Taxus Cuspidata Cells Research Articles

Ce 4+ induced down-regulation of ERK-like MAPK and activation of nucleases during the apoptosis of cultured Taxus cuspidata cells

Ce 4+ (Ce(NH 4) 2(NO 3) 6) at 1 mM induces apoptosis of suspension cultures of Taxus cuspidata cells; however, the underlying signal mechanisms are unknown. We show here that a 46-kDa ERK (extracellular signal-regulated kinase)-like MAPK appears to be down-regulated at 4 h, and remains at low levels for up to 48 h. An inhibitor of superoxide anions ( O 2 - ) generation, diphenyl iodonium (DPI) successfully blocks down-regulation of ERK-like MAPK and degradation of DNA. Moreover, a 41-kDa p38-like MAPK activity remains unchanged from 0.5 to 48 h. The p38 inhibitor SB202190 effectively inhibits p38-like MAPK activity, however, SB202190 fails to modify the apoptotic rate at concentrations up to 100 μM. Three nuclease (34-kDa, 22-kDa and 20-kDa) activities are profoundly enhanced in Ce 4+-induced T. cuspidata cells. They have an optimum pH at 6.8, and are stimulated by Ca 2+/Mg 2+. Caspase-3 inhibitor, Ac-DEVD-CHO, does not attenuate the 34-kDa nuclease activity, but inhibits the 22-kDa and the 20-kDa nuclease activities. In addition, inhibition of O 2 - generation by DPI significantly reduces the three nuclease activities. In conclusion, the present study suggests that down-regulation of ERK-like MAPK, burst of O 2 - , activation of caspase-3-like and induction of three nucleases as the key signaling events mediating apoptosis in Ce 4+-induced cultured T. cuspidata cells.

Signal role for activation of caspase-3-like protease and burst of superoxide anions during Ce4+-induced apoptosis of cultured Taxus cuspidata cells

The signal events of 1 mM Ce4+ (Ce(NH4)2(NO3)6)-induced apoptosis of cultured Taxus cuspidata cells were investigated. The percentage of apoptotic cells increased from 0.82% to 51.32% within 6 days. Caspase-3-like protease activity became notable during the second day of Ce4+-treatment, and the maximum activity was 5-fold higher than that of control cells at the fourth day. When the experiment system was pretreated with acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) at 100 microM, caspase-3-like activity resulted in distinct inhibition by 70% and 77.3% after 3 and 4 days of induction. Furthermore, 100 microM Ac-DEVD-CHO partially reduced the apoptotic cells by 58.6% and 60.8% at day 4 and 5 respectively. Ce4+ induced superoxide anions (O2*-) transient burst, and the first peak appeared at around 3.7-4 h, the second appeared at about 7 h. Both O2*- burst and cell apoptosis were effectively suppressed by application of diphenyl iodonium (NADPH oxidase inhibitor). Inhibition of O2*- production attenuated caspase-3-like activation by 49% and 53.6% during day 3 and 4 respectively. In addition, a total of 15 protein spots changed in response to caspase-3-like protease activation were identified by two-dimensional gel electrophoresis. These results suggest that Ce4+ of 1 mM induces apoptosis in suspension cultures of T. cuspidata through O2*- burst as well as caspase-3-like protease activation. The burst of O2*- exerts its activity as an upstream of caspase-3-like activation. Our results also implicate that other signal pathways independent of an O2*- burst possibly participate in mediating caspase-3-like protease activation.

Abnormal mitosis versus apoptosis of Taxus cuspidata induced by oleic acid in two-liquid-phase suspension cultures

The effects of oleic acid on abnormal mitosis and apoptosis of Taxus cuspidata cells were investigated, when oleic acid improved taxol production in two-liquid-phase suspension cultures. The results showed that the addition of oleic acid (4%, v/v) led to an increase of cell apoptosis and a decrease of mitotic activity. Moreover, the occurrence of some abnormal cells, chromosomal doubling, chromosomal bridge, binuclei cells and micronuclei further confirmed that oleic acid disrupted the normal mitosis of T. cuspidata cells in two-liquid-phase suspension cultures. And the frequency of total abnormal mitosis was three to four times than that of apoptosis. It is, thus, inferred that oleic acid-induced abnormal mitosis and consequent apoptosis. We found that the increases in abnormal mitosis, the increases in cell apoptosis and the decreases in mitotic index are closely relevant to the enhancement of taxol production of T. cuspidata in two-liquid-phase cultures.

Antioxidant Responses to Oleic Acid in Two-Liquid-Phase Suspension Cultures of <I>Taxus cuspidata</I>

Two-liquid-phase plant cell cultures employ the use of a partitioning system to redirect extracellular product into a second phase. After the addition of organic solvent, in order to understand the defense system of Taxus cuspidata cells to organic solvent in two-liquid-phase suspension cultures, we investigated cells' antioxidant metabolism. The results showed that T. cuspidata cells responded to oleic acid with oxidative bursts in both intracellular H2O2 and extracellular O2-* production. Inhibition studies with diphenylene iodonium suggested that the key enzyme responsible for oxidative bursts was primarily NADPH oxidase. Investigation of the relationship between reactive oxygen species (ROS) and defense responses induced by oleic acid indicated that 4% (v/v) oleic acid increased the levels of antioxidant enzymes of superoxide dismutase, ascorbate peroxidase, and catalase and the antioxidant capacity of reduced ascorbate and glutathione. However, when oleic acid content reached a critical value (6% [v/v]), no further increase in antioxidant enzymes and antioxidant capacity was observed, indicating that the defense responses played a role in a certain range of oleic acid content, beyond which the overall ROS scavenging machinery was not induced and the peroxidation of membrane lipids emerged.

