Figure 2

LRRK2-IN-1 elicits anticancer activity in vitro . HCT116 and AsPC-1 cells were seeded into 96-well plates at 10⁴ cells per well and allowed to attach overnight at 37°C. LRRK2-IN-1 was added in triplicate to the wells at concentrations of 0 (DMSO), 0.3, 0.6, 1.25, 2.5, 5, 10, and 20 μM and incubated at 37°C. After 48 h an MTT proliferation assay was performed and revealed significant inhibition of cell proliferation starting at 0.62 μM (A-B). A live/dead assay in AsPC-1 cells was performed 24 h post treatment to confirm LRRK2-IN-1’s cytotoxic effect. Additionally, a luminescent assay revealed that this effect co-ocurred with Caspase 3/7 activation, both p < 0.0001 by ANOVA (C). AsPC-1 cells were seeded into 6-well plates allowed to reach confluence, scratched, and treated with DMSO, 0.5, 5, or 10 μM LRRK2-IN-1 and incubated at 37°C. The wound area was imaged at baseline, 12, 24, 48, and 72 h and quantified using ImageJ (D-E). mRNA expression analysis of AsPC-1 cells treated with DMSO or LRRK2-IN-1 at concentrations of 0.5 μM or 5.0 μM for 8 h revealed a significant downregulation (p < 0.01) of apoptosis regulators BCL2, BCL2L1 (BCL-XL), and MCL1 mRNA expression (F). Western blots showed decreased BCL2 and Phosphohistone H3 at both 24 and 48 h post LRRK2-IN-1 treatment (G).