The antibodies against BMP8B, Bcl-2, Bax, Bim and β-actin were purchased from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA. Kits of caspase-3 and -9 activities, and LDH assay kit were from Beyotime Institute of Biotechnology (Haimen, China). All other reagents were from common commercial sources.

BxPC-3 and PANC-1, two human pancreatic cancer cell lines, were obtained from the American Type Culture Collection (Rockville, MD, USA). Both cell lines were routinely cultured at 37°C with RPMI-1640 medium supplemented with 10% fetal calf serum, penicillin (100 U/ml) and streptomycin (100 μg/ml) in an incubator with 95% air and 5% CO₂.

One shRNA targeting human BMP8B and one scrambled shRNA (used as a negative control) were designed and synthesized by Shanghai GenePharma Co., Ltd., Shanghai, China. The resultant lentivirus containing BMP8B shRNA sequence and negative control sequence were termed BMP8B shRNA and NC, respectively. The lentivirus of BMP8B overexpression was also purchased from Shanghai GenePharma Co., Ltd. We termed the BMP8B overexpression lentivirus and blank vector BMP8B and vector, respectively.

The assessment of cell viability was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. BxPC-3 and PANC-1 cells were split into a 96-well plate (~5×10³/well), and then subjected to growth arrest for 24 h before different treatments as the indicated groups. The cells were divided into negative control and BMP8B shRNA, as well as vector and BMP8B overexpression for 72 h. After 96 h of incubation at 37°C, the cells were incubated in a medium containing 0.5% MTT at 5 g/l and 20 μl/well for 4 h, and MTT was a yellow mitochondrial dye and prepared in phosphate-buffered saline (PBS). After adding DMSO to the medium followed by incubation for 10 min at 37°C, the MTT reaction was terminated, and then the absorbance was read by a spectrophotometer at 540 nm. All experiments were carried out in triplicate and repeated thrice.

Cells in different groups (~5×10³/well) were seeded into 6-well plates and cultured for 7 days and the medium was switched every 2–3 days. Subsequently, the cells were washed twice with pre-warmed PBS, and then cells remaining were stained for 2 h with a crystal violet solution (0.5% crystal violet, 20% methanol). After removal of the crystal violet solution, the plates were washed three times by immersion in a beaker filled with tap water. Plates were left to dry at 37°C, and then the images were photographed by camera.

Caspase-3 activity was measured by its chromogenic caspase substrate cleavage. The protein samples were prepared as the indicated groups. Then, ~50 μg total proteins of every indicated group were added to the reaction buffer containing Ac-DEVD-pNA, incubated for 4 h at 37°C, and the absorbance of yellow pNA cleavage from its corresponding precursors was measured by using a spectrometer at 405 nm. The specific caspase-3 activity was normalized to total proteins of cell lysates, and expressed as fold of the baseline caspase activities of control cells.

Caspase-9 activity was measured as per the manufacturer’s instructions. We measured the cleavage of Ac-LEHD-pNA, the chromogenic caspase substrates of caspase-9, and normalized the specific caspase-9 activity to total proteins of cell lysates. Then, the specific caspase-9 activity was expressed as fold of the baseline caspase activities of control cells.

The activity of lactate dehydrogenase (LDH) release into the culture media was measured using a cytotoxicity detection kit. Percentage of injured cells in cultures is represented by the LDH activities of medium relative to total LDH activity after complete cell lysis. Briefly, a portion of culture medium was reacted with an equal volume of LDH substrate solution for 30 min. The reaction was stopped by adding 5 volume of 0.1 M NaOH, and absorbance was then measured at 440 nm in a spectrophotometer. Sister cultures were treated with 1/100 volume of 10% Triton X-100 and incubated at 37°C for 30 min. Total LDH activities were determined using medium containing Triton-lysed cellular supernatant.

The methodology was described in previous reports (15). Briefly, 5×10⁵ cells were sonicated in RIPA buffer and homogenized. Debris was removed by centrifugation at 12,000 × g for 10 min at 4°C. The samples containing 50 μg protein were electrophoresed on polyacrylamide SDS gels, and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 3% BSA, incubated with primary antibodies, and subsequently with alkaline phosphatase-conjugated secondary antibody. They were developed with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Tiangen Biotech Co. Ltd., Beijing, China). Blots were also stained with anti-β-actin antibody as an internal control for the amounts of target proteins.

