Studies On The Mechanisms Of Oridonin-induced Human Laryngeal Squamous Carcinoma HEp-2 Cells Death

Laryngeal squamous cell carcinoma (LSCC) is the most common squamous cell carcinoma of the head and neck, with a tendency towards an increasing occurrence of new cases and deaths annually. Mutations in the p53 gene are involved in acquired and intrinsic treatment resistance in human tumors, and render tumor cells refractory to many anti-cancer therapies. Nonfunctional or mutated p53 protein has been found in LSCC. This is one of the main reasons to search for new anticancer compounds, more effective in unresponsive tumors, with reduced adverse effects, to improve the general outcome of LSCC chemotherapy. The present study is designed to examine efficacy and associated mechanism of oridonin purified from Chinese herbal medicine in human laryngeal squamous carcinoma cells HEp-2 lacking functional p53 protein. Oridonin exhibited significant cell growth inhibition by inducing HEp-2 cells to undergo G2/M phase arrest and apoptosis. Oridonin triggered the mitochondrial apoptotic pathway, as indicated by increased Bax/Bcl-2 ratios, reduction of mitochondrial membrane potential (Δψm) as well as a substantial increase in apoptosis-inducing factor (AIF) and cytochrome c. Inhibition of caspase-9 in HEp-2 cells did not protect the cells from oridonin-induced apoptosis, and cleaved caspase-9 was not detected, indicating that apoptosis occurred via a caspase-9-independent pathway. Cell cycle blockage was associated with down-regulation of cell cycle related cyclin B1, cdc2 and cdc25c levels, as well as up-regulation of p21, phospho-Cdc2 and phospho-Cdc25C levels. Our results also suggest that oridonin mediates apoptosis and G2/M phase arrest in the HEp-2 cells via a p53-independent but p21/WAF1-dependent manner. In addition, we also found that the generation of reactive oxygen species (ROS) is a critical mediator in oridonin-induced growth inhibition. Blocking ROS generation with N-Acetylcysteine (NAC) or catalase (CAT) significantly prevented G2/M arrest by reversing the changes of G2/M regulatory proteins and completely protected the cells from oridonin-induced apoptosis.Epidermal growth factor receptor (EGFR) is abnormally activated in many epithelial tumors, including LSCC. Our group had also detected the EGFR gene amplification in human laryngeal squamous carcinoma HEp-2 cells and in 11 laryngeal carcinoma tissues by fluorescence in situ hybridization (FISH). Thus, the over-activation of EGFR might contribute to the tumorigenic capacity of LSCC. Since EGFR-tyrosine kinase inhibitors cause growth inhibition of tumors and enhancement of the activity of a number of cytotoxic drugs, it would be interesting to identify the detailed mechanism whereby EGFR inhibition might enhance the apoptotic response to oridonin in HEp-2 cells, a cell line characterized by EGFR gene amplification. Here, we show that inhibition of EGFR with tyrphostin AG1478 enhances oridonin-induced cell death in HEp-2 cells. The enhanced apoptotic effect correlates with high expression and activation of Bax, FADD, caspase-8 as well as caspase-3 and decreased protein levels of Bcl2 and SIRT1, suggesting that both the extrinsic and intrinsic apoptosis pathways are involved in the apoptotic processes. However, treatment with oridonin and AG1478 greatly enhances nuclear translocation of AIF without caspase-9 activation. Here, it is the active form of caspase-8 but not caspase-9 that activates downstream effector caspase-3, resulting in the cleavage of critical cellular proteins and apoptosis. Furthermore, the combined use of AG1478 and oridonin augments the production of ROS. Incubation of cells with NAC attenuates the apoptosis and theΔψm disruption induced by the combination of oridonin and AG1478, which indicates that ROS plays a pivotal role in cell death. In conclusion, targeting EGFR combined with other conventional pro-apoptotic drugs should be a potentially very effective anti-neoplastic therapy for laryngeal cancer.Of note, caspase-9 plays a central role in apoptosis. However, in this study, oridonin-induced apoptosis activation might differ in certain aspects. Caspase-9 inhibitor (C9i) enhanced apoptosis to oridonin stimuli. The sensitization to oridonin-mediated apoptosis by the C9i is associated with the amplification of ROS production. ROS subsequently triggers the progression of apoptosis through activation of both the caspase-9-independent mitochondrial pathway and death receptor pathways in HEp-2 cells. To further explore the mechanism whereby inhibition of caspase-9 increases sensitivity to apoptotic stimuli, we have studied the role of caspase-9 in oridonin-induced autophagy. Notably, inhibition of caspase-9 decreases oridonin-induced autophagy, as well as Beclin 1 activation and the conversion from LC3-I to LC3-Ⅱ. Inhibition of autophagy by 3-methyladenine (3-MA) increases oridonin-induced apoptosis. Therefore, besides suppressing apoptosis, caspase-9 promotes autophagy in HEp-2 cell treated with oridonin, and this promotion of autophagy might contribute to down-regulation of apoptosis. In addition, HEp-2 cells made deficient in caspase-9 by RNA interference exhibit no resistance to apoptotic signals and actually show increased apoptotic sensitivity to oridonin. In summary, C9i inhibited caspase-9 enzymic activity while caspase-9 specific siRNA reduced enzyme protein synthesis. It is clear that the apoptotic enhancement observed by either approach is part of an endogenous pathway that regulates apoptosis. Thus, the combination of oridonin and those leading to a reduction of caspase-9 in tumor cells with agents, such as C9i and/or reduction in caspase-9, could represent a novel approach to human laryngeal cancer treatment.