The established human PC cell lines, BxPc-3, MIA PaCa-2, PANC-1 and SW1990, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic (Life Technologies, Carlsbad, CA, USA). The cells were maintained at 37°C in a humidified 5% CO₂ atmosphere. Antibodies against SHH (ab53281), SMO (ab5694), PTCH (ab53715), Gli1 (ab49314), Gli2 (ab26056), GAPDH (ab8245) and HO-1 (ab13248) were purchased from Abcam (Cambridge, MA, USA). Recombinant SHH, and zinc proto-porphyrin IX (ZnPPIX) were obtained from R&D Systems (Minneapolis, MN, USA).

Total RNA was extracted from the cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesized using a Prime Script RT reagent kit (Takara, Dalian, China). The real-time PCR experiments were conducted on an iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad Laboratories Inc., Hercules, CA, USA) using SYBR-Green Real-time PCR Master Mix (Takara). Amplification was carried out as follows: denaturation at 94°C for 3 min, 35 cycles of 94°C for 30 sec, 58°C for 30 sec, and 72°C for 35 sec. The sequences of the primers used were as follows: HO-1 forward, 5′-CAG GCA GAG GGT GAT AGA AGA GG-3′ and reverse, 5′-CTG GGA GCG GGT GTT GAG TG-3′; and GAPDH forward, 5′-ACCACAGTCCATGCCATCAC-3′ and reverse, 5′-TCCACCACCCTGTTGCTGTA-3′. The expression of the target gene was calculated using the 2^−ΔΔCq method (17). All experiments were repeated independently 3 times.

Protein was extracted from the cells using lysis buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, and 0.1% SDS] containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA), and protein concentrations were measured by DC Protein Assay (Bio-Rad Laboratories Inc.). Following separation on 7.5% SDS-polyacrylamide gels, the proteins (20 μl) were transferred onto nitrocellulose membranes (Millipore, Billerica, MA, USA), which were then incubated with the primary antibodies (1:1,000) at 4°C overnight. After washing 3 times with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (rabbit, 1:1,000, ab191866; mouse, 1:1000, ab193651; Abcam) for 1 h. Immunoreactive bands were visualized using an enhanced chemiluminescence kit (Millipore). Quantitative analysis was performed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). The relative protein expression levels were normalized to GAPDH. All experiments were repeated independently 3 times.

Cell proliferation rates were measured by MTT assays. Briefly, the cells were seeded in 96-well plates at a density of 1×10⁴ cells per well and incubated overnight in medium containing 10% FBS. The DMSO concentration was adjusted to 0.4%. The cells incubated in serum-free medium were used as the control group. Following incubation for 24, 48 and 72 h at 37°C, 20 μl of MTT solution [5 mg/ml in phosphate-buffered saline (PBS)] were added to each well, and the cells were incubated for an additional 4 h at 37°C. Subsequently, 100 μl DMSO were added to each well at 37°C. The optical density (OD) value was determined using a spectrophotometer (Bio-Rad Laboratories Inc.) at 490 nm. The proliferation rate was defined as OD (cell plate)/OD (blank plate). All experiments were repeated independently 3 times.

HO-1 localization in PC cells was examined by immunofluorescence. The prepared cells were washed 3 times with PBS and then fixed with 100 ml 4% paraformaldehyde in PBS. The cells were permeabilized in blocking buffer (0.1% Triton X-100 or 0.1–0.5% saponin, 10% NGS, 100 mM PBS, pH 7.4) for 1 h at room temperature and then incubated with primary antibody to HO-1 overnight at 4°C. The following day, the cells were washed and incubated with FITC-conjugated goat anti-mouse IgG (green fluorescence; 1:500, anti-mouse, #115165003; Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; #0100-20, SouthernBiotech, Birmingham, AL, USA) for 10 min. After washing with PBS 3 times, cells were blocked for 5 min. As a negative control, the primary antibody was substituted with antibody diluent. All experiments were repeated independently 3 times.

