Winter And Bean Milk Thistle Of Oridonin Inducing Epidermal Carcinoma A431 Cell Death Mechanism Research

Oridonin, a diterpenoid compound extracted and purified from Rabdosia rubescen, has been reported to induce tumor cell apoptosis. To further examine the mechanism of oridonin, we selected human epidermoid carcinoma A431cells as a test object. Besides apoptosis, oridonin also induced A431cell autophagy, and this autophagy antagonized apoptosis and played a protective role for A431cells. Reactive oxygen species (ROS) played a pivotal role in induction of cytotoxicity. Therefore, a ROS scavenger, NAC (N-Acetylcysteine) combined with oridonin was appiled. Results of morphologic observation, flow cytometric analysis and Western blot analysis showed that NAC could significantly reverse both ROS generation and down-regulation of mitochondrial membrane potential in oridonin treated cells. NAC inhibited oridonin induced apoptosis through both the intrinsic and extrinsic apoptotic pathways. NAC effectively inhibited both oridonin-induced apoptosis and autophagy by reducing intracellular oxidative stress. To further examine the mechanism of ROS, exogenous enzyme antioxidants (SOD, catalase) and non-enzyme antioxidants (GSH) were applied to detect the effect of oridonin on ROS generation. Only GSH exerted a similar role with NAC, suggesting that hydroxyl radical (OH) played the major role in oridonin-induced cell death. Oridonin could decrease the GSH level in A431cells in a dose-dependent manner. In addition, after treatment with?OH donor, Fenton reagent, the changes in A431cells were similar to the results of oridonin treatment. All the results proved that?OH played a pivotal role in oridonin induced apoptosis and autophagy in A431cells.Besides apoptosis and autophagy, tyrosine kinase pathways are also involved in these processes. A specific epidermal growth factor receptor (EGFR) inhibitor AG1478was used to examine the relationship between EGFR signal pathways and oridonin-induced apoptosis and autophagy in EGFR abundant A431cells. Inhibition of EGFR augmented oridonin-induced A431cell apoptosis through both the intrinsic and extrinsic apoptotic pathways. Pretreatment with AG1478aggravated oridonin-induced loss of mitochondrial membrane potential and increased ROS generation in A431cells, while NAC completely reversed oridonin-and AG1478-incluced ROS generation and apoptosis. Therefore, AG1478augmented oridonin-induced apoptosis by enhancing oxidative stress. Pretreatment with AG1478decreased the expression of downstream MAPK proteins ERK, JNK and P38and their phosphorylated forms to varying degrees compared with oridonin alone treatment. Then after administration of ERK, JNK and P38inhibitors, only JNK inhibitor SP600125effectively augmented oridonin-induced apoptosis and ROS generation. Therefore, in EGFR downstream pathways, JNK played a major role in preventing oridonin-induced apoptosis. Autophagy antagonized apoptosis and exerted a protective effect in A431cells, and both AG1478and SP600125decreased oridonin-induced autophagy. Inhibition of EGFR augmented oridonin-induced apoptosis, this was caused by enhanced oxidative stress, and JNK played a major protective role by increasing autophagy, leading to antagonizing apoptosis and ROS generation.Silibinin, another natural drug extracts, is a major active constituent of silymarin. Silibinin is clinically used as a hepatoprotectant for its antioxidant effect, and in recent years, it has been studied for the clinical treatment of cancer. To explore the mechanism of silibinin action on cancer cells and compare its function between oridonin, we investigated the contribution of silibinin to the induction of apoptosis and autophagy via generation of reactive oxygen species (ROS) and nitric oxide (?NO) in A431cells. Silibinin inhibited the cell growth in a dose-and time-dependent manner. Obvious autophagy was observed after treatment with different doses of silibinin. At a high dose (400μM), silibinin induced apoptosis through both the intrinsic and extrinsic apoptotic pathways. Autophagy augmented apoptosis and might have an alternative cell-death effect on A431cells. Loss of mitochondrial membrane potential by silibinin led to mitochondrial dysfunction and decreased ROS levels, suggesting that silibinin might act as an antioxidant in this process. Furthermore, silibinin induced?NO generation in a time-and dose-dependent manner. The?NO scavenger PTIO could effectively clear?NO and exerted a minor cell protection effect through partial inhibition of silibinin-induced apoptosis and autophagy.