Magnolol Suppresses Pancreatic Cancer Development In Vivo and In Vitro via Negatively Regulating TGF-β/Smad Signaling

Abstract

Magnolol, a hydroxylated biphenyl extracted from Magnolia officinalis, has recently drawn attention due to its anticancer potential. The present study was aimed to explore the effects of Magnolol on restraining the proliferation, migration and invasion of pancreatic cancer in vivo and in vitro. Magnolol showed significant anti-growth effect in an orthotopic xenograft nude mouse model, and immunohistochemical staining of the xenografts revealed that Magnolol suppressed vimentin expression and facilitated E-cadherin expression. The cytoactive detection using CCK-8 assay showed Magnolol inhibited PANC-1 and AsPC-1 concentration-dependently. Scratch healing assay and the Transwell invasion assay proved the inhibiting effects of Magnolol on cellular migration and invasion at a non-cytotoxic concentration. Western blot and rt-PCR showed that Magnolol suppressed epithelial-mesenchymal-transition by increasing the expression level of E-cadherin and decreasing those of N-cadherin and vimentin. Magnolol suppressed the TGF-β/Smad pathway by negatively regulating phosphorylation of Smad2/3. Moreover, TGF-β1 impaired the antitumor effects of Magnolol in vivo. These results demonstrated that Magnolol can inhibit proliferation, migration and invasion in vivo and in vitro by suppressing the TGF-β signal pathway and EMT. Magnolol could be a hopeful therapeutic drug for pancreatic malignancy.

Keywords: Smad; TGF-β; epithelial-mesenchymal-transition; magnolol; pancreatic cancer.

Figure 1

Magnolol (MAG) inhibits pancreatic orthotopic xenograft growth in a mouse model. (A) Schematic representation of in vivo treatment strategy. (B) Images by the living imaging analysis of mice from both the groups at different time points. (C) Tumor growth curve (total photons per second) showing tumor growth at different time points. (D) The image and (E) weight of the tumors harvested at the end point. *P < 0.05.

Figure 2

Epithelial-mesenchymal-transition (EMT) and proliferation is restrained in pancreatic xenografts of Magnolol (MAG)-treated mice. (A) HE staining was applied to confirm the xenografts formation while Immunohistochemical staining was used to access the expression of Ki67, E-cadherin and vimentin. (B–D) The expression of Ki67, E-cadherin and vimentin were analyzed. *P < 0.05.

Figure 3

Magnolol (MAG) suppressed the viability of pancreatic cancer cells (A, D) MAG suppressed the viability of Panc-1 and AsPC-1 in time- and concentration-dependent manner. (B, C, E, F) MAG (15 or 30 μM) reduced colony formation of Panc-1 and AsPC-1. *P < 0.05; **P < 0.01.

Figure 4

Magnolol Suppressed Cellular Migration and Invasion in vitro (A, B) The wound-healing of cells treated with Magnolol(0, 15, 30 μM) was shown at 0, 24 and 48 h after scratching. (C, D) The results of the scratch-wound assay were analyzed. (E) Morphological changes in Panc-1 and AsPC-1 cells after culture with Magnolol(0, 15, 30 μM). (G, H) Evaluation of cell morphology for Panc-1 and AsPC-1 cells after culture with Magnolol(0, 15, 30 μM). (F, I) Transwell invasion assay showed Magnolol treatment reduced the invasive rate of PANC-1 and AsPC-1 cells. *P < 0.05; **P < 0.01.

Figure 5

Magnolol inhibited epithelial-mesenchymal-transition (EMT) via the suppression of transforming growth factor-β1 (TGF-β1)/Smad Signalling. (A) Western blot analysis of protein levels of E-cadherin, N-cadherin, Vimentin, p-Smad2/3, Smad2/3 in Panc-1 and AsPC-1 cells treated with Magnolol(0, 15, 30 μM) for 48 h. (B, C) Histograms show the change of relative protein expression of E-cadherin, N-cadherin, Vimentin, p-Smad2/3, Smad2/3 in Panc-1 and AsPC-1 cells treated with Magnolol(0, 15, 30 μM) for 48 h. *P < 0.05.

Figure 6

Magnolol abolished transforming growth factor-β1 (TGF-β1)-Induced Migration and Invasion in vitro. (A, B) The wound-healing of cells treated with with Magnolol or TGF-β1 was shown at 0, 24, and 48 h after scratching. (D, E) The migration rate of the scratch-wound assay was analyzed. (E) Morphological changes in Panc-1 and AsPC-1 cells after culture with Magnolol or TGF-β1. (C, F, G) Transwell invasion assay showed Magnolol treatment abolished TGF-β1 induced invasion of PANC-1 and AsPC-1 cells. *P < 0.05.

Figure 7

Magnolol abolished transforming growth factor-β1 (TGF-β1)-Induced epithelial-mesenchymal-transition (EMT) amd phosphorylation of Smad2/3 in vitro. (A) Morphological changes in Panc-1 and AsPC-1 cells after culture with Magnolol or TGF-β1. (B) Evaluation of cell morphology for Panc-1and AsPC-1 treated with Magnolol or TGF-β1. (C, E) Western blot results of E-cadherin, N-cadherin, Vimentin, p-Smad2/3, Smad2/3 in Panc-1or AsPC-1 treated with Magnolol or TGF-β1. (D, F) Histograms show the changes of relative expression of E-cadherin, N-cadherin, Vimentin, p-Smad2/3, Smad2/3 in Panc-1or AsPC-1 treated with Magnolol or TGF-β1. *P < 0.05.