HO-1 expression was specifically suppressed by the introduction of 21-nucleotide duplex siRNA. The sequences of the ribonucleotides used were as follows: 5′-rGACUGCGUUCCUGCUCAACdTdT-3′ and 5′-rGUUGA GCAGGAACGCAGUCdTdT-3′ (Ambion, Inc., Austin, TX, USA). The cells seeded into small dishes were transfected with 100 nM siRNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Negative control siRNA (Ambion Inc.) was used as a negative control. In addition, plasmids for the overexpression of HO-1 were constructed according to manufacturer’s instructions (Roche, Penzberg, Germany). The untransfected cells used in the experiment were cultured under normal conditions. All experiments were repeated independently 3 times.

Recombinant SHH (#CYT597; ProSpec-Tany TechnoGene Ltd., Ness-Ziona, Israel) was applied to the cultured PC cells at 5 mg/ml. In addition, cyclopamine (Cyc) (#4449518; ApexBio, Houston, TX, USA), a SHH pathway inhibitor, was diluted to 18 μg/ml in PC cell medium. The compound, ZnPPIX, an inhibitor of HO-1, was used at 50 μM (18). Gem was applied according to the PC cell line-specific EC50 (15 μg/ml for the MIA PaCa-2 cells and 100 μg/ml for the PANC-1 cells (9,19).

The analyses of the results was carried out using the SPSS statistical software package (version 13.0). The significance of the data was determined using a Student’s t-test or one-way analysis of variance (ANOVA), followed by the Bonferroni post hoc test, where applicable. A value of P<0.05 was considered to indicate a statistically significant difference. All results are expressed as the means ± SD. All the experiments were repeated 3 times.

To determine whether HO-1 plays an important role in the progression of PC, the mRNA expression of HO-1 was identified by real-time PCR in the BxPc-3, MIA PaCa-2, PANC-1 and SW1990 cells human PC cells (Fig. 1A). The results revealed that HO-1 mRNA expression differed significantly in the different PC cells. On average, a 3-fold upregulation of HO-1 mRNA expression was observed in the PANC-1 cells compared with the MIA PaCa-2 cells (P<0.05).

Thus, we selected the MIA PaCa-2 and PANC-1 cell lines for use in the following experiments due to the fact that they expressed low and high levels of HO-1, respectively. The protein expression of HO-1 was examined by the immunofluorescence staining of the MIA PaCa-2 and PANC-1 cell lines (Fig. 1B). As a negative control, the primary antibody was substituted with antibody diluent (data not shown). The expression of HO-1 in the PC cells provided the basis for our subsequent research.

To examine the effects of HO-1 on PC cell growth, the PANC-1 (high HO-1 expression) and MIA PaCa-2 cells (low HO-1 expression) were used for transfection. The PANC-1 cells were transfected with HO-1 siRNA, and the MIA PaCa-2cells were transfected with HO-1 expression plasmids. At 12 h after transfection, the transfection efficiencies were verified by real-time PCR (Fig. 2A and B) and western blot analysis (Fig. 2C). Cell proliferation in response to HO-1 stimulation was measured by MTT assays at 24, 48 and 72 h following transfection or treatment with ZnPPIX. Our results revealed that the PANC-1 cells transfected with siRNA (PANC-1-si-HO-1) exhibited a decreased rate of proliferation compared with the control group; ZnPPIX was used as a positive control and treatment with ZnPPIX also decreased cell proliferation (P<0.05) (Fig. 2D). On the contrary, the MIA PaCa-2 cells transfected with the HO-1 expression plasmid exhibited an increased proliferation rate (P<0.05) (Fig. 2E).

