Use of SPIK as a HCC biomarker was explored after it was discovered as a protein secreted into patients’ blood.[
44,
52] However, the use of SPIK as a cancer biomarker had been impeded by the fact that serum levels of SPIK are also elevated in the presence of other diseases, especially pancreatitis.[
53,
54,
55] Lu et al found that although all cells, including HCC cells, express identical SPIK in the cytoplasmic form, the secreted form of SPIK is unique to HCC cells. For pancreatic SPIK (pan-SPIK), or normal SPIK produced by the pancreas, a 23 amino-acid fragment in the N-terminus, assumed to be a signal peptide, is removed during secretion, but for SPIK secreted by liver cancer cell lines or HCC, this segment is retained.[
26,
56,
57] We call the SPIK secreted from liver cancer cells Liver Cancer SPIK (LC-SPIK). The sequence of animo acid of LC-SPIK and SPIK were listed in
Figure 2A. The extra 23 AA sequence which only exists in LC-SPIK is underlined. The common region of LC-SPIK and SPIK (AA No. 24-79) is also listed (
Figure 2A). The size differences between LC-SPIK secreted from liver cancer cells and normal SPIK secreted from pancreatic cells were compared by Western blot with monoclonal antibodies IMCA1 and MA86, which bind to the 23 extra AA fragment and the common region shared by LC-SPIK and pan-SPIK, respectively. The culture medium from a liver cancer cell line (S2-3) and a pancreatic cell line (PanC1) were collected and analyzed by Western blot.
Figure 2B shows that only LC-SPIK from S2-3 cells but not SPIK from pancreatic cells (pan-SPIK), was recognized by antibody IMCA1, suggesting it has extra 23 AA in N-terminus. In contrast, both SPIKs were recognized by MA86, which suggests that it binds to the common region shared by LC-SPIK and pan-SPIK (
Figure 2B, MA86). The size of S2-3 generated protein (LC-SPIK) was around 8.5 kDa, which corresponds to the correct molecular weight for a full length genetic SPIK, confirming that the LC-SPIK secreted by cancer cells do have entire sequence of SPIK.[
26,
35,
58] However, the secreted protein from PanC1 cell line (pan-SPIK) showed that the size of protein is smaller than its counterpart from the cell lines S2-3 (
Figure 2B MA86), around 6kDa. This is correspondent to an attenuated SPIK with 56 amino acids, suggesting that pancreatic SPIK is proteolytically cleaved upon secretion. [
21,
59] Edman N-terminal analysis further confirmed this conclusion (
Figure 2C). The LC-SPIK secreted by liver cancer cells has animo acids
VTG in positions 2-4, confirming that LC-SPIK has entire sequence of genetic SPIK (
Figure 2C, Edman N-terminal analysis). The quantity of LC-SPIK in HCC patients’ serum was also much greater than the quantity of SPIK secreted by pancreatic cells. This was confirmed by analysis of serum samples from patients with HCC with western blot. The results showed that the SPIK with molecular weight around 8.5 KDa were existing in all 6 patients’ serum that were examined which has same molecular weight of LC-SPIK from S2-3, while the SPIK in patients with pancreatitis has small molecular at 6kDa.[
26,
60,
61] The reason cancer cells could secreted unattenuated SPIK is unclear. Considering SPIK is a serine proteinase inhibitor and signal peptidase is a serine proteinase, it is possible that over-expression of SPIK inhibits signal peptidase activity, resulting in the secretion of entire uncutting protein.