Several plant derived molecules such as
Several plant-derived molecules such as resveratrol, curcumin, zerumbone, and physalin B have been reported as modulators of Hh/Gli signaling pathway (Hosoya et al., 2008, Mohapatra et al., 2015). Our previous studies have demonstrated that a sesquiterpene lactone and diarylheptanoids from Siegesbeckia glabrescens, Alpinia officinarum and Alnus japonica can inhibit the Hh/Gli signaling pathway (Dong et al., 2017, Lee et al., 2016). Herein, we report that Z-ajoene (Fig. 1A) from garlic can also affect the Hh/Gli signaling pathway. While tightly regulated Hh pathway is responsible for coordinating cellular growth and embryo development, dysregulation of Hh pathway is involved in the formation, development, and aggressiveness of tumors, especially pancreatic cancer (Lauth and Toftgard, 2011, Xie et al., 2013). Hh pathway is activated with a series of signal cascade such as Shh ligand binding to Ptch, Smo activation and initiation of Gli-mediated transcription. Recent evidence suggests that constitutive activation of Gli is associated with tumor formation and growth (Kasper et al., 2006, Pietrobono et al., 2018). Although Smo antagonists have been reported as therapuetic candidates for various cancers (Fendrich et al., 2011, Ibuki et al., 2013), their therapeutic efficacies in preclinical and clinical cancer models are not promising (Rimkus et al., 2016). Recent studies have shown that acquired resistance to Smo inhibition is linked to mutations in Smo and aberrant activation of Gli or upregulation of synergic signals such as PI3K signaling (Benvenuto et al., 2016, Lauth and Toftgard, 2007). Resistance against Smo antagonist (vismodegib) has already been reported in patients with advanced or metastatic solid tumors (Atwood et al., 2012, Gonnissen et al., 2015, LoRusso et al., 2011). Another study has reported that GANT 61 (a Gli inhibitor) shows therapeutic effects on colon cancer oxytocin antagonist that are resistance to Smo inhibitors (cyclopamine or GDC-0449) (Agyeman, Mazumdar, & Houghton, 2012). Therefore, the inhibition of Gli has been recognized as an efficient strategy to regulate the Hh signaling pathway for cancer therapy. In our reporter assay system, C3H10T1/2-Gli1-Luc cells were incubated with Shh CM to induce Gli1-mediated luciferase activity (Fig. 1B). Z-Ajoene inhibited Shh CM-induced luciferase activity in a concentration-dependent manner as seen in Fig. 1C. Z-Ajoene also inhibited SAG (a Smo activator)-induced luciferase activity (Fig. 1D). These findings suggest that Z-ajoene could modulate the activation of Gli at the downstream of Smo in Hh pathway. Here, we found that Z-ajoene could inhibit Gli transcriptional activity and mRNA level of Gli1 in PANC-1 human pancreatic cancer cells known to have high level of Gli1 with resistance to Smo inhibitor (Guo et al., 2013, Thayer et al., 2003) (Fig. 2A and B). These results suggest that regulation of Gli-mediated transcription might contribute to the inhibitory potential of Z-ajoene on Hh signaling pathway. Our colleagues demonstrated that an ajoene analog suppressed NF-κB transcriptional activity through modulating the interaction between NF-κB and PPARγ. The analog did not affect nuclear localization and DNA binding affinity of the p65 subunit, but enhanced its physical interaction with PPARγ, which resulted in suppression of NF-κB transcriptional activity (Hwang et al., 2016). Here, we found that Z-ajoene also did not alter Gli1 protein level in nuclear fraction in PANC-1 cells (Supplementary material Fig. S2). The mode of action for inhibition of Gli transcriptional activity by Z-ajoene in PANC-1 cells might be derived from altered interaction between Gli and regulatory cofactors. The exact mechanism needs to be elucidated in a further study. Gli1, one of target genes of the Gli transcription factor, can regulate the transcription of Hh responsive genes by itself. Gli1 is upregulated in pancreatic cancer tissues. Its expression level is positively correlated with Hh signaling (Liu et al., 2015). FoxM1, another Gli target gene, acts as a cell cycle regulator and plays an important role in carcinogenesis (Wang et al., 2017, Wierstra, 2013). Z-Ajoene down-regulated Gli target proteins such as Gli1, Gli2, Ptch, and FoxM1 protein levels whereas basal levels of Gli1 and FoxM1 were elevated in PANC-1 cells (Fig. 2C). The level of FoxM1 was more susceptible than Gli1 level by treatment with 10 μM Z-ajoene. This means that Z-ajoene might have another mechanism for modulating FoxM1 protein level besides the Gli pathway. Further study is needed to determine the molecular target of Z-ajoene so that we could explain the detailed mechanism. Moreover, Z-ajoene did not alter levels of phosphorylated Akt in PANC-1 cells (Fig. 2C), suggesting that Z-ajoene could specifically regulate the Hh/Gli signaling pathway without affecting the PI3K/Akt signaling pathway.