To examine the effects of HO-1 on the SHH signaling pathway in the PC cells, we examined the expression of molecules associated with the SHH signaling pathway in the PC cells under various treatment conditions by western blot analysis (Fig. 3A). Based on this analysis, the PANC-1 cells transfected with siRNA against HO-1 (PANC-1-si-HO-1) exhibited a decreased expression of SHH and Gli1 compared with the untransfected PANC-1 cells; similar results were obtained with the PANC-1 cells treated with the positive control, ZnPPIX (P<0.050 (Fig. 3B). We also found that the MIA PaCa-2 cells transfected with the HO-1 expression plasmid (MIA PaCa-2-HO-1) exhibited an increased expression of SHH and Gli1 (P<0.050 (Fig. 3B). However, no marked differences were observed in the expression of PTCH and SMO the cell groups (P>0.05). Thus, these results indicate that HO-1 activates the SHH signaling pathway in PC cells.

To determine whether the SHH pathway is involved in the high proliferation rate induced by HO-1, as shown in Fig. 4, the PANC-1 and MIA PaCa-2 cells were treated with Cyc, which is a SHH signaling pathway inhibitor. The results revealed the dose-dependent effects of Cyc on cell proliferation (data not shown). In addition, treatment of the PANC-1 cells with Cyc in combination with transfection with the HO-1 overexpression vector significantly decreased the PANC-1 cell proliferative ability in comparison with the controls or the other intervention groups (P<0.05) (Fig. 4A). Additionally, as regards the MIA PaCa-2 cells, the results revealed that treatment of these cells with Cyc inhibited the proliferation, compared with the control group and the MIA PaCa-2 cells transfected with the expression vector and not treated with Cyc (P<0.05) (Fig. 4B). Thus, HO-1 was found to activate the SHH signaling pathway in PC cells; thus, this suggests that the activation of the SHH signaling pathway is involved in HO-1-induced PC cell proliferation. However, the mechanisms through which the SHH signaling pathway is activated by HO-1 remain unknown. Whether the activation of the SHH signaling pathway occurs through an autocrine manner or through other mechanisms, is worthy of investigation. To clarify this, we used exogenous SHH to treat the PC cells. The results revealed that differences were only found between the cells not treated with SHH and those that were treated with SHH in the control group; no significant changes in the cell proliferative ability were observed in the other 3 intervention groups in both cell lines. (P>0.05) (Fig. 4C and D).

To determine whether the chemoresistance of PC cells is due to HO-1, we examined PC cell proliferation when the MIA PaCa-2 and PANC-1 cells were transfected with either HO-1 siRNA or the HO-1 expression plasmid and treated with Gem (EC50 dose). The results of the dose-dependent experiments of Gem treatment are shown in Fig. 5A and B). We found that the PANC-1 cells transfected with HO-1 siRNA (PANC-1-si-HO-1) and treated with Gem exhibited a decreased proliferative ability (P<0.05) (Fig. 5C). By contrast, MIA PaCa-2 cells transfected with the HO-1 expression vector and treated with Gem exhibited a greater proliferative ability than the MIA PaCa-2 cells treated with Gem alone (P<0.05) (Fig. 5D). Thus, a decreased proliferative ability was observed after HO-1 inhibition and treatment with Gem.

To determine whether HO-1 affects the chemosensitivity of PC cells, we treated the PC cells with an EC50 dose of Gem for 24 h. The quantification data from real-time PCR revealed that Gem increased the mRNA expression of HO-1 in the PANC-1 and MIA PaCa-2 cells compared with the untreated controls (P<0.05) (Fig. 6A). Furthermore, the cells treated with Gem exhibited an increased protein expression of HO-1 in both cell lines (P<0.05) (Fig. 6B). To determine whether Gem is responsible for the activation of the SHH pathway, we examined the expression of SHH and Gli1 in the PC cells treated with Gem. Treatment with Gem resulted in an increase in the expression of Gli1 in the PANC-1 and MIA PaCa-2 cells (P<0.05) (Fig. 6C). However, no signifi-cant induction of SHH expression observed in both cell lines (P>0.05).