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

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

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

PA inhibits growth of pancreatic tumors <i>in vivo</i>.

<p>MIA PaCa-2 cells were subcutaneously implanted into female nude mice and treated with PA (A) 0 and 25 mg kg^-1, and (B) 0 and 50 mg kg^-1 of body weight 3 times week^-1 as described in Materials and methods. Tumor sizes were measured by microcalipers 3 times per week and calculated for additional 5 weeks. Data are mean ± SD (n = 10–15). T-test was used to compare the mean tumor volumes of both treatment groups to control at day 34 (*p = 0.004).</p

NAHA inhibits growth of breast cancer cells.

<p>(A) Structure of NAHA, <i>2-[Benzyl-(2-nitro-benzenesulfonyl)-amino]-N-hydroxy-3-methyl-N-propyl-butyramide</i>. (B) MDA-MB-231, (C) MCF-7, (D) MCF-10A, (E) HMEC cells were treated with NAHA (0–50 µM). Cell proliferation was determined by the tetrazolium salt method 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>. Cell viability was determined by trypan blue staining 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>. Data are the means ± SD. Similar results were obtained in at least two additional experiments. * p<0.05 for cell proliferation, # p<0.05 for cell viability. (F) Anchorage-independent growth (colony formation) of MDA-MB-231 cells was assessed on 1% agarose after incubation with NAHA (a – 0, b – 10 µM, c -25 µM, d – 50 µM) for 14 days 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>. (G) MDA-MB-231 cells were treated with NAHA (0–50 µM) for 24 hours and whole cell extracts were subjected to Western blot analysis with anti-Cdk2 and anti-CDC20 antibodies 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 equal protein loading was verified with anti β-actin antibodies. The rfesults are representative of three independent experiments.</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 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

Blocking ER stress inhibits PA induced apoptosis.

<p>(A) PANC-1 and (B) MIA PaCa-2 cells were treated with or without PA (30 μM) in presence of TUDCA (200 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 in presence of TUDCA (200 μM) for 24 h. Representative blots show expression of PARP cleavage (c-PARP) and β-actin was used as loading control. Effect of TUDCA on PA induced expressions of ATF4 and CHOP in (C) PANC-1 and (D) MIA PaCa-2 cells were shown as well. 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