Proteomics characterization of the cytotoxicity mechanism of ganoderic acid D and computer-automated estimation of the possible drug target network.

Triterpenes isolated from Ganoderma lucidum could inhibit the growth of numerous cancer cell lines and were thought to be the basis of the anticancer effects of G. lucidum. Ganoderic acid D (GAD) is one of the major components in Ganoderma triterpenes. GAD treatment for 48 h inhibited the proliferation of HeLa human cervical carcinoma cells with an IC(50) value of 17.3 +/- 0.3 microM. Flow cytometric analysis and DNA fragmentation analysis indicated that GAD induced G(2)/M cell cycle arrest and apoptosis. To identify the cellular targets of GAD, two-dimensional gel electrophoresis was performed, and proteins altered in expressional level after GAD exposure of cells were identified by MALDI-TOF MS/MS. The regulation of proteins was also confirmed by Western blotting. The cytotoxic effect of GAD was associated with regulated expression of 21 proteins. Furthermore these possible GAD target-related proteins were evaluated by an in silico drug target searching program, INVDOCK. The INVDOCK analysis results suggested that GAD could bind six isoforms of 14-3-3 protein family, annexin A5, and aminopeptidase B. The direct binding affinity of GAD toward 14-3-3 zeta was confirmed in vitro using surface plasmon resonance biosensor analysis. In addition, the intensive study of functional association among these 21 proteins revealed that 14 of them were closely related in the protein-protein interaction network. They had been found to either interact with each other directly or associate with each other via only one intermediate protein from previous protein-protein interaction experimental results. When the network was expanded to a further interaction outward, all 21 proteins could be included into one network. In this way, the possible network associated with GAD target-related proteins was constructed, and the possible contribution of these proteins to the cytotoxicity of GAD is discussed in this report.