Elsevier

Phytomedicine

Volume 18, Issues 2–3, 15 January 2011, Pages 163-169
Phytomedicine

Proteomic identification of proteins involved in the anticancer activities of oridonin in HepG2 cells

https://doi.org/10.1016/j.phymed.2010.06.011Get rights and content

Abstract

Oridonin is the main bioactive constituent of the Chinese medicinal herb Isodon rubescens and has been shown to have anti-neoplastic effects against a number of cancers in vitro and in vivo. Here we report the proteomic identification of proteins involved in the anticancer properties of oridonin in hepatocarcinoma HepG2 cells. Cell viability assay showed that oridonin dose-dependently inhibited cell growth with an IC50 of 41.77 μM. Treatment with oridonin at 44 μM for 24 h induced apoptosis and G2/M cell cycle arrest, which were associated with nine differentially expressed proteins identified by proteomic analysis. The proteomic expression patterns of Hsp70.1, Sti1 and hnRNP-E1 were confirmed by quantitative real-time PCR and/or immunoblotting. Eight of the nine identified proteins are shown, for the first time, to be involved in the anticancer activities of oridonin. Up-regulation of Hsp70.1, STRAP, TCTP, Sti1 and PPase, as well as the down-regulation of hnRNP-E1 could be responsible for the apoptotic and G2/M-arresting effects of oridonin observed in this study. Up-regulation of HP1 beta and GlyRS might contribute to inhibitory effects of oridonin on telomerase and tyrosine kinase, respectively. These findings shed new insights into the molecular mechanisms underlying the anticancer properties of oridonin in liver cancer cells.

Introduction

Isodon rubescens (Hemsl.) C.Y. Wu et Hsuan, native to the Yellow River valley in China, has been in use as a remedy for the treatment of respiratory and gastrointestinal bacterial infections as well as cancers (Sun et al., 2006). Oridonin (Fig. 1), a diterpenoid compound, is the major bioactive constituent of I. rubescens. In human patients oridonin exhibits curative effect for digestive system tumors such as esophageal and liver cancers (Wang, 1984, Wang and Wang, 1984). In animal experiments oridonin prolongs the lifespan of mice with Ehrlich ascites carcinoma, P388 lymphocytic leukemia or t (8;21) leukemia (Zhou et al., 2007). In various cancer cell lines, including HepG2, oridonin shows antiproliferative (Chen et al., 2007) and apoptotic effects (Ikezoe et al., 2003, Wang et al., 2008). Caspases, p53, reactive oxygen species, p38 MAPK pathway, and nuclear factor-kappa B (NF-κB) have been implicated in the apoptosis-inducing activity of oridonin (Huang et al., 2008, Cheng et al., 2009). Oridonin can also induce G2/M or G0/G1 arrest depending on cell type (Liu et al., 2004, Wang et al., 2008). Other effects of oridonin such as inhibition of telomerase (Wang et al., 2008) and tyrosine kinase (Li et al., 2007), anti-angiogenesis (Meade-Tollin et al., 2004), and anti-migration and differentiation induction (Ren et al., 2006) have also been reported. To further understand the molecular mechanism of oridonin in liver cancer therapy, in this study we identified differentially expressed proteins in oridonin-treated HepG2 cells by using two-dimensional gel electrophoresis (2-DE)-based proteomics.

Section snippets

Oridonin

Oridonin was purchased from Shanghai Shamrock Import & Export, Trading Co. Ltd., and the purity was determined to be 97% by HPLC. Stock solution of oridonin (274.5 mM) was prepared in dimethyl sulfoxide (DMSO, Sigma, France). Aliquots were stored at −20 °C.

Cell culture

HepG2 cells (ATCC, USA), grown in Dulbecco's modified Eagle's medium (DMEM, GIBCO, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, GIBCO, USA) and 1% penicillin/streptomycin (P/S, GIBCO, USA) were cultured at 37 °C in an

Cytotoxicity of oridonin

Initial MTT assay showed that treatment with oridonin for 24 h resulted in a marked decrease in cell viability in a dose-dependent manner (Fig. 2). The IC50 value was determined as 41.77 μM from the dose–response curve with GraphPad Prism 5.0 software. Thus, in subsequent assays 44 μM oridonin was used.

Cell cycle arrest and apoptosis induced by oridonin

In the cytotoxicity assay it was noted that oridonin treatment resulted in rounding up of cells when observed under the microscope (Fig. 3(A)). To verify whether oridonin caused cell cycle

Discussion

Oridonin has been shown to exhibit antiproliferative activity in various cancer cell lines. The concentrations needed to cause 50% inhibition of cell growth (IC50s) are cell type-dependent, varying from several to tens of micromoles per liter (Zhang et al., 2009, Zhou et al., 2007, Ren et al., 2006, Chen et al., 2005). In HepG2 cells oridonin has been reported to have an IC50 value of about 30 μM (at 24 h) (Huang et al., 2008), while in the present report the IC50 value is 41.77 μM. The

Acknowledgements

We thank the Proteomic Laboratory for System Biology, School of Chinese Medicine, Hong Kong Baptist University, for proteomic analyses. This work was supported by a research grant (FRG/08-09/I-08) from Hong Kong Baptist University.

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