Effects of organic solvents on membrane ofTaxus cuspidata cells in

The effects of organic solvents (oleic acid and dibutyl phthalate) on viability and membrane integrity of Taxus cuspidata cells were investigated in two-liquid-phase suspension cultures. It has been found that the cell viability, electrical conductivity and concentration of malonyl dialdehyde did not change obviously when the content of oleic acid or dibutyl phthalate was 2% (v/v), but varied markedly when the contents of oleic acid or dibutyl phthalate were raised to 6% (v/v) or more, indicating that the organic solvents at higher concentrations severely affected the cell membrane permeability.

Random sequencing of an induced Taxus cell cDNA library for identification of clones involved in Taxol biosynthesis.

Biosynthesis of the anticancer drug Taxol involves 19 enzymatic steps from the universal diterpenoid progenitor geranylgeranyl diphosphate derived by the plastidial methylerythritol phosphate pathway for isoprenoid precursor supply. To gain further insight about Taxol biosynthesis relevant to the improved production of this drug and to draw inferences about the organization, regulation, and origins of this complex natural product pathway, random sequencing of a cDNA library derived from Taxus cuspidata cells (induced for taxoid biosynthesis with methyl jasmonate) was undertaken. This effort revealed surprisingly high abundances for transcripts of several of the 12 defined genes of Taxol biosynthesis, yielded cDNAs encoding two previously uncharacterized cytochrome P450 taxoid hydroxylases, and provided candidate genes for all but one of the remaining seven steps of this extended sequence of reactions.

The final acylation step in taxol biosynthesis: cloning of the taxoid C13-side-chain N-benzoyltransferase from Taxus.

The formation of several acyl groups and an amide group of Taxol is catalyzed by regioselective CoA thioester-dependent acyltransferases. Several full-length acyltransferase sequences, obtained from a cDNA library constructed from mRNA isolated from Taxus cuspidata cells induced for Taxol production with methyl jasmonate, were individually expressed in Escherichia coli, from which a cDNA clone encoding a 3'-N-debenzoyl- 2'-deoxytaxol N-benzoyltransferase was identified. This recombinant enzyme catalyzes the stereoselective coupling of the surrogate substrate N-debenzoyl-(3'RS)-2'-deoxytaxol with benzoyl-CoA to form predominantly one 3'-epimer of 2'-deoxytaxol. The product 2'-deoxytaxol was confirmed by radio-HPLC,(1)H-NMR, and chemical ionization-MS. This enzymatic reaction constitutes the final acylation in the Taxol biosynthetic pathway. The full-length cDNA coding for the N-benzoyltransferase has an ORF of 1,323 nucleotides and encodes a 441-residue protein with a calculated molecular weight of 49,040. The recombinant enzyme expressed in E. coli has a pH optimum at 8.0, a k(cat) approximately 1.5 +/- 0.3 s(-1) and K(m) values of 0.42 mM and 0.40 mM for the N-deacylated taxoid and benzoyl-CoA, respectively. In addition to improving the production yields of Taxol in genetically engineered host systems, this enzyme provides a means of attaching modified aroyl groups to taxoid precursors for the purpose of improving drug efficacy.

Kinetics of two-liquid-phase Taxus cuspidata cell culture for production of Taxol

The effects of different organic solvents (paraffin, organic acid, alcohol and ester) and their volumetric fractions on the cell growth and Taxol production were studied in two-liquid-phase and the two-stage culture. A kinetic model, incorporated the effects of the toxicity of organic solvents was developed for two-liquid-phase culture of Taxus cuspidata in the two-stage Taxol production. The results showed that the proposed kinetic model could fit the experimental data satisfactorily. The results also showed that Taxol production could reach the optimal value when 10−log P was in the range of 2 to 5 and the volumetric fraction of the organic solvents at the corresponding the highest Taxol production should be lower when 10−log P was high.

An abietane from Taxus cuspidata cell suspension cultures

An abietane diterpenoid was isolated from an ethyl acetate extraction of Taxus cuspidata cell suspension media. The structural assignments were established by mass spectroscopy, and 1H and 13C NMR studies including COSY, HMQC and HMBC. This compound was determined to be 3,20-epoxy-12-methoxy-8,11,13-abietatriene-3,7,11-triol.

Enhancement of taxol production by in situ recovery of product

The effects of additon of adsorbents on the production of taxol and taxane compounds were investigated in Taxus cuspidata cell culture. As a suitable adsorbent, the non-ionic exchange resin XAD-4, among several adsorbents tested, showed maximum adsorptive capacity of taxol which resulted from the hydrophobic interaction between them. The biosynthesis of taxol was enhanced by 40–70% by adding XAD resin into the culture medium at 16th days after subculture. Although the productions of cephalomanin and 10-deacetyl baccatin III were greatly increased by adding adsorbents, the production of baccatin III was not increased which may be due to the weak hydrophobic interaction between adsorbents and baccatin III. The refreshment of medium during the cell culture also enhanced the biosynthesis of 10-deacetyl baccatin III, but did not enhance the cephalomanin production.