Another contributor for the G M arrest in A and

Another contributor for the G2/M arrest in A549 and H1299 cells might be p21WAF1/CIP1 which was up-regulated upon ovatodiolide treatment. p21WAF1/CIP1 is a common CDKs inhibitor which blocks Cabozantinib G2/M phase progression in various types of cancer cells (Kim et al., 2015, Liberio et al., 2015). It has been reported that DNA damage signaling up-regulates p21WAF1/CIP1 expression via both p53-dependent and -independent pathways to trigger cell cycle arrest in G2 phase (Dvory-Sobol et al., 2006, Han et al., 2012, Sugimoto et al., 2006). In response to DNA damage, p21WAF1/CIP1 is a key mediator of cell cycle G2 arrest by inactivating Cyclin B1/CDK1 kinase through degradation of Cyclin B1 (Gillis et al., 2009). The data presented here indicated that ovatodiolide-induced DNA damage and p21WAF1/CIP1 expression was accompanied by decreasing Cyclin B1 protein level and inactivating CDK1, as well as G2/M cell cycle arrest; however, the level of Cyclin B1 mRNA was not affected (data not shown), suggesting that ovatodiolide-induced G2/M arrest was partly through p21WAF1/CIP1-mediated inactivation of Cyclin B1/CDK1 kinase by promoting Cyclin B1 degradation. Overall, our results indicated that ovatodiolide induced G2 arrest in A549 and H1299 cells by down-regulation of Cyclin B1 expression and inactivation of CDK1 activity, which was maintained through up-regulation of p21WAF1/CIP1 and down-regulation of CDC25C. Worthy of note, overexpression of p21WAF1/CIP1 and induction of G2/M arrest were observed in both A549 (p53 wild type) and H1299 (p53 null) cells, this suggests that ovatodiolide-mediated p21WAF1/CIP1 up-regulation and G2/M arrest irrespective of the p53 gene status. Apoptosis is a highly conserved cell suicide process which is triggered by a lot of physiological and pathological stimuli, including chemotherapy and radiation therapy (Hassan et al., 2014). Mammalian apoptosis is mainly categorized into two pathways, extrinsic (death receptor-dependent) and intrinsic (mitochondria-dependent), that are regulated by a variety of molecular mechanisms, which attribute to both pro-apoptotic proteins, including Bax, PUMA, Bak, Fas, TNF-R and TRAIL-R (DR4 and DR5), and anti-apoptotic proteins, such as Bcl-2 and Mcl-1 (Elmore, 2007). It is well documented that apoptosis plays an important role in tumorigenesis; its impairment in cancer cells may lead to tumor progression. Therefore, the therapeutic strategy targeting the apoptotic signaling pathway may play a key role in the fight against cancer. Ovatodiolide has been reported induces apoptosis, as indicated by caspase (-3, -8, and -9) activation, DNA fragmentation, and PARP cleavage in human oral squamous cell carcinoma Ca9-22 cells (Hou et al., 2009). Similarly, our results also showed that caspase-3, caspase-8 and caspase-9 activation was contributed to ovatodiolide-induced apoptosis in both A549 and H1299 cells. Moreover, our data demonstrated that ovatodiolide increased the levels of pro-apoptotic proteins, Bax, PUMA and DR5, and reduced the level of anti-apoptotic proteins, Bcl-2 and Mcl-1 (Fig. 3C). All of these findings indicated ovatodiolide as a powerful pro-apoptotic agent, which could trigger different pathways leading to both intrinsic and extrinsic apoptotic cell death in human cancer cells. Induction of intracellular reactive oxygen species generation is one of the important mechanisms by which most anticancer drugs or radiation therapy kill cancer cells (Verrax et al., 2009). Reactive oxygen species-mediated oxidative stress can damage cellular components including DNA and proteins. DNA damage induction is the primary mechanism of action of most lung cancer therapeutics (Helleday et al., 2008). The cumulative failure of the cell to repair DNA lesions by their corresponding repair mechanisms may lead to their conversion to DNA double-strand breaks, eventually triggering cell cycle blockade and cell death. Growing evidence demonstrates that reactive oxygen species can influence cell cycle progression and apoptosis through activating a variety of oxidative stress-sensitive intracellular signaling pathways, such as ATM/ATR, CHK1/2 and JNK (Sahu et al., 2009). Elevating intracellular levels of reactive oxygen species has been associated with reducing cancer cell proliferation by induction of G2/M cell cycle arrest; increasing phosphorylation of ATMSer1981, ATRSer428, CHK1Ser345, CHK2Thr68; and decreasing CDC25C (O'Reilly et al., 2007, Sun et al., 2007). Ovatodiolide has been shown to trigger cell cycle G2/M arrest and apoptosis through disturbance of intracellular redox balance in oral squamous carcinoma cells (Hou et al., 2009). In this study, we found that ovatodiolide-induced cell cycle G2/M retardation and apoptosis was accompanied by reactive oxygen species accumulation. Moreover, ovatodiolide induced DNA damage (Fig. 4B), and subsequent phosphorylation of ATMSer1981, ATRSer428, CHK1Ser345, CHK2Thr68 (Fig. 4C). Functionally, we found that ATM/ATR signaling was responsible for ovatodiolide-mediated cell cycle retardation and apoptosis. Because, inhibition of ATM/ATR by pharmacological inhibitors caffeine effectively blocked ovatodiolide-mediated cell cycle arrest and apoptosis in A549 and H1299 cell lines. Besides, the principal role of reactive oxygen species generation in ovatodiolide-mediated cellular and molecular events was confirmed by the capability of the antioxidant N-acetylcysteine to inhibit ATM/ATR and CHK1/2 activation, reduce p21WAF1/CIP1, CDC25C, PUMA, Bax, and DR5 expression, as well as completely block G2/M arrest and apoptosis. Therefore, we conclude that stimulated reactive oxygen species production plays a key and regulatory role in ovatodiolide-induced cell cycle G2/M arrest and apoptotic cell death caused by ATM/ATR and their downstream molecules in human lung cancer A549 and H1299 cells.