Proteomics Characterization of the Cytotoxicity Mechanism of Ganoderic Acid D and Computer-automated Estimation of the Possible Drug Target Network*□S

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 IC50 value of 17.3 0.3 M. Flow cytometric analysis and DNA fragmentation analysis indicated that GAD induced G2/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 MALDITOF 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 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. Molecular & Cellular Proteomics 7:949–961, 2008. Ganoderma lucidum is a medicinal mushroom known to the Chinese as “Lingzhi.” It has been used as a home remedy in traditional Chinese medicine (TCM) for over 2000 years (1). In TCM, it was believed to preserve the human vitality and to promote longevity. More recently, it has been used for the prevention or treatment of a variety of diseases including cancer. And in Western countries, the dried powder of G. lucidum is also popularly used as a dietary supplement (2). Among the reported biological/pharmacological properties of G. lucidum, their antitumor activities are of particular interest. Investigations into the anticancer activity of G. lucidum have been performed in both in vitro and in vivo studies, supporting its application for cancer treatment and prevention (for reviews, see Refs. 3 and 4). Polysaccharides and triterpenes are two major categories of the bioactive ingredients from G. lucidum, and it has been found previously that polysaccharides exert their anticancer effect mainly via an immune-modulatory mechanism, whereas triterpenes directly suppress growth and invasive behavior of cancer cells (5). Triterpenes were reported to be able to inhibit growth, induce apoptosis, and cause cell cycle arrest of cancer cells (6–9). However, the cytotoxicity mechanism of Ganoderma triterpenes is still far from clear. In the present study, ganoderic acid D (GAD), a main component of Ganoderma triterpenes, with purity greater than 99% was used. We checked the GADmediated response on the proliferation of HeLa human cervical carcinoma cells. Then for a comprehensive analysis of the molecular targets of GAD, a proteomics approach was used for identifying proteins altered in steady-state levels after exposure of HeLa cells to GAD for 48 h. 2-DE was conducted, and then differentially expressed proteins were identified by