Figure 5

LRRK2-IN-1 induces anti-oncogenic molecular changes and LRRK2-IN-1 induced cell death depends on DCLK1 kinase activity. AsPC-1 cells treated with LRRK2-IN-1 show decreased expression of stem (A), oncogenic (B), and EMT-related gene expression (C) (Vehicle = white; 0.5 μM = gray; 5.0 μM = striped bars. p < 0.05). Consistent with the changes seen in EMT-related gene expression, LRRK2-IN-1 significantly decreases invasion in AsPC-1 cells at 5 μM (D). DCLK1 isoform 1 is the most highly expressed referenced variant in colon cancer (E – top panel). Lentiviral overexpression of this isoform and its kinase dead form demonstrates approximately equal levels of DCLK1 transcript as determined by real-time RT-PCR of infected HCT116 cells (E – bottom panel). HCT116(vector), HCT116-DCLK1, and HCT116-DCLK1-KD cell lines were treated with LRRK2-IN-1 at various concentrations. After 48 h an MTT proliferation assay was performed. Compared to vector control cells, HCT116-DCLK1-KD cells demonstrated no change from 0.63 – 2.5 μM and decreased resistance at 5 and 10 μM, while HCT116-DCLK1 cells demonstrated significantly increased resistance to LRRK2-IN-1 cytotoxicity at 0.63 and 1.25 μM concentrations leading to a shift in relative cytotoxicity at low doses (F). AsPC-1(vector), AsPC-1-DCLK1, and AsPC-1-DCLK1-KD cells were treated with vehicle, 0.5, or 5 μM of LRRK2-IN-1 and allowed to form colonies for 9 days. Colonies were stained with crystal violet and plates were divided into grids and counted. AsPC-1-DCLK1 cells demonstrated significant resistance at 0.5 μM compared to vector control cells, while AsPC-1-DCLK1-KD cells demonstrated a significant decrease in resistance compared to vector control cells (G).