Simultaneous gene silencing of Bcl-2, XIAP and Survivin re-sensitizes pancreatic cancer cells towards apoptosis

Keywords for the search were "apoptosis", "cell death", "cell death pathway", "cell death receptor" together with the term "pancreatic carcinoma" or "pancreatic cancer". Genes were considered if dysregulation was demonstrated on the level of the transcriptome and the proteome. The literature search comprised publications until October 2008.

The MiaPaCa-2 cell line (ATCC: CRL-1420), derived from a primary pancreatic carcinoma, was used for this study. Cells were grown in DMEM with high glucose, 1.5 g/l sodium bicarbonate and 4 mM GlutaMAX (Invitrogen, Karlsruhe, Germany) with 10% FCS and 2.5% horse serum. We also used the AsPC-1 cell line (ATCC: CRL-1682), derived from malignant ascites. Cells were grown in RPMI-1640 medium with 2 mM GlutaMAX, 1 mM sodium pyruvate, 4.5 g/l glucose (Invitrogen, Karlsruhe, Germany) and 10% FCS. Both cell lines were cultured in a humidified atmosphere containing 5% CO₂ at 37°C. Next, 5 × 10⁴ cells were transfected using Oligofectamine (Invitrogen GmbH, Karlsruhe, Germany). Target sense sequences that effectively mediated silencing were as followed: Bcl-2 [14]: GUACAUCCAUUAUAAGCUG (NCBI probe ID: Pr196034.1), XIAP [15]: GUGGUAGUCCUGUUUCAGC, Survivin: GAAUUUGAGGAAACUGCGA [16] (all from MWG Biotech, Ebersberg, Germany). As negative control, we used siRNA against Firefly Luciferase (CGUACGCGGAAUACUUCGA), the Allstars siRNA (Qiagen) and carrier solution. A total of 50,000 cells/well were single-transfected with 0.046 nM of two negative controls (nonsense siRNA); one group was treated with vehicle only. For the SGS, cells were co-transfected with 0.015 nM of each of the three target genes. Additional controls were performed using single transfection of the different target genes, either with low-dose siRNA (0.015 nM) or standard-dose siRNA (0.046 nM). The low-dose single transfection (lowST) was undertaken with 0.015 nM Bcl-2, Survivin or XIAP siRNA, each with 0.03 nM of nonsense siRNA to exclude bias caused by the absolute amount of siRNA. Standard-dose single transfection (ST) was undertaken with 0.046 nM of the correspondent target gene. Cells were cultured for 72 h. At the end of incubation, all cells were harvested. After treatment of the cells, flow cytometry and a caspase activity assay were performed to determine the apoptosis rate. Silencing was confirmed by western blot.

For quantitative RT-PCR, total RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany), cDNA was synthesized using random primer and SuperScript II (Invitrogen GmbH, Karlsruhe, Germany). The genes were amplified with the Power SybrGreen PCR Master Mix according to the manufacturer's instructions. Gene expression was quantified by the comparative cT-Method, normalizing cT-values to a housekeeping gene (β-Actin) and calculating the relative expression values. Primers used in this study are shown in table 1.

Cells were lysed with LDS sample buffer (Invitrogen, Karlsruhe, Germany). Proteins were electrophoresed under reducing conditions on 4-12% acrylamide gels (Invitrogen, Karlsruhe, Germany) and then transferred to a nitrocellulose membrane (Hybond ECL, GE Healthcare, Munich, Germany). To block nonspecific binding, the membrane was incubated overnight in TBS-T 0.1% containing 5% BSA for Bcl-2 or 5% milk for Survivin and XIAP at 4°C. Subsequently, the membrane was either incubated with an antibody to Survivin (1:1000, NB 500-201, Novus Biologicals, Acris Antibodies, Hiddenhausen, Germany), Bcl-2 (1:200, sc-509, Santa Cruz, Heidelberg, Germany), XIAP (1:500, 610716, Becton Dickinson, Heidelberg, Germany) or GAPDH (1:5000, ab 8245-100, abcam, Cambridge, UK) in TBS-T 0.1% with blocking as mentioned above for 1 h. After washing in TBS, the protein was visualized using the ECL detection kit (GE Healthcare, Munich, Germany) with a peroxidase-labeled anti-mouse antibody (1:25,000, NIF825, GE Healthcare, Munich, Germany) or an anti-rabbit antibody (1:5,000, NIF824, GE Healthcare, Munich, Germany).

For a loading control, the membranes were blocked again and probed with monoclonal antibodies for GAPDH according to standard protocols. Protein expression was determined as the ratio of target gene staining intensity to GAPDH staining intensity using AIDA evaluation software (Raytest, Straubenhardt, Germany). To compare blots of the different transfections, staining intensities of the different transfections were normalized to the SGS treated group. All blots were done in triplicate.

