol (2-ME), an endogenous derivative of 17β-estradiol, has been reported to elicit antiproliferative responses in various tumors. In this study, we investigated the effects of 2-ME on cell viability, proliferation, cell cycle, and apoptosis in human urothelial carcinoma (UC) cell lines. We used two high-grade human bladder UC cell lines (NTUB1 and T24). After treatment with 2-ME, the cell viability and apoptosis were measured by MTT assay and flow cytometry (fluorescence-activated cell sorting), with annexin V-FITC staining and propidium iodide (PI) labeling. DNA fragmentation was analyzed by agarose gel electrophoresis. Flow cytometry with PI labeling was used for the cell cycle analyses. The protein levels of caspase activations, poly (ADP-ribose) polymerase (PARP) cleavage, phospho-histone H2A.X, phospho-Bad, and cell cycle regulatory molecules were measured by Western blot. The effects of the drug combinations were analyzed using the computer software, CalcuSyn. We demonstrated that 2-ME effectively induces dose-dependent cytotoxicity and apoptosis in human UC cells after 24 h exposure. DNA fragmentation, PARP cleavage, and caspase-3, 7, 8, 9 activations can be observed with 2-ME-induced apoptosis. The decreased phospho-Bad (Ser136 and Ser155) and mitotic arrest of the cell cycle in the process of apoptosis after 2-ME treatment was remarkable. In response to mitotic arrest, the mitotic forms of cdc25C, phospho-cdc2, cyclin B1, and phospho-histone H3 (Ser10) were activated. In combination with arsenic trioxide (As₂O₃), 2-ME elicited synergistic cytotoxicity (combination index <1) in UC cells. We concluded that 2-ME significantly induces apoptosis through decreased phospho-Bad and arrests bladder UC cells at the mitotic phase. The synergistic antitumor effect with As₂O₃ provides a novel implication in clinical treatment of UC.     

Entry into the stationary phase is associated with a rapid loss of viability and an apoptotic-like phenotype in the opportunistic pathogen Aspergillus fumigatus

When the opportunistic pathogen Aspergillus fumigatus entered the stationary phase, there was a rapid loss in cell viability which was associated with the appearance of markers characteristic of apoptosis, namely annexin V-FITC binding to the cytoplasmic membrane, demonstrating exposure of phosphatidylserine to the outer leaflet of the membrane; and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) staining of the nuclei, indicating DNA fragmentation. This was followed later by a loss of membrane integrity as revealed by propidium iodide staining. The development of the apoptotic phenotype was blocked when the protein synthesis inhibitor cycloheximide was added to the culture 1h prior to the onset of the stationary phase, demonstrating active participation of the cell. In addition, intracellular activity against substrates specific for caspase-1 and -8 also increased on stationary phase entry and the development of the apoptotic phenotype was blocked when the cell permeant caspase inhibitor Z-FAD-fmk was present in the medium. Cell death in A. fumigatus during the stationary phase therefore appears to share similarities to apoptotic cell death in higher eukaryotes and to be dependent on a caspase-like activity.     

Studies of the cell cycle of in vitro cultured skin fibroblasts in goats: work in progress

The effect of serum starvation and olomoucine treatment on the cell cycle and apoptosis of goat skin fibroblasts cultured in vitro is reported in this paper. The cells were obtained from the ear of a female goat 1.5 years of age. Analysis of cell cycle distribution by fluorescence-activated cell sorting (FACS) showed that 3.4, 60.8 and 15.1% of normally cycling cells were at G1, G0 and S phase, respectively. Serum starvation for 1, 3 and 5 days arrested 70.1, 70.2 and 83.4% cells, respectively, at G0/G1 phase. Seventy-eight percent of confluent cells were at G0/G1 stage, but in contrast to the serum starved group, this high percentage of G0/G1 cells was mainly associated with G1 cells. Of cells not deprived of serum, 73.6% were arrested at G1/G0 when treated with 100 microM olomoucine for 9 h compared to 85.5% of cells that had been starved of serum for 2 days (co-inhibition) (P<0.01). After co-inhibition, 45% of cells entered S phase when re-cultured in normal medium for 5 h, indicating that the inhibition was reversible. Under normal culture conditions, 1.2% of cells underwent apoptosis. Serum starvation for 1, 2, 3, 5 and 10 days caused apoptosis in 1.7, 3.9, 4.5, 11.7 and 90.3% of cells, respectively. Treatment with 100 microM olomoucine for 9h did not increase the number of apoptotic cells significantly (1.9%, P>0.05). When cells were co-inhibited, 4.1% of cells underwent apoptosis. In conclusion, although serum withdrawal for 5 days or more effectively arrested cells at G0/G1 stages, it increased apoptosis of cells significantly. However, co-inhibition by serum withdrawal and olomoucine treatment was found to be an appropriate treatment to obtain more healthy G0/G1 cells based on the low percentage of apoptotic cells after treatment.     

