OSU-A9 inhibits pancreatic cancer cell lines by modulating p38-JAK-STAT3 signaling

Abstract

Pancreatic cancer is an aggressive malignancy that is the fourth leading cause of death worldwide. Since there is a dire need for novel and effective therapies to improve the poor survival rates of advanced pancreatic cancer patients, we analyzed the antitumor effects of OSU-A9, an indole-3-carbinol derivative, on pancreatic cancer cell lines in vitro and in vivo. OSU-A9 exhibited a stronger antitumor effect than gemcitabine on two pancreatic cancer cell lines, including gemcitabine-resistant PANC-1 cells. OSU-A9 treatment induced apoptosis, the down-regulation of Akt phosphorylation, up-regulation of p38 phosphorylation and decreased phosphorylation of JAK and STAT3. Cell migration and invasiveness assays showed that OSU-A9 reduced cancer cell aggressiveness and inhibited BxPC-3 xenograft growth in nude mice. These results suggest that OSU-A9 modulates the p38-JAK-STAT3 signaling module, thereby inducing cytotoxicity in pancreatic cancer cells. Continued evaluation of OSU-A9 as a potential therapeutic agent for pancreatic cancer thus appears warrented.

Keywords: JAK; OSU-A9; STAT3; p38; pancreatic cancer.

Figure 1. Effects of OSU-A9 on cell viability of pancreatic cancer cell lines

MTT assay analyzing the effect of OSU-A9 and gemcitabine treatment on (A) BxPC-3 and (B) PANC-1 cell viability. (C) Trypan Blue exclusion assay to determine cell viability of BxPC-3 and PANC-1 cell lines treated with OSU-A9 for 24 h. Data are presented as mean ± S.D. from three individual experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 compared to the control group.

Figure 2. OSU-A9 induces apoptosis in BxPC-3 and PANC-1 cell lines

Histograms showing quantitation of cell apoptosis in (A, top left panel) BxPC-3 and (B, bottom left panel) PANC-1 cell lines when treated with different doses of OSU-A9. Cells were treated with OSU-A9 at the indicated concentrations for 24 h, followed by Annexin V/PI staining and FACS analysis. Fold increase in apoptosis due to increasing doses of OSU-A9 treatment of BxPC-3 (A, top right panel) and PANC-1 cell lines (B, bottom right panel) in comparison to control (control value = 1) are represented by the bar graph. Data represent mean ± SEM from six independent experiments. * P < 0.05, ** P < 0.01 compared to the control group.

Figure 3. Effects of OSU-A9 on Akt and MAPK signaling pathways in pancreatic cancer cell lines

Phosphorylation and expression status of Akt and MAPK signaling module (ERK, JNK, p38 and MAPKAPK2) were analyzed in BxPC-3 (left panel) and PANC-1 (right panel) cells that were treated with OSU-A9 (0-5 μM) in 5% FBS-supplemented RPMI1640 medium for 24 h. Cell lysates were immunoblotted as described in Material and Methods.

Figure 4. OSU-A9 down-regulates JAK-STAT3 signaling

(A) Dose-dependent effects of OSU-A9 on JAK and STAT3 phosphorylation and expression in BxPC-3 and PANC-1 cells. (B) p38 inhibitor SB203580 (SB) restores cell growth inhibited by OSU-A9 in BxPC-3 cells. Cells were treated with DMSO or OSU-A9 (5 μM) in the presence of 1, 5 or 10 μM SB for 24 h and cell viability was determined by MTT assays. Data are presented as mean ± S.D. from three individual experiments. ** P < 0.01. (C) Effects of SB203580 (SB) on the phosphorylation and expression of MAPKAPK-2, JAK and STAT3 in BxPC-3 cells. Cells were treated with DMSO or OSU-A9 (5 μM) in the presence of 1, 5 or 10 μM SB for 24 h. (D) Western blot analysis showing phosphorylation and expression of PARP, COX-2, survivin, and STAT3 in PANC-1 cells transfected with p-CMV-Flag or STAT3-CA-Flag and treated with 0-10 μM OSU-A9 for 24 h. (E) STAT3-CA partially protects against OSU-A9-mediated cytotoxicity in PANC-1 cells. Percent cell viability of PANC-1 cells expressing p-CMV-Flag or STAT3-CA-Flag treated with 0-10 μM OSU-A9 are plotted. Data are presented as mean ± S.D. from three individual experiments. ** P < 0.01, *** P < 0.001.

Figure 5. OSU-A9 mitigates aggressiveness of BxPC-3 and PANC-1 cells

(A) BxPC-3 and PANC-1 cell migration images following treatment with 0-3 μM OSU-A9 for 24 h. Plots showing percentages of migrating (B) and invasive (C) BxPC-3 and PANC-1 cells treated with 0-3 μM OSU-A9 for 24 h. Data are presented as mean ± S.E.M. from three individual experiments. *P < 0.05, *** P < 0.001 compared to the control group. (D) Gelatin zymography analysis showing secreted MMP-2 and MMP-9 activity for both BxPC-3 and PANC-1 cell lines treated with 0-5 μM OSU-A9 for 24 h in conditioned media. (+) and (−) are the positive and negative control for MMP-2 and MMP-9 activity, respectively. (E) Bar diagrams represent fold changes in MMP-2 and MMP-9 (control value = 1) in BxPC-3 and PANC-1cell lines treated with 0-5 μM OSU-A9 for 24 h in conditioned media. Data are presented as mean ± S.E.M. from three individual experiments. *P < 0.05, ** P < 0.01, *** P < 0.001 compared to the control group.

Figure 6. In vivo efficacy of OSU-A9 in BxPC-3 xenografted mice

(A) Tumor volume data plotted for mice with BxPC-3 xenografts treated with either DMSO or OSU-A9 (25 mg/kg) per day via oral gavage analyzed at different time points from 0-42 days. (B) Plot showing change in body weight of xenografted mice in the DMSO- and OSU-A9-treated groups. (C) Western blot showing phosphorylation status and expression of Akt, STAT3, p38, and MAPKAPK-2 proteins as analyzed from tumor protein extracts of 3 mice in each treatment group.