The clonogenic assay was performed using different doses of CaLa. The expression level of c-Myc and Cyclin D1 was measured in addition to the confirmation of 尾-catenin expression in the CRC cells. Glycogen synthase kinase (GSK)-3尾 expression was also confirmed in order to investigate the mechanism of 尾-catenin degradation. Tumorigenic ability was confirmed using a xenograft animal model.
The number of colonies was significantly decreased after 2.5 mM CaLa treatment. CaLa-treated CRC cells showed a decrease in the 尾-catenin expression. The quantitative level of the 尾-catenin protein was significantly decreased in the CRC cell lysates, hence the expression level of c-Myc and cyclin D1 was significantly decreased following 2.5 mM CaLa treatment. We also confirmed that an increased expression of GSK-3尾 by CaLa is a key pathway in 尾-catenin degradation. In the xenograft study, tumorigenicity was significantly inhibited to a maximum of 45% in the CaLa-treated group as compared with the control.
These results support the idea that calcium supplementation via CaLa contributes to 尾-catenin degradation and is hypothesized to reduce the risk of CRC. In addition, it indicates the possibility of CaLa being a potential incorporating agent with existing therapeutics against CRC.