Shift in variability of 2,4-dichlorophenoxyacetic acid-deprived pear cell suspensions

Quiescent 2,4D-dependent pear fruit cells (Pyrus communis L.) were cultured in a bioreactor in the absence of 2,4-Dichlorophenoxyacetic acid. Effects on the ability to divide following transfer into different concentrations of auxin (0; 0.45; 2.3; 4.5; 6.8; 9 M) are studied and discussed. Changes in cell number and in cell viability during subculture were examined (at all 2,4-D concentrations). In the presence of 2.3, 4.5 and 6.8 M of 2,4-D, in the standard medium, pear cells undergo a classical growth cycle and cell mortality increased only after 16 days. The level of ploidy estimated by cytophotometry shows, after previous starvation, a significant shift only for 4.5 M 2,4-D concentration, the value of the usual culture medium of the studied strain.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid     

Inhibition of macrophage migration inhibitory factor or its receptor (CD74) attenuates growth and invasion of DU-145 prostate cancer cells

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is overexpressed in prostate cancer, but the mechanism by which MIF exerts effects on tumor cells remains undetermined. MIF interacts with its identified membrane receptor, CD74, in association with CD44, resulting in ERK 1/2 activation. Therefore, we hypothesized that increased expression or surface localization of CD74 and MIF overexpression by prostate cancer cells regulated tumor cell viability. Prostate cancer cell lines (LNCaP and DU-145) had increased MIF gene expression and protein levels compared with normal human prostate or benign prostate epithelial cells (p < 0.01). Although MIF, CD74, and CD44 variant 9 expression were increased in both androgen-dependent (LNCaP) and androgen-independent (DU-145) prostate cancer cells, cell surface of CD74 was only detected in androgen-independent (DU-145) prostate cancer cells. Therefore, treatments aimed at blocking CD74 and/or MIF (e.g., inhibition of MIF or CD74 expression by RNA interference or treatment with anti-MIF- or anti-CD74- neutralizing Abs or MIF-specific inhibitor, ISO-1) were only effective in androgen-independent prostate cancer cells (DU-145), resulting in decreased cell proliferation, MIF protein secretion, and invasion. In DU-145 xenografts, ISO-1 significantly decreased tumor volume and tumor angiogenesis. Our results showed greater cell surface CD74 in DU-145 prostate cancer cells that bind to MIF and, thus, mediate MIF-activated signal transduction. DU-145 prostate cancer cell growth and invasion required MIF activated signal transduction pathways that were not necessary for growth or viability of androgen-dependent prostate cells. Thus, blocking MIF either at the ligand (MIF) or receptor (CD74) may provide new, targeted specific therapies for androgen-independent prostate cancer.     

Endocytosis in rat cultured astrocytes is inhibited by unconjugated bilirubin

Excessive hyperbilirubinemia can cause irreversible neurological damage in the neonatal period. However, the complete understanding of the pathogenesis of unconjugated bilirubin (UCB) encephalopathy remains a matter of debate. This study investigates whether UCB inhibits the endocytosis of cationized ferritin (CF) by cultured rat astrocytes. The relationship between endocytosis and MTT reduction, as well as changes on tubulin and glial fibrillary acidic protein (GFAP) assembly, were also evaluated. Inhibition of endocytosis was complete in the presence of 171 M UCB, while a marked decrease of CF labeling was noticed for 86 M UCB. In addition, MTT reduction was inhibited by 60 to 76% as UCB concentrations changed from 17 to 171 M, while alterations on both GFAP and microtubule morphology were only achieved by cell exposure to 171 M UCB. These findings indicate that inhibition of CF endocytosis in rat cortical astrocytes by UCB is a concentration-dependent process that appears to be primarily related to a direct effect on the cell membrane and not to any alteration of cytoskeletal microtubules and intermediate filaments.     

The antineoplasic agent estramustine and the derivative estramustine-phosphate inhibit secretion of interleukin-3 in leukemic cells. Possible roles of MAPs

