Identification And Verification Of Candidate Glycoprotein Biomarkers In Colon Cancer And Effects And Mechnisms Of A Novel Tylophorine Analog NK-007in Colitis

Colon cancer is a major public health issue worldwide, and novel tumor biomarkers may contribute to its efficient management by helping in early detection, prognosis or surveillance of disease. The current study evaluated the glycoproteomic profile of tissues from colon cancer patients for discovery of potential glycoprotein biomarkers. The lectin microarray was first performed to compare the glycoprotein profiles between colon cancer and matched normal tissues. The results showed that compared with normal tissues, colon cancer had a wide range of expression of glycosylated proteins and a higher level of glycosylation. In order to confirm the results of lectin microarrays, lectin histochemistry was used. Level of N-acetylglucosamine (GlcNAc) that Solanum Tuberosum lectin (STL) bound was found to be elevated in colon cancer, which was consistent with the results of lectin microarrays. The subsequent glycoproteomic analysis based on STL enrichment of glycoproteins followed by label-free quantitative nanoLC-MS/MS analysis identified72proteins in high confidence. Among these proteins,17were exclusively detected in cancer tissues, and14were significantly upregulated in tumor tissues. Annexin A1and HSP90β were chosen for further investigation by immunoprecipitation coupled with lectin blots, western blots and tissue microarrays. Both HSP90β and Annexin A1were GlcNAcylated, and their protein expressions were elevated in colon cancer, compared to normal tissues. Moreover, specific changes of GlcNAc abundances in Annexin A1and HSP90β suggested that tumor-specific glycan patterns could serve as candidate biomarkers of colon cancer for distinguishing cancer patients from healthy individuals.Colitis-associated Cancer (CAC) is one type of colon cancer, which proceeded with inflammatory bowel disease (IBD) clinically. Studies have shown that IBD is one of the factors that induce the CAC. Therefore, prevention and treatment of IBD become an effective means of prevention of colorectal cancer. In this study, we synthesized (±)-tylophorine malate (NK-007), an analog of tylophorine (DCB3503), and analyzed its anti-inflammatory effect in vivo using a dextran sulfate sodium (DSS)-induced colitis model and an acetic acid-induced colitis model. As indicated by disease activity index (DAI) and degree of macroscopic colonic damage, NK-007can significantly suppress colitis. To delineate the underlying mechanism, we have explored the influence of NK-007on the production of TNF-a by murine primary bone marrow derived cells (BMDCs) as well as monocyte/macrophage cell line raw264.7triggered by lipopolysaccharide (LPS). For both types of innate immune cells, NK-007showed a potent TNF-a inhibitory effect, and has in addition reduced the expression of IL-12in BMDCs. Moreover, raw cells treated with NK-007also showed decreased phosphorylation of NF-kB, which may explain the protective immune-regulatory effect of NK-007for experimental colitis.