epidermal-growth-factor and oridonin

As the active anticancer component of Rabdosia Rubescens, oridonin has been proved to show strong anticancer activity in cancer cells, which is also found to be closely related to its specific inhibition effects on the EGFR tyrosine kinase activity. In this study, atomic force microscopy based single molecule force spectroscopy (AFM-SMFS) was used for real-time and in-situ detection of EGF-EGFR interactions in living esophageal cancer KYSE-150 cells to evaluate the anticancer activity of oridonin for the first time. Oridonin was found to induce apoptosis and also reduce EGFR expression in KYSE-150 cells. AFM-SMFS results demonstrated that oridonin could inhibit the binding between EGF and EGFR in KYSE-150 cells by decreasing the unbinding force and binding probability for EGF-EGFR complexes, which was further proved to be closely associated with the intracellular ROS level. More precise mechanism studies based on AFM-SMFS demonstrated that oridonin treatment could decrease the energy barrier width, increase the dissociation off rate constant and decrease the activation energy of EGF-EGFR complexes in ROS dependent way, suggesting oridonin as a strong anticancer agent targeting EGF-EGFR interactions in cancer cells through ROS dependent mechanism. Our results not only suggested oridonin as a strong anticancer agent targeting EGF-EGFR interactions in ROS dependent mechanism, but also highlighted AFM-SMFS as a powerful technique for pharmacodynamic studies by detecting ligand-receptor interactions, which was also expected to be developed into a promising tool for the screening and mechanism studies of drugs.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Diterpenes, Kaurane; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Esophagus; Humans; Isodon; Microscopy, Atomic Force; Reactive Oxygen Species

We have reported that oridonin, a diterpenoid isolated from the plant Rabdosia rubescens, had apoptosis-inducing activities in many cell lines (e.g., human melanoma A375-S2, human cervical cancer HeLa, human breast adenocarcinoma MCF-7, and murine fibrosarcoma L929). In this study, we further investigated signaling events involved in oridonin-induced apoptosis in human epidermoid carcinoma A431 cells. It was found that the total tyrosine kinase activity was inhibited and the protein expressions of epidermal growth factor receptor (EGFR) and phosphorylated EGFR were decreased in oridonin-induced A431 cell apoptosis. Expression of EGFR downstream effector proteins, Grb2, Ras, Raf-1, and extracellular signal-regulated kinase (ERK), was also downregulated by oridonin. Moreover, the oridonin-induced apoptosis was augmented by the Ras inhibitor manumycin A, Raf-1 inhibitor GW5074, or ERK inhibitor PD98059, suggesting that inactivation of Ras, Raf, or ERK participates in oridonin-induced apoptosis. Taken together, oridonin-induced apoptosis in A431 cells might be through blocking EGFR and its downstream Ras/Raf/ERK signal pathway.

Topics: Apoptosis; Cells, Cultured; Diterpenes; Diterpenes, Kaurane; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Genistein; GRB2 Adaptor Protein; Humans; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-raf; ras Proteins; Signal Transduction

Oridonin, an active component isolated from the plant Rabdosia rubescens, has been reported to exhibit antitumor effects, but little is known about its molecular mechanism of action. In this study, we first investigated the mechanism involved in oridonin-induced cell death in human epidermoid carcinoma A431 cells, which overexpress epidermal growth factor receptor (EGFR). After treatment with various doses of oridonin for 24 h, the majority of A431 cells underwent apoptosis in a time- and dose-dependent manner as measured by an LDH activity-based assay. Treatment with oridonin at various concentrations for 24 h caused significant inhibition on the total tyrosine kinase activities and downregulation of EGFR expression or EGFR phosphorylation. Oridonin significantly affected the localization of EGFR and phosphorylated EGFR on the cell membrane. However, genistein (a well-known tyrosine kinase inhibitor) did not induce apoptotic A431 cell death. Importantly, oridonin exhibited much stronger inhibitory effect on the total tyrosine kinase activities or EGFR tyrosine phosphorylation as well as much stronger suppression on EGFR and phosphorylated EGFR localization than genistein in A431 cells. Taken together, oridonin exerted a potential inhibitory effect on the tyrosine kinase activity of A431 cells. The decrease in the tyrosine kinase activity and the blockage of EGFR tyrosine phosphorylation might be one of the causes of oridonin-induced A431 cell death.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Survival; Diterpenes; Diterpenes, Kaurane; DNA Fragmentation; Epidermal Growth Factor; ErbB Receptors; Humans; L-Lactate Dehydrogenase; Necrosis; Phosphorylation; Protein Kinase Inhibitors; Protein-Tyrosine Kinases