The antineoplasic drug estramustine is an adduct of estradiol and nor-nitrogen mustard. It has been shown that this drug interferes with microtubule assembly, an effect mediated by estramustine interaction with microtubule-associated proteins (MAPS). In the present report we demonstrate that estramustine and the phosphorylated derivative of the drug, estramustine-phosphate, inhibit the secretion of interleukin-3 by WEHI-3B cells. These studies also show that the estramustine derivative specifically interacts with a MAPs component found in these cells, which exhibited characteristics ressembling those of tau protein isoforms. Western blots using a unique monoclonal antibody MTB6.22 that recognizes microtubule-binding domains on MAPs, indicated that this WEHI protein factor contained the antigenic determinant that are functionally significant for microtubule assembly. ELISA assays using this antibody, also showed a decrease in the levels of the immunoreactive protein in WEHI cells after treatment with EMP. Interestingly, it has been recently described that the action of estramustine-phosphate is mediated by a direct interaction with MAP-binding sites on the microtubule surface, which are recognized by the site-specific monoclonal antibody. These findings together with immuno-precipitation experiments using anti-interleukin-3 antibodies and the inhibitory effect of the estramustine derivative on WEHI secretion process suggest that this anti-mitotic agent may block IL-3 secretion by a mechanism involving its interaction with a tau-like MAPs component present in these cells.     

The zebrafish/tumor xenograft angiogenesis assay as a tool for screening anti-angiogenic miRNAs

The zebrafish/tumor xenograft angiogenesis assay is used to approach tumor angiogenesis, a pivotal step in cancer progression and target for anti-tumor therapies. Here, we evaluated whether the assay could allow the identification of microRNAs having an anti-angiogenic potential. For that, we transfected DU-145 prostate cancer cells with four microRNAs (miR-125a, miR-320, miR-487b, miR-492) responsive to both anti- and pro-angiogenic stimuli applied to human umbilical vein endothelial cells. After transfection, DU-145 cells were injected close to the developing subintestinal vessels of transgenic Tg(Kdrl:eGFP)s843 zebrafish embryos that express green fluorescent protein under the control of Kdrl promoter. At 72 h post-fertilization, we observed that green fluorescent protein–positive neo-vessels infiltrated the graft of DU-145 transfected with miR-125a, miR-320, and miR-487b. Vice versa, neo-vessel formation and tumor cell infiltration were inhibited when DU-145 cells transfected with miR-492 were used. These results indicated that the zebrafish/tumor xenograft assay was adequate to identify microRNAs able to suppress the release of angiogenic growth factors by angiogenic tumor cells.

Electronic supplementary material

The online version of this article (doi:10.1007/s10616-014-9735-y) contains supplementary material, which is available to authorized users.     

Amiprophos-methyl (APM): A rapid,reversible, anti-microtuble agent for plant cell cultures

Summary In plant cell suspension cultures sensitive to the herbicide amiprophos-methyl (APM), 1 to 3 M APM completely depolymerized both cortical and mitotic microtubule (MT) arrays in 1 hour. In comparison, a 2 hour application of 3 mM colchicine had no effect on MT arrays. Recovery from APM treatment occurred as early as 5 minutes after removal of APM. Short, cortical MTs were visible in 3 hours and complete MT arrays were found within 22 hours after drug removal.Sensitivity to APM-induced MT depolymerization varied according to species but was increased or decreased by varying the mitotic rate in cultures. The results indicated APM sensitivity was related to lowered stability of MT arrays in rapidly cycling cells. APM treatment may help distinguish stabilized cortical MTs in elongating cells and nonstabilized cortical MTs in rapidly dividing cells.Abbreviations MT microtubule - APM amiprophos-methyl - DMSO dimethyl sulfoxide - PBS phosphate buffered saline     

Evaluation of in vitro antioxidant and anticancer activity of <Emphasis Type="Italic">Allophylus cobbe</Emphasis> leaf extracts on DU-145 and PC-3 human prostate cancer cell lines

Allophylus cobbe (L.) Raeusch. belonging to the family Sapindaceae, is a commonly distributed small shrub in Western Ghats of India previously reported for its traditional medicinal properties. It is used for the treatment of various ailments. The present study is aimed at investigating preliminary phytochemicals, inducing the determination of the total phenolic contents, antioxidant assays and anticancer activity of A. cobbe leaf extracts on (DU-145) and (PC-3) cell lines. Preliminary phytochemical screening showed a broad spectrum of secondary metabolites. Highest amount of phenolic content was present in aqueous extract (91.96 ± 0.61 mg/g GAE) and it also proved to have the most potent antioxidant activity at a concentration of 100 mg/ml (64.71 ± 0.15%). IC₅₀ value was (431.10 ± 15.05 µg/mL) for DU-145 and (362.08 ± 24.17 µg/mL) for PC-3 cell lines while the standard drug paclitaxel showed an IC₅₀ value of 0.3 µM/mL. Morphological changes was observed in cancerous cells undergoing apoptosis in human prostate cancer cell lines (DU-145 and PC-3) while the extract showed no cytotoxicity towards normal cells (MEF-L929). It can be concluded that the tested extracts holds significant antioxidant and anticancer activities. However further investigation on lead compounds of A. cobbe will enable its therapeutic use.     