Real-time PCR (qPCR) was performed with an Applied Biosystems 7300 Fast Real-Time PCR System. Primers were specifically designed using Applied Biosystems Primer Express 3.0. The primer used for BMP8B was: forward, 5′-AGGTGGCTTCCTTATCTGCG-3′ and reverse, 5′-ATGTGCCAACTCTGCTTCGT-3′, with a product length of 165 bp The specificity of the primers was confirmed with a BLAST program. Each 20 μl reaction contained 1X SYBR^® Premix Ex Taq™ II, 10 μM forward and reverse primers, 0.4 μl ROX reference dye and 2 μl of cDNA. ABI 7300 Sequence Detector (Perkin-Elmer Applied Biosystems) was programmed for the PCR conditions: 95°C for 30 sec, 40 cycles of 95°C for 5 sec and 60°C for 31 sec, followed by routine melting curve analysis. Relative quantitation (RQ) of target gene expression was calculated by the 2^−ΔΔCT method (16). The first step in the RQ analysis is to normalize target gene expression level to β-actin (ΔCt). The second step is to compare the difference between normalized target gene expression between different samples (ΔΔCt). Each experiment was repeated 2–3 times in 3–4 samples.

The results are expressed as the mean values ± standard deviation, and a Student’s t-test was used to evaluate statistical significance. A value of <0.05 (p<0.05) was considered to indicate statistically significant differences.

To determine whether BMP8B is involved in the progression of pancreatic cancer, we first examined the expression of BMP8B in the patients of pancreatic cancer with western blotting and qPCR. We found that the protein and mRNA expression of BMP8B was significantly lower in pancreatic cancer tissue compared with the normal tissue adjacent to the tumors (Fig. 1A and B, P<0.05). The results showed that BMP8B may play important roles in regulating the development of pancreatic cancer.

We then constructed the stably transfected cell lines of BMP8B overexpression in PANC-1 in order to further demonstrate that BMP-8 is associated with pancreatic cancer cell growth and survival. To confirm the transfection efficiency and to achieve the overexpressed cells of BMP8B, we examined the expression of BMP8B. As shown in Fig. 2A and B, the protein and mRNA expression of BMP8B were considerably increased in PANC-1.

We found the protein and mRNA expression of BMP8B was significantly decreased after applying BMP8B-shRNA in BxPC-3 compared to the negative control (NC) group (Fig. 2C and D, n=3, p<0.05), indicating the adequate knocking down of the BMP-8 gene by BMP8B-shRNA.

MTT was applied to determine the effects of BMP8B on cell growth. The results showed that the cell viability of PANC-1 was inhibited after overexpression of BMP8B (Fig. 3A, n=3, p<0.05). Moreover, the release of LDH, which occurs with cell death, was increased in BMP8B-overexpressed cells (Fig. 3B, n=3, p<0.05). The colony formation of tumor cells reflects the tumorigenicity of tumor cells. We found that overexpression of BMP8B resulted in the decreased colony formation activities in PANC-1 (Fig. 3C, n=3, p<0.05). These results demonstrated that overexpression of BMP8B blocked the growth of pancreatic cancer cells.

We examined the cell growth and colony formation in BxPC-3 after silencing the BMP8B gene expression with BMP8B shRNA. As shown in Fig. 4, knocking down BMP8B expression increased cell growth and promoted colony formation, and inhibited the release of LDH in BxPC-3. The results were in accordance with the findings of Fig. 3.

Bim, Bcl-2 and Bax are important molecules localized on mitochondrial outer membrane and control the stability of mitochondria. Bim and Bax are pro-apoptotic proteins and Bcl-2 is an anti-apoptotic protein. In our experiments, we found the expression of Bim and Bax was upregulated by BMP8B overexpression, and the overexpression of BMP8B blocked the expression of Bcl-2 in PANC-1 (Fig. 5A and B, n=3, p<0.05). Furthermore, caspase-3 and -9 are the key performers during the mitochondrial pathway apoptosis. We then examined whether BMP8B participated in regulating the activation of caspase-3 and -9. The results showed that the activation of caspase-3 and -9 was increased by BMP8B overexpression in PANC-1 (Fig. 5C, n=3, p<0.05). These results demonstrate that the overexpression of BMP8B triggers the mitochondrial-dependent apoptosis through regulating Bim/Bcl-2/Bax expression.

We also examined the activation of caspase-3 and -9 and the expression of mitochondrial membrane proteins after knocking down BMP8B gene expression in BxPC-3 to further support our conclusion. Our results showed that silencing BMP8B gene induced Bcl-2 expression and decreased the expression of Bim and Bax (Fig. 6A and B, n=3, p<0.05). Moreover, the activities of caspase-3 and -9 were suppressed by BMP8B shRNA in BxPC-3 (Fig. 6C, n=3, p<0.05), indicating that the apoptosis is inhibited by knocking down BMP8B expression.