A sesquiterpene lactone from Siegesbeckia glabrescens suppresses Hedgehog/Gli-mediated transcription in pancreatic cancer cells

Abstract

Pancreatic cancer is aggressive and therefore difficult to treat; however, continued efforts have been made with the aim of developing an effective therapy against the disease. The Hedgehog (Hh) signaling pathway is reportedly involved in the proliferation and survival of pancreatic cancer cells. The transcription factor glioma-associated oncogene (Gli) is a key component of the Hh signaling pathway and the primary effector of pancreatic cancer development. Inhibiting Gli is a proven therapeutic strategy for this disease. The present study examined the regulation of Gli and the expression of its target genes to identify an inhibitor of the Sonic Hh (Shh) pathway. A germacranolide sesquiterpene lactone (GSL) was isolated from Siegesbeckia glabrescens as an inhibitor of Gli-mediated transcription. The results demonstrated that GSL inhibited Shh-induced osteoblast differentiation and Gli homolog 1 (Gli1)-mediated transcriptional activity in mesenchymal C3H10T1/2 stem cells. Furthermore, GSL suppressed Gli-mediated transcriptional activity in human pancreatic cancer PANC-1 and AsPC-1 cells, which resulted in reduced cancer cell proliferation and downregulated expression of the Gli-target genes, Gli1 and cyclin D1. A sesquiterpene lactone from S. glabrescens may therefore serve as a candidate for the treatment of Hh/Gli-dependent pancreatic cancer.

Keywords: Gli; Hedgehog pathway; Siegesbeckia glabrescens; compositae; pancreatic cancer; sesquiterpene lactone.

Figure 1.

(A) Structure of GSL from Siegesbeckia glabrescens. (B) Effect of GSL on Shh-CM-induced ALP activity. C3H10T1/2 cells were treated with GSL and/or Shh-CM for 96 h and ALP activity was measured. Cyclopamine (5 µM) was used as the positive control. . (C) Effect of GSL on Gli1-mediated transcriptional activity. C3H10T1/2-Gli1-luc cells were seeded onto a 96-well plate and were subsequently induced with Shh-CM. At the same time, various concentrations (0,5,10 and 20 µM) of GSL were added for 30 h. Cyclopamine (5 µM) was used as the positive control. The ratio of luciferase reporter activity of the GSL-treated cells/vehicle-treated cells was calculated as the inhibitory effect on Shh-CM-stimulated Gli1-mediated transcriptional activity. *P<0.05 vs. Shh-CM. GSL, germacranolide sesquiterpene lactone; Shh-CM, Sonic Hedgehog conditioned medium; ALP, alkaline phosphatase; Gli1, glioma-associated oncogene homolog 1.

Figure 2.

Effect of GSL on Gli-mediated transcriptional activity in human pancreatic cancer PANC-1 cells. PANC-1-Gli-luc cells were seeded onto a 96-well plate and treated with various concentrations (0, 1, 5, 10 and 20 µM) of GSL. GANT61 (30 µM) was used as the positive control. Luciferase activity was measured following 20 h using a microplate luminometer. The ratio of luciferase reporter activity of the GSL-treated cells/vehicle-treated cells was calculated as the inhibitory effect on Gli-mediated transcriptional activity. *P<0.05 vs. control. GSL, germacranolide sesquiterpene lactone; Gli, glioma-associated oncogene.

Figure 3.

Effect of GSL on Gli1 and cyclin D1 protein levels in human pancreatic cancer cells. Cells were treated with GSL at the indicated concentrations for 20 h. Cell lysates were prepared, and the Gli1, cyclin D1 and β-actin protein levels were determined by western blotting. Images are representative of three independent experiments with similar results. GSL, germacranolide sesquiterpene lactone; Gli1, glioma-associated oncogene homolog 1; p-, phospho.