Tanshinone IIA Could Inhibit Pancreatic Cancer BxPC-3 Cells through Increasing PERK , ATF 6 , Caspase-12 and CHOP Expression to Induce Apoptosis

Tanshinone IIA (Tan-IIA) is extracted from Dan-Shen. Tan-IIA could inhibit human pancreatic cancer BxPC-3 cells through decreasing TCTP, Mcl-1 and Bcl-xl expression in vitro. Our previous study showed that Tan-IIA can inhibit hepatocellular carcinoma hep-J5 cells and human breast cancer BT-20 cells through inducing endoplasmic reticulum (ER) stress. In the present study, we investigated the ER stress related protein expressions in human pancreatic cancer BxPC3 cells were treated with Tan-IIA. The ER stress related protein expressions in human pancreatic cancer BxPC-3 cells were evaluated by western blotting. The results showed that Tan-IIA can increase the protein expressions of PERK, ATF6, Caspase-12 and CHOP, but decrease Bip, PDI, Calnexin, Calreticulin and Bcl-2 expression. These findings indicated that Tan-IIA can inhibit human pancreatic cancer BxPC-3 cells by inducing ER stress to induce apoptosis.


Introduction
In 2012, pancreatic cancer is the 4th leading cause of cancer death in the US [1].Gemcitabine is the standard treatment for pancreatic cancer patients, but the survival rate for one year was only 18% [2].Although many ef-forts have been made to improve the clinical efficacy, but current chemotherapeutic medicines for pancreatic cancer are unsatisfactory [3]- [5] and need to identify new treatments.Tanshinone IIA (C 19 H 18 O 3 ) is one of the active components in Radix Salviae miltiorrhizae [6] [7].It was well documented that Tanshinone IIA (Tan-IIA) has anti-inflammatory activities [8] [9] and antioxidant properties [10] [11].It was well documented that Tan-IIA can inhibit many human cancer cell lines through different molecular mechanisms, such as Tan-IIA downregulates ErbB-2 and up-regulates TNF-alpha expression to inhibit colon cancer colo205 cells [12], Tanshinone IIA inhibits human breast cancer MDA-MB-231 cells through increasing Bax to Bcl-xL ratios [13], Tanshinone IIA inhibits human non-small cell lung cancer A549 cells through the induction of reactive oxygen species and decreasing the mitochondrial membrane potential [14], Tan-IIA could inhibit small cell lung cancer H146 cells by up-regulating the Bax/Bcl-2 ratio and decreasing mitochondrial membrane potential [15], Tan-IIA possesses cytotoxic effects in human pancreatic (MIAPaCa-2) tumor cell lines was documented, but the mechanism has not been established [16].Because our previous studies showed Tan-IIA could inhibit hepatocellular carcinoma Hep-J5 cells [17] and breast cancer BT-20 cells [18] through inducing ER stress.Our previous study also showed Tan-IIA can inhibit human pancreatic cancer BxPC-3 cells through decreasing TCTP, Mcl-1 and Bcl-xl expression in vitro [19].In the present study, we investigated the ER stress related protein expressions in human pancreatic cancer BxPC3 cells were treated with Tan-IIA.
Cell culture: The BxPC-3 cells are established from a 61-year-old female pancreatic epithelial cell adenocarcinoma was obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan), were maintained in RPMI-1640 medium containing 10% FBS, 1% penicillin/streptomycin (10,000 U/ml penicillin, 10 mg/ml streptomycin) at 37˚C in a humidified atmosphere containing 5% CO 2 .

Cytotoxicity Assay
The cytotoxicity of Tan-IIA for BxPC-3 cells was evaluated by MTT assay in triplicate as document described [20].Briefly, the BxPC-3 cells were plated in 96-well plates at a density of 1 × 10 4 cells/well and treated with various concentrations of Tan-IIA for different durations (24, 48 and 72 hours).Subsequently, the cells were incubated with 100 μl MTT (1 mg/ml) in fresh complete RPMI medium for 2 hrs.The surviving cells converted MTT to formazan by forming a blue-purple color when dissolved in dimethyl sulfoxide.Absorbance was measured using an ELISA microplate reader at 590 nm.The relative percentage of cell viability was calculated by dividing the absorbance of treated cells by that of the control in each experiment, using the following formula: Proliferation rate (%) = (ODtest − ODblank) × 100, where ODtest and ODblank are the optical density of the test substances and the blank control, respectively.
BxPC-3 cells were treated with Tan-IIA (8.5 μM) for different durations (0, 24, 48 and 72 h) and then the proteins expression levels of Bip, PDI, Calnexin, Calreticulin, IRE1α, PERK, elF2α, ATF6, ATF4, Caspase-12, Caspase-9, CHOP and Bcl-2 were evaluated by western blotting.BxPC-3 cells were treated with Tan-IIA (8.5 μM) for different durations (0, 2, 4, 6, 8, 12, 24 and 48 h) and then the proteins expression levels of Bip, PDI, Calnexin, Calreticulin were evaluated by western blotting.The western blot procedures as document described [21] [22].Briefly, BxPC-3 cells were treated with various concentrations of Tan-IIA for different durations; the cells were lysed in the ice-cold whole cell extract buffer containing the protease inhibitors.The lysate was vibrated for 30 min at 4˚C and centrifuged at 10,000 rpm for 10 minutes.Protein concentration was measured by BCA protein assay kit (Pierce, Rockford, IL).Equal amounts of proteins (40 μg) were subjected to electrophoresis using 10% -15% sodium dodecyl sulfate-polyacrylamide gels.To verify equal protein loading and transfer, proteins were then transferred to polyvinylidene difluoride membranes and then the membranes were blocked overnight at 4˚C using blocking buffer (5% non-fat dried milk in solution containing 50 mM Tris/HCl (pH 8.0), 2 mM CaCl 2 , 80 mM sodium chloride, 0.05% Tween 20 and 0.02% sodium azide).The membranes were then incubated with specific primary antibody.After incubated with primary Abs for 2 hours at 25˚C, the membranes were washed thrice with TBST buffer and followed by anti-rabbit or anti-mouse immunoglobulin G-horseradish peroxidase conjugated secondary antibodies.The membranes were washed three times for 10 min with washing solution.Finally, the immunoreactive protein bands were visualized on the X-ray film and analyzed using the enhanced chemiluminescence detection system (Perkin Elmer Life and Analytical Sciences, Boston, MA).The detection of β-actin was used as an internal control in all of the data for Western blotting.

