Herbal medicines have been used to

Herbal medicines have been used to treat various types of diseases and disorders for thousands of years worldwide (Li and Brown, 2009). Natural products have been the remedy of cancer chemotherapy for the past several decades. Anisomeles indica (L.) Kuntze. (Labiatae), commonly known as 'yu-chen-tsao', has been traditionally used as an anti-inflammatory agent (Rao et al., 2009), it also has been shown to have activity against gastrointestinal disease (Liao et al., 2012), and used for immune system deficiencies, as well as liver disorders (Huang et al., 2003). The compound ovatodiolide, a macrocyclic diterpenoid isolated from Anisomeles indica (Rao et al., 2012), has been previously reported to exhibit the biological activities, including, antibacterial activity (Rao et al., 2012), anti-HIV activity (Shahidul Alam et al., 2000), inducing SCH-900776 arrest and apoptosis in human oral squamous cell carcinoma cells (Hou et al., 2009), anti-metastatic effect in human breast cancer cells (Lin et al., 2011), and suppressing tumorigenesis and overcoming drug resistance in renal cell carcinoma (Ho et al., 2013). However, the anti-tumor effect and its molecular mechanisms of ovatodiolide have not been extensively explored. In this study, we examined the anti-tumor property of ovatodiolide on lung cancer cells. Our results showed that ovatodiolide stimulated intracellular reactive oxygen species generation, caused DNA damage, activated ATM/ATR and CHK1/2 signaling pathways, consequently induced cell cycle G2/M arrest and apoptosis in A549 and H1299 cells.
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Discussion Cancer prevention and therapy using traditional Chinese medicines have attracted growing interest (Zhang et al., 2013). Anisomeles indica, is an aromatic plant and used in traditional medicine, as a tonic and carminative as well as in stomach pain, dyspepsia and intermittent fever (Cross et al., 1987), also known to have anti-inflammatory properties (Jones, 1987, Rao et al., 2009). Several active components have been isolated from Anisomeles indica including β-sitosterol, ovatodiolide, apigenin, and β-sitosterol-3-O-β-D-glucoside (Chen et al., 2008, Liao et al., 2012). Recently, Anisomeles indica and its purified compounds exert anti-metastatic activity through inhibition of NF-kappaB/AP-1- dependent MMP-9 activation in human breast cancer (Liao et al., 2012). Among them, ovatodiolide has been shown as a potential inhibitor of Helicobacter pylori-induced inflammation (Jones, 1987) and also acts as an immune modulator (Rao et al., 2013). Only a few studies have investigated the anti-cancer activities of ovatodiolide and provided limited evidence to state its molecular events. A previous report demonstrates that ovatodiolide inhibits the metastasis by decreasing MMP-9 activity via suppressing PI3K/Akt, p38-MAPK, JNK, and NF-κB signaling pathways in human breast cancer MDA-MB-231 cells (Lin et al., 2011). Targeting β-catenin signaling pathway by ovatodiolide can suppress tumorigenesis and overcoming drug resistance in human renal cancer cells (Ho et al., 2013). In human oral cancer Ca9-22 cells, ovatodiolide can induce cell cycle G2/M arrest and apoptosis (Hou et al., 2009). In this study, we showed that ovatodiolide triggered a reactive oxygen species-mediated cell cycle G2/M arrest and apoptosis through activating ATM/ATR and CHK1/2 signaling pathways in human lung cancer A549 and H1299 cells. Disruption of cell cycle progression is regarded as an important strategy for eliminating cancer cells. The eukaryotic cell cycle progression is stringently controlled by the reciprocal actions between activators (Cyclin, CDK, CDC25, etc.) and inhibitors (INK and CIP/KIP family, p53, etc) (Boonstra and Post, 2004). The CDK1/Cyclin B1 kinase is a main regulator to progress cell cycle from G2 into M phase. During G2 phase, CDK1 is activated by CDC25C through dephosphorylation at Thr14/15 and inactivated by WEE1 and MYT1 protein kinases through phosphorylation at Thr14/15 (Bulavin et al., 2003, Weinert, 1997). CDC25C is a key protein controlling the cell cycle G2 /M transition, and also an essential factor of the checkpoint pathway which can be activated in response to DNA damage (Abraham, 2001). Activation of ATM triggered by DNA damage mediates the CHK1, CHK2 activation and inhibition and degradation of CDC25C, moreover, inhibition of CDK1 by CDC25C to cause cell cycle G2/M blockade (Abraham, 2001). In this study, exposure of A549 and H1299 cells to ovatodiolide markedly increased ATM, ATR, CHK1 and CHK2 activation by phosphorylation at Ser-1981, Ser-428, Ser-345 and Thr-68, respectively. Activation of ATM/ATR and CHK1/2 signaling axis led to phosphorylate CDC25CSer216 that inhibited CDK1/Cyclin B1 kinase activity and then triggered cell cycle arrest at G2/M phase.