Cells were harvested 72 h after transfection. Cells were trypsinized and treated with 50 μg/mL propidium iodide and Annexin V-FITC (Becton Dickinson, Heidelberg, Germany). The cells were analyzed and quantified with flow cytometry (10,000 cells analyzed) (FACS Calibur; Becton Dickinson, Heidelberg, Germany). For analysis of Apoptosis we counted Annexin-positive cells of the upper and lower right quadrants. For further statistical analysis, we formed a quotient with untreated cells for every group. Caspase 3 and 7 activity was measured on deep frozen cell pellets containing 20,000 cells harvested 72 h after transfection using the Caspase Glo-Assay (Promega, Mannheim, Germany) according to the manufacturer's instructions. Results of the caspase assay were also normalized to untreated cells.

For statistical analysis, the t test and the Mann-Whitney U test of "SPSS 13.0" for Windows were used.

A comprehensive literature search identified several dysregulated apoptosis-associated genes in pancreatic cancer. The ideal characteristics of our candidate genes for SGS were upregulation at the RNA and protein levels and synergistic anti-apoptotic action in the course of the cell death pathway. Because the intrinsic pathway has a central role in pancreatic cancer cells we only considered members of this pathway for our study. XIAP [13, 17, 18], Survivin [11, 13, 19] and Bcl-2 [20‐22] met all these criteria and are characterized as upregulated in PDAC cell lines as well as native tumor tissue.

To elucidate the effect of simultaneous gene-silencing in pancreatic cancer, we established a protocol for simultaneous post transcriptional gene silencing using siRNA. For SGS we chose the human pancreatic cancer cell line MiaPaCa-2, which expresses XIAP, Bcl-2 and Survivin and AsPC-1 cells, which express XIAP and Survivin (Figure 1; Additional file 1). We determined the maximal dose without toxic effects to be 0.046 nM siRNA per 50,000 cells (data not shown). Using qRT-PCR and western blot we verified the target genes to be silenced at the mRNA and protein levels in transfected cells. QRT-PCR showed the expression of all three target genes in SGS, lowST and ST to be strongly reduced compared with controls. We validated knock-down of the target genes by SGS in western blot.

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The reduction of Bcl-2, XIAP and Survivin protein expression by SGS was statistically significant as compared to controls in MiaPaCa-2 cells (p < 0.05). We also found a statistically significant reduction of the expression of XIAP and Survivin by SGS in AsPC-1 cells in western blot (p < 0.05) (Figure 1).

LowST and ST transfection also resulted in a significant reduction of protein expression of the target genes in both cell lines in western blot.

To examine off-target effects of gene-silencing in our cells, we performed a qRT-PCR of Interferon beta and STAT1. The genes showed no differential expression in any of our transfections (Additional file 2).

To determine the optimal time point for harvesting and to show that all groups displayed a similar kinetic response to transfection, a dynamic measurement of caspases 3 and 7 activity was conducted every 12 hours after transfection; activation was maximal after 72 h (Additional file 1).

Simultaneous silenced MiaPaCa-2 cells showed a 3.43-fold increase in the rate of apoptotic cells in flow cytometry compared to controls (12.81 ± 2.09 vs. 3.73 ± 0.55; p < 0.001). Standard single transfected cells (ST) of the Survivin group also resulted in a significantly elevated apoptosis rate compared to the controls (7.77 ± 1.82 vs. 3.73 ± 0.55; p = 0.043) (Figure 2A, E).

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Moreover, transfection of XIAP led to significant caspase activation in the ST group (7.0 ± 0.35; p = 0.011) (Figure 2C) (Table 2).

In AsPC-1 cells, SGS resulted in a 2.01-fold increase in the rate of apoptotic cells compared to controls as measured by flow cytometry (4.49 ± 0.30 vs. 2.23 ± 0.22; p = 0.009). Survivin showed increased numbers of apoptotic cells in flow cytometry in the lowST (3.17 ± 0.20; p = 0.0028) as well as in the ST group (3.94 ± 0.27; p = 0.009). ST of XIAP led to a significantly elevated apoptosis rate compared to controls (3.19 ± 0.11; p = 0.009) (Figure 2B, E).

Analogously, activation of caspases 3 and 7 in AsPC-1 cells was significantly elevated in the SGS group (9.90 ±1.92 vs. 1.17 ± 0.31; p = 0.021). However, none of the control groups showed statistically significant activation, although there was a tendency in the XIAP (2.55 ± 0.41) and Survivin ST groups (3.27 ± 0.96) (Figure 2D) (Table 2).

Corresponding to the higher apoptosis rate we saw a reduced number of viable cells in the SGS group in both cell lines, with a 6.7-fold reduction of vital MiaPaCa-2 cells (p = 0.021) and a 4.75-fold reduction in AsPC-1 cells (p = 0.05) as compared to controls (Table 2).