Functional proteomic and structural insights into molecular targets related to the growth inhibitory effect of tanshinone IIA on HeLa cells

Certain antitumor agents have recently been extracted from the roots of Salvia miltiorrhiza Bunge. The diterpene derivative, tanshinone IIA, possesses cytotoxic activity against several human carcinoma cell lines. It also inhibits invasion and metastasis of cancer cells. In the present study, we isolated tanshinone IIA from S. miltiorrhiza, and it exhibited strong growth inhibition against human cervical cancer cells in dose- and time-dependent manners with a 50% cell growth inhibition value of 2.5 mu g/mL (8.49 mu M). Flow cytometric analysis of cell cycle progression revealed that G(2)/M arrest was initiated after a 24h exposure to the drug. It also resulted in DNA fragmentation and degradation of poly (ADP-ribose) polymerase indicating that tanshinone IIA may be a potential antitumor agent. Furthermore, we performed a comprehensive proteomic analysis to survey global protein changes induced by tanshinone IIA treatment on HeLa cells. Significant changes in the levels of cytoskeleton proteins as well as stress-associated proteins were observed. Immunoblot analysis and immunofluorescence staining were used to confirm the levels of protein expression. Overexpression of the vimentin rescued these tanshinone IIA-induced events. Computational docking methods indicated that tanshinone IIA could stably bind to the beta-subunit of the microtubule protein. An interaction network analysis of these 12 proteins using MetaCore (TM) software suggested that tanshinone IIA treatment regulated the expressions of proteins involved in apoptotic processes, spindle assembly, and p53 activation, including vimentin, Maspin, alpha- and beta-tubulin, and GRP75. Taken together, our results suggest that tanshinone IIA strongly inhibited the growth of cervical cancer cells through interfering in the process of microtubule assembly, leading to G(2)/M phase arrest and Sequent apoptosis. The success of this large-scale effort was assessed by a bioinformatics analysis of proteins through predictions of protein domains and possible functional roles. The possible contributions of these proteins to the cytotoxicity of tanshinone IIA provide potential opportunities for the development of cancer therapeutics.