The main reason for the failure of chemotherapy therapies based on 5-Fluorouracil (5-Fu) is the development of resistance to 5-Fu in cancer patients, particularly those with colorectal cancer. Tissue inhibitor of metalloproteinases 2 (TIMP-2) has been shown to be associated with colorectal cancer (CRC), but its correlation with 5-Fu resistance in colorectal cancer has not been thoroughly studied. We screen the expression of different cytokines through Cytokine array. CCK-8 assay was conducted to evaluate the IC 50 of 5-Fu and cell proliferation. ELISA and RT-qPCR were performed to detect TIMP-2 expression levels in cells and patient serum. Western blotting was utilised to analyse the differences in the expression of proteins related to signalling pathways in cells. Through cytokine array screening, we found that the expression of TIMP-2 was significantly increased in CRC drug-resistant cell lines. In addition, the expression of TIMP-2 in the serum of patients with CRC resistance to 5-Fu was significantly increased. Subsequent mechanistic experiments showed that TIMP-2 regulated the resistance of CRC cells to 5-Futhrough the JAK–STAT signalling pathway. Moreover, anti-TIMP-2 antibody or small molecule drug LY2784544 targeting the JAK–STAT signalling pathway can effectively reverse the resistance of CRC cells to 5-Fu. It is exactly TIMP-2 that mediates the resistance of CRC to 5-Fu through the JAK–STAT signalling pathway. Targeting drugs for TIMP-2 or the JAK–STAT signalling pathway are expected to be opportunities to reverse 5-Fu resistance in CRC.
This study aimed to investigate the long-term scar hypertrophy in the rabbit transplanted oral mucosa and scrotal skin with changed matrix environment, as well as the scar location expression, quantitative analysis of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) and biomechanical changes in the transplanted tissues. The split-thickness skin grafts were collected from the oral mucosas and scrotal skins of 30 male rabbits, and prepared into reelpipes for autologous transplantation into the rabbit back muscular tissues. Samples were collected to carry out elastic tensile mechanical detection and histological observation. The maximum longitudinal tensile displacement of scrotal skin before 8 weeks of transplantation was greater than that after 8 weeks of transplantation (P < 0.05). The expression intensities of MMP-2 and TIMP-2 in the oral mucosa and in scrotal skin at 2 W time point were higher than those at T o time point (P < 0.05). The expression quantities of TIMP-2 in oral mucosa and scrotal skin during 8–24 W were higher than those of MMP-2 (P < 0.05). At 8 W time point, the TIMP-2/MMP-2 ratio in scrotal skin was higher than that in oral mucosa (P < 0.05). MMP-2 and TIMP-2 expression in normal oral mucosa and scrotal skin is weak, but their expression is remarkably up-regulated after 2 weeks of transplantation, revealing that scar formation was related to the high expression of MMP-2 and TIMP-2. At the 8th–24th weeks, the AOD values of TIMP-2 in oral mucosa and scrotal skin are apparently higher than those of MMP-2; moreover, the TIMP-2/MMP-2 ratio in scrotal skin at the 8th week was higher than that in oral mucosa, which can well explain the earlier scar formation in scrotal skin than in oral mucosa, and it also suggests that the different expression levels between TIMP-2 and MMP-2 may account for the important cause of scar formation.
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