alpha-Bisabolol, a nontoxic natural compound, strongly induces apoptosis in glioma cells

In this study, alpha-bisabolol, a sesquiterpene alcohol present in natural essential oil, was found to have a strong time- and dose-dependent cytotoxic effect on human and rat glioma cells. After 24 h of treatment with 2.5-3.5 microM alpha-bisabolol, the viability of these cells was reduced by 50% with respect to untreated cells. Furthermore, the viability of normal rat glial cells was not affected by treatment with alpha-bisabolol at the same concentrations as above. Glioma cells treated with high concentration of alpha-bisabolol (10 microM) resulted in a 100% cell death. Judging from hypo-G1 accumulation, poly(ADP-ribose) polymerase cleavage, and DNA ladder formation, the cytotoxicity triggered by alpha-bisabolol resulted from apoptosis induction. Moreover, the dissipation of mitochondrial-inner transmembrane potential and the release of cytochrome c from mitochondria indicated that, in these glioma cells, apoptosis occurred through an intrinsic pathway. As pointed out by the experimental results, alpha-bisabolol may be considered a novel compound able to inhibit glioma cell growth and survival.     

Dual Role of Dextran Sulfate 5000 Da as Anti-Apoptotic and Pro-Autophagy Agent

Dextran sulfate 5,000 Da (DS), a sulfated polysaccharide, has been used in recombinant mammalian cell cultures to prevent cell aggregation, thereby increasing cell viability. Previous studies using Chinese hamster ovary (CHO) suspension cultures had shown that low concentrations of DS are related to an inhibition of apoptosis. In this study, DS was used on anchorage-dependent CHO cells producing erythropoietin (EPO), in order to investigate the effect of this molecule on anti-apoptotic and pro-survival cellular pathways. DS 5,000 Da treatment was shown to prolong the life of cells and increase productivity of EPO by 1.8-fold comparing with controls, in standard batch conditions. At a molecular level, we show that DS inhibits apoptosis by DNA fragmentation delay and decrease of annexin V-labeled cells, causes a G0/G1 cell cycle arrest, decreases p53 expression and increases the pro-survival factor Hsc70 expression. DS treatment also resulted in an enhanced LC3-I to LC3-II conversion and increased autophagosomes formation employing tagged-LC3. Our data show, for the first time, that low doses of DS may promote autophagy in different cell lines. These findings suggest that a better understanding and manipulation of phenomenon of autophagy could be of crucial importance in the bio-pharmaceutical industry, in particular in the field of protein production.     

Cytotoxicity induced by nivalenol, deoxynivalenol, and fumonisin B1 in the SF-9 insect cell line

The toxicity of the mycotoxins nivalenol (NIV), deoxynivalenol (DON), and fumonisin B1 (FB1) was studied in the lepidopteran Spodoptera frugiperda (SF-9) cells, by the trypan blue dye-exclusion and 3-(4,5-dimethylthiozole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) tests, uptake analyses of cytotoxicity, and cell metabolism, respectively. Deoxyribonucleic acid analysis by flow cytometry was used to identify apoptosis and cell cycle distribution. After 48 h of exposure, the MTT and trypan blue dye-exclusion tests indicated that NIV was significantly more toxic than DON, and both were significantly more toxic than FB1. The IC50 (mycotoxin concentration resulting in 50% inhibition of proliferation) values for NIV and DON were 4.5 and 41 microM, and the CC50 (mycotoxin concentration that caused 50% cytotoxicity) values were 9.5 and 45 microM, respectively. At the highest concentration of FB1 (100 microM), there was 80% viability. With the same incubation time, cell cycle distribution showed an arrest of cells in the G0/G1 phase in the presence of NIV (up to 0.3 microM), DON (up to 3 microM), and FB1 (up to 10 microM). Morphological evidence of apoptosis was related to the toxicity of the substances in that the more toxic NIV induced late apoptosis, whereas DON and FB1 produced less-severe morphological changes characteristic of early apoptosis. This study suggests that NIV is more toxic than DON, which in turn is more toxic than FB1. These mycotoxins can modify the normal progression of the cell cycle and induce an apoptotic process.