Pyruvate blocks zinc-induced neurotoxicity in immortalized hypothalamic neurons

Zinc is an essential trace element and present at high concentrations in the central nervous system. Recent studies have revealed that excess amount of extracellular zinc is neurotoxic, and that the disruption of zinc homeostasis may be related to various neurodegenerative diseases. Zinc (25–100 M) caused significant death of immortalized hypothalamic neuronal cells (GT1-7 cells) in a dose- and time-dependent manner. LD₅₀ was estimated to be 34 M. The degenerated cells were TUNEL-positive and exhibited apoptosis-like characteristics. Preadministration of sodium pyruvate (1–2 mM), a downstream energy substrate, inhibited the zinc-induced neurotoxicity in GT1-7 cells. GT1-7 cells can be used as a good tool for the investigation of zinc neurotoxicity in the hypothalamus.     

Sites of microtubule reorganization in tobacco BY-2 cells during cell-cycle progression

Summary The sites of microtubule (MT) reorganization were examined in synchronized tobacco BY-2 cells. The MTs of these cells were completely destroyed by a combined cold and drug treatment at 0 °C with 100 M propyzamide for 3 h. After the cells were washed and cultured at 30 °C, the reorganization of MTs was observed in detail. Sites for MT reorganization at each stage of the cell cycle were identified on the cell cortex and nuclei, the mitotic apparatus, the nuclei (or the nuclei and cell cortex), and the cell cortex in the S-G₂ phase, M phase, M/G₁ interface, and g₁ phase, respectively. The polypeptide synthesis elongation factor (EF)-1 is co-localized with these sites of MT reorganization. At some stages, microfilaments (MFs) were found to be involved in the reorganization of MTs. Based on these results, the mode of MT reorganization during cell cycle progression is discussed.Abbreviations EF-1 elongation factor 1 - MAP microtubule-associated protein - MF microfilament - MIs mitotic indices - MT microtubule     

Shoot and root organogenesis of Camellia sasanqua

In vitro-derived shoot tips, (10 mm) taken from primary cultures of Camellia sasanqua L., were evaluated for organogenesis when cultured on a half-strength MS medium supplemented with various concentrations of NAA, IBA, BA and GA₃. Maximum shoot proliferation and growth for juvenile and mature tissue was obtained when 0.54 M NAA, 8.8 M BA plus 14.4 to 28.9 M GA₃ was added to the culture media, with a pH between 4.5 and 5.0. In vitro-derived shoots (20 mm) from mature C. sasanqua Day Dream and juvenile C. sasanqua cultures initiated roots in vitro after immersion in 2.5 mM IBA for 30 min. Sixty percent of the mature shoots and 90% of the juvenile shoots initiated roots within 3 weeks of treatment with IBA.Abbreviations MS Murashige and Skoog (1962) - IBA lH-indole-3-butanoic acid - BA N-(phenyl-methyl)-lH-purine-6-amine - GA₃ gibberellic acid - kinetin N-(Z-furanyl-methyl)-lH-purine-6-amine - NAA l-naphthaleneacetic acid - L Linear - Q Quadratic     

Lead‐related effects on rat fibroblasts

Lead (Pb) is an environmental toxicant that can induce structural and functional abnormalities of multiple organ systems, including the central nervous and the immune systems. The aim of this study was to evaluate the effects of extracellular Pb supplementation on the cellular content of the metal and on the proliferation and the survival of normal rat fibroblasts.We found that the concentration of Pb in the culture medium was 0.060 M and the normal Pb concentration in rat fibroblasts was 3.1 ± 0.1 ng/10⁷ cells. Then we exposed the cells to increasing concentration of Pb (as Pb acetate) from 0.078–320 M. We observed a dosedependent inhibition of cell proliferation after 48 h, which was already apparent at a concentration of 0.312 M (p = 0.122) and became statistically significant for concentration higher than 0.625 M (p = 0.0003 at 5 M). Cell proliferation was completely compromised at 320 M Pb total inhibition of cell proliferation.To investigate the mechanisms of Pbmediated inhibition of cell proliferation, we evaluated the occurrence of apoptosis in the same cells and found that cytosolic DNA fragments, hallmark of apoptotic cell death, increased significantly at Pb concentrations from 2.5–10.0 M. The occurrence of apoptosis was also confirmed by FACS analysis which showed the appearance of a subdiploid peak at Pb concentrations from 5–20 M. The distribution of cells in the cell cycle showed a dosedependent accumulation of cells in the G₀/G₁ phase mainly compensated by a decrease in the percentage of cells in the S phase. In conclusion, our results demonstrate that induction of apoptosis contributes to the Pbinduced inhibition of cell proliferation in rat fibroblasts.     

