Studies On The Mechanisms For Mitotic Catastrophe And Senescence Induced By Pseudolaric Acid B In L929Cells

Pseudolaric acid B (PAB), the primary biologically active compound isolated from the root bark of P. kaempferi Gordon. Previous studies demonstrated that PAB exhibited multiple pharmacological effects including antifungal, antimicrotubule, antibiosis and antitumor. It is reported that PAB is a novel microtubule-destabilizing agent that exhibits antitumor activity in vivo and in vitro. In addition, PAB shows no obvious toxicities to the liver and kidney. PAB exhibits anti-tumor effect in vitro primarily via cell cycle arrest and apoptosis. In this study, we chose L929cells to study the mechanisms of mitotic catastrophe, senescence, autophagy and ROS generation.PAB induced mitotic catastrophe in L929cells. The cells became flat, enlarged and adherent after treated with PAB by72h. PAB interfered with the function of the mitotic spindle apparatus by inhibiting microtubule polymerization, resulting in G2/M phase arrest. The level of histone H3phosphorylation, a specific mitotic marker, increased up to24h after treatment and then decreased in a time-dependent manner. In addition, the mitotic index increased during a24h treatment with PAB, suggesting that PAB arrested cell cycle progression at mitosis. The M phase arrested cells undergone mitotic slippage due to the inactivation of the cylinBl-cdc2complex, resulting in multinucleated cells. To investigate whether the mitotic slippage and subsequent apoptosis observed was specific for the L929cell line or the species, the effects of PAB on primary mouse dermal fibroblast cells and colorectal carcinoma HCT-116cells were examined. The results indicated that the mitotic slippage and subsequent apoptosis induced by PAB was not common but instead was specific for L929cells.Besides apoptosis, a majority of the cells undergoing mitotic catastrophe entered a period of senescence. PAB induced senescence through p19-p53-p21and p16-Rb pathways in L929cells. Autophagy inhibitors (3-Methyladenine (3MA), chloroquine (CQ) or ATG5siRNA significantly delayed the senescence process, indicating that autophagy facilitated senescence. Further data indicated that PAB triggered serious mitochondrial dysfunction including reactive oxygen species (ROS) generation, the increase in mitochondrial mass, damage to the respiratory chain and ATP production. Moreover, ROS scavenger significantly decreased the autophagic level and improved mitochondrial function. Additionally, autophagy inhibitors effectively reduced ROS levels and ameliorated mitochondrial function, In conclusion, autophagy promoted senescence via enhancement of ROS generation and mitochondrial dysfunction in PAB-treated L929cells. We also investigated the fate of the senescent cells. The results showed that the cells were not stationary senescent, on the contrary, the ratio of dead cells significantly increased after a5-7days exposure of PAB.PAB was also found to trigger autophagy via inhibiting Akt/mTOR activity in L929cells, In addition, autophagy was demonstrated to reinforce senescence through regulating the senescence pathways. Thus, we focused on the detailed molecular mechanisms whereby autophagy promoted senescence. PAB triggered a ROS-JNK-p53positive feedback loop played a crucial role in autophagy via repressing the activation of mTOR in L929cells. Furthermore, ROS-JNK-p53positive feedback loop was demonstrated to regulate senescence. TSC1/TSC2heterodimer, a downstream target of growth factor-phosphoinositide3-kinase-Akt signaling, negatively regulates mTOR activity. Activation of mTOR by insulin or inhibition of endogenous TSC2levels by siRNA obviously delayed PAB-induced senescence, In conclusion, mTOR inactivation by ROS-JNK-p53pathway played an important role in autophagy-dependent senescence in PAB-treated L929cells. Finally, mTOR inactivation was also demonstrated to be involved in autophagy-dependent senescence in PAB-treated MCF-7cells.