Role of the inflammatory protein serine protease inhibitor Kazal in preventing cytolytic granule granzyme A-mediated apoptosis

Abstract

Serine protease inhibitor Kazal (SPIK) is an inflammatory protein whose levels are elevated in numerous cancers. However, the role of this protein in cancer development is unknown. We have recently found that SPIK suppresses serine protease-dependent cell apoptosis. Here, we report that anti-SPIK antibodies can co-immmunoprecipitate serine protease granzyme A (GzmA), a cytolytic granule secreted by cytotoxic T lymphocytes and natural killer cells during immune surveillance, and that SPIK suppresses GzmA-induced cell apoptosis. Deletion studies show that the C3-C4 region of SPIK is critical for this suppression. These studies suggest that over-expression of SPIK may prevent GzmA-mediated immune-killing, thereby establishing the tolerance of cancer cells to the body's immune surveillance system. Suppression of over-expressed SPIK can restore the susceptibility of these cells to apoptotic death triggered by GzmA. This finding implies that it is possible to overcome tolerance of cancer cells to the body's immune surveillance system and restore the GzmA-mediated immune-killing by suppressing the over-expression of SPIK.

Figure 1

Serine protease inhibitor Kazal (SPIK) gene expression is limited or inactivated outside the pancreas; 20 μm mRNA samples from different human tissues were resolved in 1% denatured agarose gel and transferred to a nylon membrane for Northern blot analysis. The membrane was then hybridized with a SPIK-specific probe. The SPIK mRNA in hepatocellular carcinoma (HCC) cells is presented for comparison. Ribosomal RNA is used as loading control.

Figure 2

Serine protease inhibitor Kazal (SPIK) secreted by hepatocellular carcinoma (HCC) cells has nine extra amino acids in the N-terminus. Culture medium or cell lysate from S2–3 and G54 cells or human pancreatic cells (Panc-1) was run on an SDS–PAGE gel. After transfer to a PVDF membrane, SPIK protein was visualized by staining with a monoclonal anti-SPIK antibody and then with an anti-mouse-horseradish peroxidase secondary antibody. An ECL Advance kit was used to visualize the image. (a) The Western blot used to detect SPIK either in cell lysates or in culture medium. (b) Edman degradation, used to determine the N-terminus sequence of pancreatic SPIK. The N-terminal sequence of SPIK secreted from pancreatic cells was analysed by Edman N-terminal degrading analysis, which was performed by Alphalyse Inc. (Palo Alto, CA) after excision of the pancreatic SPIK band from the PVDF membrane. The sequence predicted by Edman degradation in the N-terminal of secreted pancreatic SPIK is bold and italicized. (c) The sequence of intact SPIK. The missing sequence in secreted pancreatic SPIK is underlined.

Figure 3

Granzyme A (GzmA) was co-immmunoprecipitated with serine protease inhibitor Kazal (SPIK). The medium from cells expressing high levels of SPIK, such as pancreatic cells, S2–3 cells, and G54 cells, was incubated with 200 ng recombinant GzmA and then incubated at 4° overnight with magnetic beads covalently linked with anti-SPIK monoclonal antibody. SPIK and binding proteins were released from beads by treatment with pH 2·5 buffer and resolved in SDS–PAGE. The proteins were then transferred to the membrane and analysed by Western blot with monoclonal anti-GzmA and anti-SPIK antibodies, respectively. (a) Experimental procedure. (b) Western blots showing SPIK and binding proteins after immune co-precipitation with anti-SPIK antibody. GzmA and SPIK are indicated on blots. (c) Immune co-precipitation with anti-GzmA antibody.

Figure 4

Over-expression of serine protease inhibitor Kazal (SPIK) results in cellular resistance to Granzyme A (GzmA) -induced apoptosis. S2–3 and SP23 cells were incubated with GzmA and perforin (PFR), individually or combined. Apoptosis was assessed. (a) Hoechst staining of apoptotic cells. (b) Cell apoptosis was analysed by flow cytometry after staining with annexin V-FITC. (c) Cell apoptosis was quantified by flow cytometry after double staining with propidium iodide (PI) and annexin V-FITC. The lower-right quadrant indicates the apoptotic cells. (d) Percentage of apoptotic cells from flow cytometry with double staining.

Figure 5

Silencing of over-expressed serine protease inhibitor Kazal (SPIK) in S2–3 cells restores cell sensitivity to Granzyme A (GzmA) -induced cell apoptosis. S2–3 cells were transfected with SPIK small interfering (si) RNA plasmids L71 and L183 or with their vector for mock transfection. The plasmid for woodchuck hepatitis B virus (WHBV) siRNA was used as a control for specificity. SP23 cells, expressing only background levels of SPIK, were used as a control. Cell apoptosis was induced by treatment of cells with GzmA/PFR 3 days after transfection. (a) The SPIK RNA levels were examined by Northern blot with the probe specified for SPIK. (b) SPIK protein was examined by Western blot with monoclonal anti-SPIK antibody. (c) Cell apoptosis was analysed by flow cytometry after staining with annexin V-FITC. (d) Cell apoptosis was quantified by flow cytometry after double staining with propidium iodide (PI) and annexin V-FITC. The lower-right quadrant indicates the apoptotic cells. (e) Percentage of apoptotic cells from flow cytometry with double staining.

Figure 6

The C3–C4 region is critical for the ability of serine protease inhibitor Kazal (SPIK) to suppress Granzyme A (GzmA) -induced apoptosis. Wild-type SPIK plasmid PWT was constructed with pCMVscript vector with a myc tag sequence at the N-terminus. Three mutants PD1, PD2 and PD3 were constructed based on PWT. (a). The structure of PWT and its deletion mutants. (b). HeLa cells were transfected with either wild-type plasmid or its mutants (PD1, PD2 and PD3) or mock transfected with vector as a control (none). Cellular SPIK levels were assessed with Western blot using anti-myc antibody. Actin was used as loading control. (c) Cell apoptosis was analysed by flow cytometry after staining with annexin V-FITC. (d) Cell apoptosis was quantified by flow cytometry after double staining with propidium iodide (PI) and annexin V-FITC. The lower-right quadrant indicates the apoptotic cells. (e) Percentage of apoptotic cells from flow cytometry with double staining.