Synthesis of benzo[d]imidazo[2,1-b]thiazole-chalcone conjugates as microtubule targeting and apoptosis inducing agents

A series of benzo[d]imidazo[2,1-b]thiazole-chalcone conjugates (5a-aa) were designed, synthesized and evaluated for their cytotoxic potency against a panel of human cancer cell lines like lung (A-549), breast (MDA MB-231), prostrate (DU-145) and colon cancer (HT-29). Preliminary results revealed that some of these conjugates like 5d and 5u exhibited significant antiproliferative effect against human breast cancer (MDA MB-231) with IC₅₀ values of 1.3 and 1.2 µM respectively. To investigate the mechanistic aspects underlying the activity, the detailed biological studies of these promising conjugates (5d and 5u) were carried out on the MDA MB-231 cancer cells. Flow cytometric analysis revealed that these conjugates induce cell-cycle arrest in the G2/M phase. The tubulin polymerization assay suggests that these conjugates effectively inhibit microtubule assembly. In addition, morphological changes, reactive oxygen species (ROS) detection by 2′, 7′–dichlorofluorescin diacetate (DCFDA) and annexin V–FITC/PI assays indicate that 5d and 5u induces apoptosis. Furthermore, in silico computational studies, including molecular docking studies have been carried out to rationalise the binding modes of these conjugates with the tubulin protein.     

cDNA array analysis of alterations in gene expression in the promyelocytic leukemia cell line,HL-60, after apoptosis induction with etoposide

Alterations in gene expression during apoptosis in HL-60 cells were identified by a cDNA based array analysis. Apoptosis was induced in the human promyelocytic leukemia cell line, HL-60, by incubation with 30 M etoposide for 5 hours. Changes in gene expression occurring during apoptosis in these cells were detected using the ATLAS cDNA Expression Array technique. 40 genes were identified as differentially expressed in the apoptotic cells by at least a factor of two. 30 of these genes were down-regulated during apoptosis. Many of the down-regulated genes reflected decreased proliferative activity in the cells as well as decreased activity of survival pathways. Most of the genes, which were up-regulated during apoptosis, were genes involved in pathways leading to cell death and suppression of proliferation. Based on the up-regulations observed at the mRNA level, it is speculated that etoposide-induced apoptosis in the HL-60 cells proceeds via pathways involving factors such as TNF, IGFBP3, SAPK1, AP-1 and GADD153/CHOP10. Four genes, which showed changes at the mRNA level, were also analyzed by Western blotting in order to confirm the observed differences at the protein level.     

Inhibition of cell survival signal protein kinase B/Akt by curcumin in human prostate cancer cells

Although curcumin has been shown to inhibit prostate tumor growth in animal models, its mechanism of action is not clear. To better understand the anti-cancer effects of curcumin, we investigated the effects of curcumin on cell survival factor Akt in human prostate cancer cell lines, LNCaP, PC-3, and DU-145. Our results demonstrated differential activation of Akt. Akt was constitutively activated in LNCaP and PC-3 cells. Curcumin inhibited completely Akt activation in both LNCaP and PC-3 cells. The presence of 10% serum decreased the inhibitory effect of curcumin in PC-3 cells whereas complete inhibition was observed in 0.5% serum. Very little or no activation of Akt was observed in serum starved DU-145 cells (0.5% serum). The presence of 10% serum activated Akt in DU-145 cells and was not inhibited by curcumin. Results suggest that one of the mechanisms of curcumin inhibition of prostate cancer may be via inhibition of Akt. To our knowledge this is the first report on the curcumin inhibition of Akt activation in LNCaP and PC-3 but not in DU-145 cells.     

Structure and assembly of the cortical microtubule cytoskeleton in the green alga,Boodlea coacta

