Interferon gamma inhibits growth of human pancreatic carcinoma cells via caspase-1 dependent induction of apoptosis

Abstract

Background and aims: The poor prognosis of pancreatic cancer is partly due to resistance to a broad spectrum of apoptotic stimuli. To identify intact proapoptotic pathways of potential clinical relevance, we characterised the effects of interferon gamma (IFN-gamma) on growth and survival in human pancreatic cancer cells.

Methods: IFN-gamma receptor expression and signal transduction were examined by reverse transcriptase-polymerase chain reaction (RT-PCR), immunoprecipitation, western blot analysis, and transactivation assays. Effects on cell growth and survival were evaluated in terms of cell numbers, colony formation, cell cycle analysis, DNA fragmentation, and poly(ADP ribose) polymerase (PARP) cleavage.

Results: All four pancreatic cancer cell lines examined expressed functional IFN-gamma receptors and downstream effectors, including the putative tumour suppressor interferon regulatory factor 1 (IRF-1). IFN-gamma treatment profoundly inhibited anchorage dependent and independent growth of pancreatic cancer cells. Cell cycle analyses revealed subdiploid cells suggesting apoptosis, which was confirmed by demonstration of DNA fragmentation and PARP cleavage. Time and dose dependency of apoptosis induction and growth inhibition correlated closely, identifying apoptosis as the main, if not exclusive, mechanism responsible for growth inhibition. Apoptosis was preceded by upregulation of procaspase-1 and accompanied by proteolytic activation. Furthermore, the caspase inhibitor z-vad-fmk completely prevented IFN-gamma mediated apoptosis.

Conclusions: These results identify an intact proapoptotic pathway in pancreatic cancer cells and suggest that IRF-1 and/or procaspase-1 may represent potential therapeutic targets to be further explored.

Figure 1

Pancreatic cancer cells express interferon γ receptor (IFN-γ-R) mRNA transcripts. Total mRNA was extracted, reverse transcribed into cDNA, and amplified using polymerase chain reaction with specific primers directed against the IFN-γ-R α chain. Alternating lanes represent reaction with (+) or without (−) addition of reverse transcriptase (RT). The expected size of the amplification product was 559 bp and a 100 bp DNA standard was included for size determination (lane 1).

Figure 2

Interferon γ (IFN-γ) activates the Jak-Stat signal transduction pathway in human pancreatic cancer cells. Cells were stimulated with 500 IU/ml IFN-γ for the indicated time periods and Jak-1 (A), Jak-2 (B), and Stat-1 (C) were immunoprecipitated (IP) from cell lysates. Immunoblots were performed to determine IFN-γ induced changes in the phosphotyrosine content of Jak and Stat complexes (top panels). To ensure that equal amounts of protein had been examined, the western blots (WB) were subsequently stripped and reprobed with Jak-1, Jak-2, or Stat-1 antibody, respectively (bottom panels). Molecular masses were deduced from a molecular size marker electrophoresed in parallel.

Figure 3

Interferon γ (IFN-γ) stimulation results in transactivation of a GAS driven reporter construct. AsPc-1 cells were transiently transfected with pGL2-GAS and relative luciferase activity was measured after a 24 hour period of IFN-γ stimulation with the indicated doses. Data represent mean (SEM) values from at least three separate experiments conducted in triplicate.

Figure 4

Interferon γ (IFN-γ) inhibits anchorage dependent and independent growth of human pancreatic cancer cells. Subconfluent cells were treated with 500 IU/ml IFN-γ or vehicle for the indicated time periods (A), or with the indicated final concentrations of IFN-γ for 96 hours (B) and cell numbers were determined. (C) A single cell agar suspension containing 1×10³ cells was incubated with the indicated doses of IFN-γ for 10 days and colony formation was evaluated. Data represent mean (SEM) values from at least three separate experiments, each conducted in triplicate (*p<0.01).

Figure 5

Interferon γ (IFN-γ) treated pancreatic tumour cells contain a subdiploid DNA complement. (A) Time course of representative FACS analyses illustrating the cell cycle distribution of pancreatic cancer cells under control conditions (top panel) or in the presence of 500 IU/ml IFN-γ (bottom panel). (B) Summary of IFN-γ induced cell cycle redistribution. Mean (SEM) percentage of cells with subdiploid DNA content were determined in three independent experiments. *p<0.01 versus time matched untreated controls. (C) Alignment of growth inhibition and apoptosis.

Figure 6

Interferon γ (IFN-γ) induced DNA fragmentation and poly(ADP ribose) polymerase (PARP) cleavage in human pancreatic cancer cell lines. (A) Cells were incubated for four days with 500 IU/ml IFN-γ (lanes 3, 5, 7, and 9) or left untreated (lanes 2, 4, 6, and 8) and genomic DNA was subsequently analysed for oligonucleosomal fragmentation. For size determination a 100 bp DNA standard was used (lane 1). (B) Cells were treated with vehicle or IFN-γ (500 IU/ml) for the indicated time periods and examined using a specific histone DNA ELISA to quantitate DNA fragmentation. Mean (SEM) absorbance at 405 nm relative to untreated controls was determined in three independent experiments, each performed in triplicate. (C) Immunoblot demonstrating expression of PARP (p 116) and its apoptosis related cleavage product p 85 in untreated controls and cells that had been treated with 500 IU/ml IFN-γ for the time periods indicated. Alternating lanes represent IFN-γ treated (+) and untreated (−) cells. Whole cell lysates were separated by 7.5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

Figure 7

Interferon γ (IFN-γ) treatment regulates retinoblastoma protein (pRb) phosphorylation and abundance in human pancreatic cancer cells. Immunoblot analysis demonstrating the time course of changes in pRb expression and phosphorylation status in control and IFN-γ stimulated human pancreatic cancer cells. In each lane, 20 µg of whole cell lysates were separated by 7.5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Shown is a representative of two experiments yielding similar results. pRb, ppRb, hypo- and hyperphosphorylated forms of Retinoblastoma protein, respectively.

Figure 8

Interferon γ (IFN-γ) mediated apoptosis is prevented by the caspase inhibitor z-vad-fmk. Cells were stimulated for three days with either IFN-γ (500 IU/ml), a combination of IFN-γ and z-vad-fmk at various doses, or were left untreated. Subsequently, cells were analysed by flow cytometry and the fraction of cells with subdiploid DNA content was quantitated. Mean (SEM) percentage of cells with subdiploid DNA content was determined in three independent experiments.

Figure 9

Interferon γ (IFN-γ) causes upregulation of procaspase-1 and interferon regulatory factor 1 (IRF-1) in pancreatic cancer cell lines. (A) Cells were incubated with vehicle or IFN-γ (500 IU/ml) for the indicated time periods. Aliquots of 20 µg of whole cell lysate were separated by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS PAGE) and subsequently incubated with an anticaspase antibody that recognises a double band of the procaspase precursor form at approximately 45 kDa. (B) Immunoblot revealing caspase-1 processing intermediates with the 35 kDa form indicative of activation. Lysates were from AsPc-1 and Dan-G cells treated for 48 and 72 hours with 500 IU/ml IFN-γ or time matched control cells. In Dan-G cells, top and bottom panels represent light and darker exposures of the same blot. (C) Immunoblot analysis for IRF-1 expression. Cells were incubated with vehicle or IFN-γ (500 IU/ml) for the indicated time periods. In each lane, 20 µg of whole cell lysates were separated by 12% SDS-PAGE and IRF-1 expression was determined using a monospecific antibody. Shown are representative blots of at least two experiments.