Pachymic Acid Inhibits Growth and Induces Apoptosis of Pancreatic Cancer In Vitro and In Vivo by Targeting ER Stress

<div><p>Pachymic acid (PA) is a purified triterpene extracted from medicinal fungus <i>Poria cocos</i>. In this paper, we investigated the anticancer effect of PA on human chemotherapy resistant pancreatic cancer. PA triggered apoptosis in gemcitabine-resistant pancreatic cancer cells PANC-1 and MIA PaCa-2. Comparative gene expression array analysis demonstrated that endoplasmic reticulum (ER) stress was induced by PA through activation of heat shock response and unfolded protein response related genes. Induced ER stress was confirmed by increasing expression of XBP-1s, ATF4, Hsp70, CHOP and phospho-eIF2α. Moreover, ER stress inhibitor tauroursodeoxycholic acid (TUDCA) blocked PA induced apoptosis. In addition, 25 mg kg^-1 of PA significantly suppressed MIA PaCa-2 tumor growth <i>in vivo</i> without toxicity, which correlated with induction of apoptosis and expression of ER stress related proteins in tumor tissues. Taken together, growth inhibition and induction of apoptosis by PA in gemcitabine-resistant pancreatic cancer cells were associated with ER stress activation both <i>in vitro</i> and <i>in vivo</i>. PA may be potentially exploited for the use in treatment of chemotherapy resistant pancreatic cancer.</p></div

NAHA inhibits invasive behavior of breast cancer cells and capillary morphogenesis of endothelial cells.

<p>(A) Cell adhesion. MDA-MB-231 cells were treated with NAHA (0–50 µM) for 24 hours and cell adhesion to vitronectin determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. Each bar represents the mean ± SD of three experiments. * p<0.05. (B) Cell migration. Cell migration of MDA-MB-231 cells was determined after 5 hours of incubation in the presence of NAHA (0–50 µM), as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. Each bar represents the mean ± SD of three experiments. * p<0.05. (C) Cell invasion. Invasion of MDA-MB-231 cells through Matrigel was determined after 24 hours of incubation in the presence of NAHA (0–50 µM) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. Each bar represents the mean ± SD of three experiments. * p<0.05. (D) uPA secretion. MDA-MB-231 cells were treated with NAHA (0–50 µM) for 24 hours, and the expression of uPA detected in conditioned media from the same amount of cells with anti-uPA antibody by Western blot analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. The results are representative of three independent experiments. (E) MDA-MB-231 cells were treated with NAHA (0–50 µM) for 24 hours, media collected and secretion of VEGF determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. Each bar represents the mean ± SD of three experiments. * p<0.05. (F) HAECs were treated with NAHA (0–50 µM) for 16 hours. Capillary morphogenesis was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. Each bar represents the mean ± SD of three experiments. * p<0.05.</p

Effect of PA on the body weight of mice.

<p>Animal experiments were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122270#pone.0122270.g004" target="_blank">Fig 4</a>. Body weights were measured 3 times per week for (A) 0 and 25 mg kg-1, and (B) 0 and 50 mg kg−1 PA treatment. The graphical data represent mean +/- SD.</p

Model for the involvement of ER in PA-induced apoptosis and inhibition of tumor growth.

<p>Target molecules XBP-1s, ATF4, phospho-eIF2α, HSPA and CHOP are in red.</p

PA induces apoptosis in chemotherapy resistant pancreatic cancer cells.

<p>(A) PANC-1 and (B) MIA PaCa-2 cells were treated with PA (0–30 μM) for 24 h. Apoptosis was detected by Cell Death Detection ELISA and expressed relative to vehicle-treated cells (set equal to 1). Data were compared by ANOVA with Bonferroni correction for the significant level. Here, *p≤0.025 was considered significant for the individual t-tests. Apoptosis was confirmed in C) PANC-1 and D) MIA PaCa-2 cells treated with same concentrations of PA for 24 h. Representative blots show expression of PARP cleavage (c-PARP) and β-actin was used as loading control. Three independent experiments were done for the western blot studies and quantitative data composed of all the experiments in E) PANC-1 and F) MIA PaCa-2 cells with statistical analysis were shown below the representative blot image. *P<0.050 was considered to be significant when compared to control (n = 3) by Student t-test. The graphical data represent mean +/- SD.</p

Evaluation of organ pathology with PA treatment.

<p>Animal experiments were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122270#pone.0122270.g004" target="_blank">Fig 4</a>. Liver, kidney, spleen, lung and heart tissues were stained with H&E. Images are representative of control and PA treatment (25 mg kg^-1 and 50 mg kg^-1) groups.</p

PA induces expression of ER stress response proteins.

<p>(A) PANC-1 and (B) MIA PaCa-2 cells were treated with PA (0–30 μM) for 24 h, respectively. Whole protein extracts isolated from cells were prepared and western blot analysis with anti-XBP-1s, anti-ATF6, anti-ATF4, anti-Hsp70 and anti-CHOP antibodies were performed as described in Materials and methods. (E) PANC-1 and (F) MIA PaCa-2 cells were treated with PA (0–30 μM) for 10 min and 30 min, respectively. Whole protein extracts isolated from cells were prepared and western blot analysis with anti-phospho-eIF2α and anti-eIF2α antibodies were performed as described in Materials and methods. β-actin was used as loading control. Representative blots from three experiments were shown and quantitative data composed of all the experiments in C), G) PANC-1 and D), H) MIA PaCa-2 cells with statistical analysis were below the representative blot image. *P<0.050 was considered to be significant when compared to control (n = 3) by Student t-test. The graphical data represent mean +/- SD.</p

Effect of PA on liver enzyme profile of mice.

<p>Animal experiments were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122270#pone.0122270.g004" target="_blank">Fig 4</a>. Values are Mean ± SD (n = 10–13). ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; T protein, total protein. Blood was collected and serum was analyzed at the IU Health Pathology Laboratory.</p><p>*P < 0.050 for 50 mg kg^-1 PA vs. control by ANOVA.</p><p>Effect of PA on liver enzyme profile of mice.</p

Histology of MDA-MB-231 xenografts.

<p>Tumors dissected from animals were sectioned and stained with (A) H&E, and (B) mitotic figures (arrowheads), and (C) apoptotic bodies (arrows) evaluated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>.</p

PA upregulates expression of ER stress related genes.

<p>Expressions of different genes were detected by the Human Prime View Array technology in MIA PaCa-2 cells after 24 hours PA treatment with PA (0 and 30 μM) in quadruplicates. Gene expression levels were normalized and analyzed using Microarray Data Portal (MDP) by 2-way ANOVA using cell line and treatment as factors plus the interaction of cell line and treatment. A q-value program from Storey and Tibshirani was used to calculate false discovery rates. Fold change P < 1E-05.</p><p>PA upregulates expression of ER stress related genes.</p