Summary Examination was made of the structure and assembly of the cortical microtubule (MT) cytoskeleton in the coenocytic green algaBoodlea coacta (Dickie) Murray et De Toni by immunofluorescence microscopy. Cortical MTs inBoodlea protoplasts are arranged randomly but some show a meridional arrangement within 6 h after protoplast formation. At 6–9 h such MTs become highly concentrated and parallel to each other in certain areas. At 12 h the concentration is uniformly high throughout the cell, indicating the completion of high density meridional arrangement of cortical MTs. Cortical MTs exhibiting a high density, meridional arrangement show characteristic disassembly by treatment with 10 M amiprophos-methyl (APM) or cold treatment (0 °C). Disassembly occurs by each MT unit at positions skipping 30–40 m in the transverse direction, and neighboring MTs subsequently disassemble to form MT groups. Each group becomes slender and then disappears completely within the following 24 h. The meridional arrangement of cortical MTs is disrupted by N-ethylmaleimide (NEM) accompanied by a remarkable reduction in density. The remaining MTs form groups at 30–40 m intervals from each other, as also occurs with drug or cold treatment, but disruption and density return to normal levels following removal of NEM. It appears that there are meridionally oriented channels, anchor-rich and anchor-poor, in the plasma membrane. The channels could be distributed alternately and anchors could be deposited in a cross-linking manner with cortical MTs to form a stable cortical MT-cytoskeleton. MTs comprising the cortical MT cytoskeleton could be oriented by meridionally oriented channels of anchors which are distributed following establishment of cell polarity.Abbreviations APM amiprophos-methyl - MT microtubule - MTOC microtubule organizing center - NEM N-ethylrnaleimide     

MicroRNA-Dependent Regulation of <Emphasis Type="Italic">IGF1R</Emphasis> Gene Expression in Hormone-Sensitive and Hormone-Resistant Prostate Cancer Cells

Using multiple parallel sequencing on Illumina platform, we identified eight microRNAs that showed significant opposite changes of gene expression in cells of the hormone-sensitive LNCaP prostate cancer cell line and in cells of the hormone-resistant DU-145 cell line, in comparison to the microRNA expression in the normal prostate tissue cells. We found that the insulin-like growth factor 1 receptor (IGF1R) gene is a target of five microRNAs whose expression is increased in LNCaP cells and reduced in DU-145 cells.     

Taxol maintains organized microtubule patterns in protoplasts which lead to the resynthesis of organized cell wall microfibrils

Summary We have investigated the effects of microtubule stabilizing conditions upon microtubule patterns in protoplasts and developed a new method for producing protoplasts which have non-random cortical microtubule arrays. Segments of elongating pea epicotyl tissue were treated with the microtubule stabilizing drug taxol for 1 h before enzymatic digestion of the cell walls in the presence of the drug. Anti-tubulin immunofluorescence showed that 40 M taxol preserved regions of ordered microtubules. The microtubules in these regions were arranged in parallel arrays, although the arrays did not always show the transverse orientation seen in the intact tissue. Protoplasts prepared without taxol had microtubules which were random in distribution. Addition of taxol to protoplasts with random microtubule arrangements did not result in organized microtubule arrays. Taxol-treated protoplasts were used to determine whether or not organized microtubule arrays would affect the organization of cell wall microfibrils as new walls were regenerated. We found that protoplasts from taxol-treated tissue which were allowed to regenerate cell walls produced organized arrays of microfibrils whose patterns matched those of the underlying microtubules. Protoplasts from untreated tissue synthesized microfibrils which were disordered. The synthesis of organized microfibrils by protoplasts with ordered microtubules arrays shows that microtubule arrangements in protoplasts influence the arrangement of newly synthesized microfibrils.Abbreviations DIC differential interference contrast - DMSO dimethyl sulfoxide - FITC fluorescein isothiocyanate - IgG immunoglobulin G - PIPES piperazine-N,N-bis[2-ethane-sulfonic acid] - PBS phosphate buffered saline     

c-jun-NH2JNK mediates invasive potential and EGFR activation by regulating the expression of HB-EGF in a urokinase-stimulated pathway

In this study, we demonstrated that tyrosine phosphorylation of EGFR and the autocrine expression of uPA and HB-EGF depend on the activity of c-jun amino-terminal kinase (JNK) in human prostatic DU-145 cells. These cells overexpress EGFR and produce a high amount of uPA. Treatment with either SP600125, a specific chemical inhibitor of JNK, or the expression of a dominant-negative JNK form inhibited autocrine production of uPA and HB-EGF, which block EGFR phosphorylation and mitigates invasive capacity. Our data provided evidence that in DU-145 cells, the maintenance of the activation level of EGFR, which determines the cellular invasive potential, operates through an autocrine loop involving the JNK-dependent production of uPA and HB-EGF activity. Moreover, we found that exogenously added uPA stimulates autocrine production of HB-EGF, and that blocking HB-EGF activity curbed DU-145 cell invasive potential.     

Crocetin treatment inhibits proliferation of colon cancer cells through down-regulation of genes involved in the inflammation

Background

The current study was designed to investigate the effect of crocetin on the proliferation inhibition of colon cancer cells and the underlying mechanism.