In our reporter assay system C H T Gli Luc

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 Fomepizole 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. FoxM1, a downstream target of Gli, has been mainly related to cell cycle regulation and cellular proliferation in tumor (Alvarez-Fernandez and Medema, 2013, Halasi and Gartel, 2013, Teh et al., 2002). Especially, FoxM1 is a regulator of G1/S and G2/M transitions and M-phase progression (Wonsey & Follettie, 2005). It controls cell cycle-related molecules such as c-Myc (Wierstra & Alves, 2006), cyclin B1 (Leung et al., 2001) and survivin (Nestal de Moraes et al., 2015). c-Myc, an oncoprotein, is responsible for cellular proliferation and cell cycle regulation of cancer cells. c-Myc expression is elevated and deregulated in neoplastic cells (Albihn et al., 2010, DePinho et al., 1991). Cyclin B1, another target of FoxM1, is usually expressed at very low levels. It accumulates sharply only at G2/M cell cycle transition under normal conditions. In cancer cells, cyclin B1 is overexpressed throughout cell cycle progression (Ye, Wang, Wu, Li, & Chai, 2017). Survivin, an anti-apoptotic protein, plays a role in the regulation of cell division and spindle formation. It is essential for cell cycle progression (Mita, Mita, Nawrocki, & Giles, 2008). Overexpression of survivin is frequently associated with poor prognosis and drug resistance in neoplastic mammary cells (Hinnis, Luckett, & Walker, 2007). As shown in Fig. 3, Z-ajoene suppressed target gene expression of FoxM1, c-Myc, cyclin B1, and survivin, together with depressed proliferation of PANC-1 pancreatic cancer cells (Fig. 4A). These results are in agreement with down-regulation of FoxM1 by Z-ajoene through inhibition of Gli activation. Furthermore, cell cycle analysis demonstrated that Z-ajoene significantly elevated the percentage of G2/M phase cells (Fig. 4B). These results suggest that Z-ajoene can reduce the proliferation of PANC-1 pancreatic cancer cells through inducing cell cycle arrest in G2/M phase. Taken together, Z-ajoene exhibits anti-proliferative activity in pancreatic cancer cells by down-regulating Gli in aberrantly activated Hh signaling pathway. Therefore, inhibition of Gli is a well-organized approach to regulate the Hh signaling pathway for cancer therapy.