Nat Prod Sci.  2019 Dec;25(4):298-303. 10.20307/nps.2019.25.4.298.

Asiatic Acid Induces Apoptosis and Autophagy and Reduces MiR-17 and MiR-21 Expression in Pancreatic Cancer Cell Lines

• Jo YG
• Kim M
• Shin H
• Lee KY
• Lee EJ

• KMID: 2468062
• DOI: http://doi.org/10.20307/nps.2019.25.4.298

Abstract

This study investigated the cytotoxic effects and mechanism of action of asiatic acid in pancreatic cancer cell lines. First, we confirmed the cell viability of MIA PaCa-2 and PANC-1 cells after asiatic acid administration for 48 and 72 h. The viability of MIA PaCa-2 and PANC-1 cells decreased in a dose-dependent manner following asiatic acid administration. To investigate the underlying mechanism, we performed a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, annexin V assay, and western blotting. Asiatic acid induced apoptosis and autophagy through activation of AMP-activated protein kinase (AMPK) and inhibition of mammalian target of rapamycin (mTOR) in MIA PaCa-2 cells. Finally, the expression of miR-17 and miR-21, known as oncogenes in pancreatic cancer, was decreased by asiatic acid. These results indicate that asiatic acid has potential as a new therapeutic agent against pancreatic cancer.

Keyword

Asiatic acid; Pancreatic cancer; Apoptosis; Autophagy; MicroRNA

MeSH Terms

AMP-Activated Protein Kinases
Annexin A5
Apoptosis*
Autophagy*
Blotting, Western
Cell Line*
Cell Survival
DNA Nucleotidylexotransferase
MicroRNAs
Oncogenes
Pancreatic Neoplasms*
Sirolimus
AMP-Activated Protein Kinases
Annexin A5
DNA Nucleotidylexotransferase
MicroRNAs
Sirolimus

Figure

• 

  Fig. 1 The chemical structure of asiatic acid.

• 

  Fig. 2 Asiatic acid inhibits the growth of pancreatic cancer cells. MIA PaCa-2 cells (A) and PANC-1 cells (B) were seeded onto a 96-well plate and incubated with 0, 20, 40, and 60 µM of asiatic acid for 48 (filled circle) and 72 h (unfilled circle). Cell viability was analyzed using the WST-1 assay and mean values were calculated from triplicate wells. This experiment was repeated three times (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001).

• 

  Fig. 3 Asiatic acid induces apoptosis in MIA PaCa-2 cells. (A) The effect of asiatic acid in MIA PaCa-2 cells using the TUNEL assay. Positive control was HSW345. Cells were photographed at magnifications of 10 and 20X. (B) The effect of asiatic acid on the expression of apoptosis-related proteins in MIA PaCa-2 cells. (C) The effect of asiatic acid on apoptosis in MIA PaCa-2 cells was estimated by annexin V assay.

• 

  Fig. 4 The effect of asiatic acid on the expression of autophagy-related proteins in MIA PaCa-2 cells. Western blotting was performed to detect the protein expression of LC-3II, p-AMPK, AMPK, p-p38, p38, p-mTOR, mTOR, and tubulin. Asiatic acid was dissolved in DMSO. MIA PaCa-2 cells were treated with different concentrations of asiatic acid (0, 20, 40 and 60 µM) for 48 h. Tubulin was used as a loading control and band density was calculated using Image J.

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  Fig. 5 Expression level of microRNAs in MIA PaCa-2 cells treated with asiatic acid. Cells were treated with 60 µM of asiatic acid for 48 and 72 h. The CT mean was calculated from three wells and RNU6B was used as a control to determine the relative miRNA expression. The 2−ΔΔCT method was used for the relative quantitation of miR-17 (A) and miR-21 (B).

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