[Mechanism of vitamin K2 in modulating matrix Gla protein to inhibit tumor growth in inflammation-associated colorectal cancer model mice]

Abstract

Objective: To explore the mechanism of vitamin K2 (VitK2) regulating matrix Gla protein (MGP) in inhibiting tumor growth in inflammation-associated colorectal cancer (CAC) model mice. Methods: Twenty-six C57BL/6 male mice, 6-8 weeks old and weighing 20-25 g, were divided into 4 groups according to the random number table method (each group received different treatments): normal group (treated with olive oil gavage and physiological saline intraperitoneal injection) (n=5), model group [treated with olive oil gavage, azoxymethane (AOM) intraperitoneal injection, and dextran sulfate sodium (DSS) solution drinking treatment)] (n=5), 30 mg group (treated with 30 mg·kg^-1·d^-1 of vitamin K2 gavage, AOM intraperitoneal injection, and DSS solution drinking treatment) (n=8), and 60 mg group (treated with 60 mg·kg^-1·d^-1 of vitamin K2 gavage, AOM intraperitoneal injection, and DSS solution drinking treatment) (n=8). The mice were sacrificed at the end of the 12th week, and the number and length diameter of colon tumors in each group were compared. The expression level of nuclear proliferation antigen (Ki-67) in colon tissues was assessed by immunohistochemistry (IHC), while the levels of MGP protein and Smad1/5 pathway-associated proteins were determined by Western blotting (WB). Additionally, the expression of MGP mRNA was quantified using real-time quantitative PCR (RT-qPCR). According to different treatment methods, colon cancer epithelial cell line SW480 cells were divided into control group [treated with dimethyl sulfoxide (DMSO)], 100 μmol/L group (treated with 100 μ mol/L VitK2), 200 μmol/L group (treated with 200 μ mol/L VitK2), 400 μmol/L group (treated with 400 μ mol/L VitK2), empty vector group (SW480 cells were transfected with empty plasmid) and MGP overexpressing group (SW480 cells were transfected with MGP overexpressing plasmid). After 48 hours of treatment, the expression of MGP protein, changes in the Smad1/5 pathway, and cell proliferation at different time points (24, 48, 72, and 96 hours) after treatment were detected. Results: All mice in the model group, 30 mg group, and 60 mg group developed colorectal tumors, with a tumorigenesis rate of 100% (17/17). There was no significant difference in the number of colon tumors between the 30 mg and 60 mg groups and the model group (both P>0.05), but the long diameter of tumors in the 30 mg or 60 mg groups was smaller than those in the model group (both P<0.05). There was no significant difference in tumor number or long diameter between the 30 mg and 60 mg groups (both P>0.05). The Ki-67 protein expression levels in the 30 mg and 60 mg groups were both lower than those in the model group (both P<0.05). Meanwhile, the MGP protein expression levels and pSmad1/5 protein expression levels in the 30 mg and 60 mg groups were both higher than those in the model group (all P<0.05). There was no statistically significant difference in MGP mRNA levels between the 30 mg and 60 mg groups and the model group (both P>0.05). Furthermore, there was no statistically significant difference in the expression levels of Ki-67 protein, MGP protein, pSmad1/5 protein, or MGP mRNA between the 30 mg and 60 mg groups (all P>0.05). In the cell experiments, the MGP and pSmad1/5 protein expression levels in the 100 μmol/L group showed no statistically significant difference compared to the control group (both P>0.05). The MGP and pSmad1/5 expression levels in the 200 μmol/L and 400 μmol/L groups were both higher than those in the control group (both P<0.001). After 72 and 96 hours, the cell proliferation capacity in the 200 μmol/L and 400 μmol/L groups was both lower than that in the control group (both P<0.001). The pSmad1/5 protein expression level in the MGP overexpression group was higher than that in the empty vector group (P<0.001). After 24, 48, 72, and 96 hours, the cell proliferation capacity in the MGP overexpression group was lower than that in the empty vector group (all P<0.001). Conclusion: VitK2 can inhibit the growth of CAC model mice by promoting MGP expression and activating Smad1/5 pathway.