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Background Somatic mutations in an MLK family member, Mixed Lineage Kinase 4 (MLK4), have been reported to activate its kinase activity in colorectal cancers (CRCs). Analysis of the TCGA (The Cancer Genome Atlas) database revealed that increased MLK4 expression correlates with poor overall survival in colon cancers. Novel MLK4 inhibitors are being developed using various computational techniques and in vitro models to target different types of cancers. We aim to evaluate the effects of CEP-1347, a pan-MLK inhibitor, on MLK4 expression, apoptosis, and tumorigenesis in colon cancer cells.
Methods Colon cancer cell line, HCT 116 cells were treated with CEP-1347 (500nM) every 24 hours for two days and untreated and DMSO (Dimethyl Sulfoxide) treated cells were used as controls. Cell extracts were primarily incubated with MLK4 antibody, and a western blot analysis was performed. Apoptosis was assessed by measuring Bax, Bcl-2, PARP, and caspase-3 expression using western blot analysis. Additionally, we conducted a wound healing assay on HCT 116 cells treated with CEP-1347(500nM) and cell migration evaluated every 24 hours for 7 days with untreated and DMSO treated cells as controls.
Results The western blot analysis showed significantly reduced MLK4 expression in cells treated with CEP-1347 (p < 0.05). Our apoptosis assay demonstrated a significantly increased Bax/Bcl-2 ratio (p < 0.0005) alongside other apoptotic markers like cleaved PARP (p < 0.0005) and cleaved caspase-3 (p < 0.005) in CEP-1347 treated cells when compared to control or DMSO treated cells. The cell migration assay showed delayed wound closure in CEP-1347-treated cells, with control cells achieving full closure by day 5, whereas treated cells retained ~10% of the initial wound area by day 6.
Conclusion Our results demonstrate that CEP-1347 effectively inhibits MLK4 kinase activity in colon cancer cells, leading to increased apoptosis and reduced migratory potential. These findings suggest that MLK4 inhibition may be a promising therapeutic strategy for treating intractable colon cancers unresponsive to current therapeutic approaches and warrant further investigation.
