Herbal Melanin Inhibits Colorectal Cancer Cell Motility, Invasiveness and Epithelial-Mesenchymal Transition, Associated with U-PAR Downregulation Through JNK and ERK Pathways

Herbal Melanin (HM) is a potent anticancer drug, biological effects and underlying molecular mechanisms was investigated, and the related signaling pathways, using human colon cell lines. To evaluate the impact of various concentrations of HM on cell migration, invasion and tumorigenicity, a real-time cell analyzer instrument and colony formation assays were employed, respectively. Angiogenesis-related protein array was also used and the levels of protein expression contributing to colony formation and extracellular proteolysis-driven cell migration and invasion, such as E-cadherin, N-cadherin and urokinase-type plasminogen activator receptor (uPAR) were monitored using Western blotting and RT-qPCR technologies. HM significantly decreased CRC cell motility, invasiveness and formation of colonies, associated with E-cadherin upregulation and N-cadherin downregulation. In addition, HM specifically inhibited uPAR expression levels, which were also decreased by the pharmacological mitogen-activated protein kinase (MEK) inhibitor UO126 and Jun N-terminal kinase (JNK) inhibitor SP600125, in both CRC cell lines. Addition of HM to cells pretreated with JNK and MEK inhibitors attenuated the blockade of JNK and ERK phosphorylation, alleviated HM-downregulated uPAR expression and HM-inhibited mCRC cell migration. In conclusion, our in vitro studies demonstrate that HM exhibits an inhibitory effect on CRC migration and invasiveness, associated with uPAR downregulation through JNK and ERK pathways.