Immunocytochemistry Staining
The cells were cultured in 6-well plate dish at a density of 5 × 10 5 per well for 16 -20 hours.After Tan-IIA treatment for 24 or 48 hours, the cells were washed with PBS.Then fixation with 50% acetone and 50% methanol solution overnight at 4˚C, the cells were washed three times with PBS, and non-specific binding sites were blocked in PBS containing 0.1% BSA for 1 h at room temperature.Thereafter, the cells were separately incubated with rabbit anti-caspase 3 (1:20) antibody in PBS containing 0.1% BSA overnight at 4˚C, and washed three times with PBS.Then the cells were incubated with anti-rabbit FITC (1:200) in PBS containing 0.1% BSA for 1 h at room temperature, and washed three times with PBS.The nuclei were stained with the 5 μg/ml PI, respectively.After staining, the samples were immediately examined under Olympus IX81 microscope (Olympus, Tokyo, Japan).

Statistical Analysis
Values are presented as mean ± SD.The student's t-test was used to analyze the statistical significance.* p value < 0.05 was considered significant for all tests.

The Effect of Tan-IIA on the Protein Expressions of Bip, PDI, Calnexin, and Calreticulin in BxPC-3 Cells
BxPC-3 cells were treated with various concentrations (0, 4.2 and 8.5 μM) of Tan-IIA for 24 hours and the proteins expression levels were evaluated by western blotting.The results showed that Tan-IIA decreased the protein expression levels of Bip, PDI, Calnexin and Calreticulin (Figure 2(a)).BxPC-3 cells were treated with various concentrations (0, 2.0 and 4.0 μM) of Tan-IIA for 48 hours and the proteins expression levels were evaluated by western blotting.The results showed that Tan-IIA decreased the protein expression levels of Bip, PDI, Calnexin and Calreticulin (Figure 2(b)).BxPC-3 cells were treated with Tan-IIA (8.5 μM) for different durations (0, 24, 48 and 72 h) and then the proteins expression levels were evaluated by western blotting.The results showed that Tan-IIA decreased the protein expression levels of Bip, PDI and Calnexin but mild increased Calreticulin expression (Figure 3).BxPC-3 cells were treated with Tan-IIA (8.5 μM) for different durations (0, 2, 4, 6, 8, 12, 24 and 48 h) and then the proteins expression levels of Bip, PDI, Calnexin, Calreticulin were evaluated by western blotting.The results showed that the protein expression levels of Bip (Figure 4

The Protein Expression of Caspase-3 in BxPC-3 Cells
BxPC-3 cells were treated with various concentrations (0, 4.2 and 8.5 μM) of Tan-IIA for 24 hours.The protein expression of Caspase-3 in BxPC-3 cells was observed by immunocytochemistry as described in materials and methods.The results showed that Tan-IIA could induce high level of Caspase-3 (Figure 7).

Discussion
Our results showed that Tan-IIA can inhibit the proliferation of human pancreatic cancer BxPC-3 cells with time-and dose-dependent in vitro (Figure 1).This is agreement with other documents [16] [19].It is well documented that decrease the protein expressions related to unfolding protein response (UPR) will inducing ER stress [23] [24].Our results showed that the protein expression levels of Bip (Figure 4   Caspase-3 (Figure 7).These findings indicate that Tan-IIA could induce apoptosis through inducing ER stress in BxPC-3 cells in vitro.This is the first report for Tan-IIA could inhibit the proliferations of human pancreatic cancer BxPC-3 cells through ER stress pathway.Tan-IIA with chemotherapeutic potential for human pancreatic cancer BxPC-3 cells in vitro.It is warrants further study in the future.The proposed molecular mechanisms for Tan-IIA to inhibited BxPC-3 cells as follow (Figure 8): Tan-IIA induce UPR through decreasing the protein expression of Bip, PDI, Calnexin and Calreticulin, then increase PERK, elF2α, ATF4, IRE1α, Caspase-12 and ATF6 expression, and then stimuli ER stress downstream CHOP over expression.In addition, CHOP decreased Bcl-2 protein expression and induced mitochondria dysfunction to induce apoptosis.

Figure 7 .
Figure7.The protein expression of Caspase-3 in BxPC-3 cells BxPC-3 cells were treated with Tan-IIA (0, 4.2 and 8.5 μM) for 24 hours.The protein expression of Caspase-3 in BxPC-3 cells was observed by immunocytochemistry as described in materials and methods.The results showed that Tan-IIA could induce high level of Caspase-3 (Figure7).

Figure 8 .
Figure 8.The proposed molecular mechanisms for Tan-IIA to inhibited BxPC-3 cells Tan-IIA induce UPR through decreasing the protein expression of Bip, PDI, Calnexin and Calreticulin, then increase PERK, elF2α, ATF4, IRE1α, Caspase-12 and ATF6 expression and then stimuli ER stress downstream CHOP over expression.In addition, CHOP decreased Bcl-2 protein expression and induced mitochondria dysfunction to induce apoptosis (Figure 8).