Studies On Mechanism Of Defense Responses Of Taxus Cuspidata Cells To Taxol

The suspension cultures of Taxus spp. induced by taxol and taxotere were established to study the defense-response mechanisms of suspension-cultured Taxus spp., using taxotere as taxol indicator. The results showed that taxol as elicitor-like stimulated defense responses.The effects of DMSO and ethanol, used for dissolving taxol or taxotere, on the metabolisms and membrane permeability of Taxus cuspidata cells in the suspension cultures were studied to lessen themselves toxicity to the cells. It was found that DMSO and ethanol over some additives levels inhibited cell growth, decreased the cell viability, increased the cell membrane permeability, and that DMSO was less poisonous to cells than ethanol at the same level. DMSO below 1% (v/v) did not affect cell metabolism and membrane permeability of Taxus spp. cells, only altered enzyme activity related ROS and alkalization of medium in reverse short-time manner. But ethanol in additional of the same level significantly influenced cell metabolism and membrane permeability. So 1% (v/v) DMSO is an ideal solvent and additive amounts. The effects of taxol and taxotere with various concentrations on the suspension cultures in different growth stage were investigated. The results showed that taxol and taxotere remarkablely inhibited the cell growth at the early exponential phase of suspension cultures but did not affect at the late one. So a novel viewpoint was first put forward that the functions of taxol (10 μM) and taxotere(35 μM)on the cells at late exponential phase were as a non-specific elicitor. The defense responses mechanism of Taxus spp. cells to taxol or taxotere were systemic studied, on the base of investigating the extracellular pH and ROS accumulation for the early defense responses and determining PAL activity, taxol concentration, protein contents for the late defense responses. The results showed the appropriate concentration of taxol (10 μM) or taxotere (35 μM) activate the influxes of H+, ROS accumulation, PAL activity and biosynthesis of taxol, as well as apoptosis, however, the high concentration taxol or taxotere (140 μM) did not show above results. Ten plots of protein were only observed in taxol-induced cells and there were differences at expression level of some plots between control and taxol-induced cells using two dimension electrophoresis, which reflect those differential protein were highly relevant to signal moleculars of cell defense responses, suggesting cell apoptosis and taxol biosynthesis might be parallel or two events in the same pathway of defense responses.With application of pharmacological inhibitor and of determining the changes of enzymes of CAT, POD, SOD, the source and main composition of ROS were studied. The results showed that taxol (10 μM) and taxotere (35 μM) could reduced ROS accumulation and that NADPH oxidase was the main mechanism of ROS accumulation , H2O2 is the leading component of ROS. Taxol in the cells, which pre-incubated with exogenous, released into fresh medium was studied with application of solvents altering membrane permeability of Taxus spp. cells, The results showed that increasing membrane permeability promoted release of intracellular taxol to some extent. It is first supposed that the active transport system of taxol in Taxus cells be exist, by comparing and analyzing the changes of taxol or taxotere in cells or free-cells systems.