Yonsei Med J.  2018 Jan;59(1):20-27. 10.3349/ymj.2018.59.1.20.

Pseudolaric Acid B Inhibits Proliferation, Invasion and Epithelial-to-Mesenchymal Transition in Human Pancreatic Cancer Cell

• Li X
• Zhao X
• Song W
• Tian Z
• Yang L
• Niu Q
• Zhang Q
• Xie M
• Zhou B
• Xu Y
• Wu J
• Zhang C

• KMID: 2418844
• DOI: http://doi.org/10.3349/ymj.2018.59.1.20

Abstract

PURPOSE
This study was aimed to investigate the effect of pseudolaric acid B (PAB) on proliferation, invasion and epithelial-to-mesenchymal transition (EMT) in pancreatic cancer cells and to explore the possible mechanism.
MATERIALS AND METHODS
The pancreatic cancer cell line SW1990 was cultured and treated with PAB dose- and time-dependent manners. Cell proliferation and invasion ability were measured by MTT assay and Matrigel/Transwell test, respectively. Semi-quantitative real-time polymerase chain reaction and Western blotting were conducted to detect the expression of EMT markers and the key molecules. Finally, nude mice subcutaneous transplantation tumor model was used to confirm the therapy efficacy of PAB.
RESULTS
PAB could inhibit SW1990 cell proliferation and invasion in time- and dose-dependent manners. Vimentin, fibronectin, N-cadherin, Snail, Slug, YAP, TEAD1, and Survivin were down-regulated (p < 0.01), while E-cadherin, caspase-9, MST1, and pYAP were up-regulated (p < 0.05). Combined PAB and gemcitabine treatment markedly restricted the tumor growth compared with gencitabin or PAB alone groups.
CONCLUSION
PAB could inhibit the proliferation and invasion ability of pancreatic cancer cells through activating Hippo-YAP pathway and inhibiting the process of EMT.

Keyword

PAB; PDAC; EMT; cell proliferation; Hippo-YAP pathway

MeSH Terms

Animals
Antineoplastic Agents/pharmacology/therapeutic use
Biomarkers, Tumor/metabolism
Cadherins
Cell Line, Tumor
Cell Movement
Cell Proliferation/drug effects
Cytokines
Deoxycytidine/analogs & derivatives/pharmacology/therapeutic use
Diterpenes/pharmacology/*therapeutic use
Epithelial-Mesenchymal Transition/*drug effects
Female
Humans
Mice, Nude
Neoplasm Invasiveness
Pancreatic Neoplasms/*diet therapy/*pathology
Real-Time Polymerase Chain Reaction
Signal Transduction/drug effects
Vimentin/metabolism
Antineoplastic Agents
Biomarkers, Tumor
Cadherins
Cytokines
Diterpenes
Vimentin
Deoxycytidine

Figure

• 

  Fig. 1 The effect of PAB on proliferation and invasion ability of pancreatic cancer cells. (A) The effect of PAB on proliferation rate of SW1990 cells. The proliferation of pancreatic cancer cell line was markedly reduced by PAB in dose- and time-dependent manners. (B) The number of cells passed through the Transwell micropores. The number of cells was significantly decreased when treated with PAB. (C) The cell morphology detected by crystal violet staining (×100). *p<0.01 when compared to the control group. PAB, pseudolaric acid B.

• 

  Fig. 2 The effect of PAB on the expression levels of EMT markers (vimentin, fibronectin, N-cadherin, Snail, Slug, and E-cadherin). (A) PAB down-regulated the expression levels of vimentin, fibronectin, N-cadherin, Snail, and Slug, and up-regulated the expression level of E-cadherin time-dependent manner. (B) Different concentrations of PAB down-regulated the expression levels of vimentin, fibronectin, N-cadherin, Snail, and Slug, and up-regulated the expression level of E-cadherin. *p<0.01 when compared to that of 24 h, or compared to that of 0.5 µmol/L, †p<0.01 level when compared to that of 8 µmol/L. PAB, pseudolaric acid B; EMT, epithelial-to-mesenchymal transition.

• 

  Fig. 3 The effect of PAB on Hippo-YAP pathway and synergized with gemcitabine against pancreatic cancer. (A) The relative level of Hippo-YAP pathway-related genes at different time points. Significant differences of MST and Caspase-9 were found at different treated time points. (B) The relative level of Hippo-YAP pathway-related genes when treated with various concentrations of PAB. (C) Expression level of YAP and pYAP detected by Western blotting. Significant differences of MST and Caspase-9 were found by different concentrations of PAB. (D) Expression level of YAP detected by RT-PCR. (E) Expression level of pYAP detected by RT-PCR. *p<0.01 when compared to the control group. PAB, pseudolaric acid B; RT-PCR, realtime polymerase chain reaction.

• 

  Fig. 4 The effect of PAB and gemcitabine on tumor volume. (A) Tumor morphology. (B) Tumor volume. *p<0.05 when compared to the control group. PAB, pseudolaric acid B.

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