Synergistic effects of interferon-alpha in combination with chemoradiation on human pancreatic adenocarcinoma

Abstract

Aim: To determine whether IFN-alpha is the agent that turns a slightly effective treatment (radiochemotherapy) into a potent therapy, we tested IFN-alpha for its synergistic properties.

Methods: Eight pancreatic carcinoma cell lines were treated with the single agents and combinations of these. The role of IFN-alpha regarding a) direct inhibitory effects; b) radio and chemosensitizing effects; c) anti-angiogenic properties and d) enhancement of immunogenicity was investigated.

Results: Our results show that IFN-alpha has direct inhibitory properties and some synergistic influence as determined by AnnexinV/PI stain and cell count. IFN-alpha is also able to prevent the increase in proliferation rate and VEGF secretion of CDDP resistant cells. Having taken the results from immunogenicity experiments together, we found cells that can be influenced by IFN-alpha but were less susceptible against T cells. Furthermore, high expression of MHC molecules, CD118, EGF-R and Fas was predictive for a good response.

Conclusion: In conclusion, IFN-alpha has direct cytotoxic effects, acts as a radiosensitizer and circumvents tumor cell-regrowth after CDDP treatment. These mechanisms may be responsible for the good clinical outcome of CapRI.

Figure 1

Influence on cell survival Equal amount of cells were seeded out on day +1. A: Numbers of viable cells were determined after five-day treatment. Cell numbers of untreated cells were set to ‘1’ and cell survival index was calculated. B: Shows the influence of IFN-α in combination treatments. Values of treatments without IFN-α were subtracted from values after combination treatment. Data is shown as mean±SD error from eight cell lines with at least three separate experiments. ^aP<0.05 was labeled with an asterisk; ^bP<0.01 with two asterisks and ^dP<0.001 with three asterisks.

Figure 2

Induction of apoptosis Apoptosis was determined after five-day treatment by staining for AnnexinV⁺ using flow cytometry (A). Late apoptosis is defined as AnnexinV⁺/PI⁺, necrosis as PI⁺. B) shows the influence of IFN-α in combination treatments. Values of treatments without IFN-α were subtracted from values after combination treatment. Data is shown as mean±SD error from eight cell lines with at least three separate experiments. ^aP<0.05 was labeled with an asterisk; ^bP<0.01 with two asterisks and ^dP<0.001 with three asterisks.

Figure 3

Inhibition of proliferation Proliferation rate was determined after five-day treatment by using a non-radioactive MTT assay. Proliferation rate of untreated cells was set to ‘1’ and percentage of proliferation was calculated (A). B) shows the influence of IFN-α in combination treatments. Values of treatments without IFN-α were subtracted from values after combination treatment. Data is shown as mean±standard error from eight cell lines with at least three separate experiments. ^aP<0.05 was labeled with an asterisk; ^bP<0.01 with two asterisks and ^dP<0.001 with three asterisks.

Figure 4

Secretion of VEGF Cells were seeded out after four-day treatment in a density of 1×10⁵ living cells in 2 mL. Supernatants from treated and untreated tumor cells were collected after 24 h and stored at -80 °C. VEGF concentration was determined by ELISA (A). B) shows the influence of IFN-α in combination treatments. Values of treatments without IFN-α were subtracted from values after combination treatment. Data from three cell lines are shown as mean±standard error from at least three separate experiments. ^aP<0.05 was labeled with an asterisk and ^bP<0.01 with two asterisks.

Figure 5

Immunophenotyping of tumor cells Tumor cells were treated as described. Expression of MHC molecules was analyzed after five-day treatment by flow cytometry (A). B) shows the influence of IFN-α in combination treatments. Values of treatments without IFN-α were subtracted from values after combination treatment. Data is shown as mean±SD error from eight cell lines each with at least four separate experiments. ^aP<0.05 was labeled with an asterisk and ^bP<0.01 with two asterisks.

Figure 6

Expression of surface receptors Tumor cells were treated as described. Expressions of IFN and EGF receptors were analyzed after five-day treatment by flow cytometry. Data is shown as mean±SD error from eight cell lines each with at least four separate experiments. ^aP<0.05 was labeled with an asterisk; ^bP<0.01 with two asterisks and ^dP<0.001 with three asterisks.

Figure 7

Statistical analysis Expression level on untreated cells were correlated with results from AnnexinV/PI stain, proliferation and cell survival index after treatment with the CapRI scheme and analyzed for use as predictive marker (A-F).