Tylophorine analogs exhibit potent cytotoxic activity against a broad range of human cancer cells. The previous data showed that WLY-1, a C9-substituted phenanthrene-based tylophorine derivatives (PBTs), exhibited high potent cytoxicity to various cancer cell lines. In this study, we further explored the mechanism of WLY-1 inducing cancer cell death as a potential anti-cancer agent. The results showed WLY-1 provided high cytotoxic activity against certain lung cancer cell lines including CL1-0, CL1-5 and A549 with IC50 values ranging from 0.48 to 0.84 μM by MST assay. The CL1-0 cells treated with WLY-1 induce cell shrinkage and rounding, and then membrane become broken while increasing concentration of the drug. DNA flow cytometric analysis indicated that the treatment of WLY-1 resulted in cell cycle G2M arrest in a time-dependent manner and the sub-G1 peak appeared at 16 hours under WLY-1 treatment. Western blots analysis indicated that the treatment of WLY-1 resulted in cyclin B1 accumulation and p21 decrease but no effect on cyclin A, cyclin E and p27 protein levels. In addition, Annexin V and western blot analysis indicated that the treatment of WLY-1 induce lung cancer cells apoptosis through activating pro-apoptotic protein. The treatment of WLY-1 also suppressed the colony formation in a dose-dependent manner. To realize the molecular mechanism of WLY-1 inducing cell apoptosis, we analyzed the changes of gene expression after WLY-1 treatment by using cDNA microarray with Gene Spring software. The data showed that MAPK signaling and apoptosis pathway were dramatically activated after the treatment of WLY-1. On the contrary, focal adhesion and NF-kB pathway were suppressed by the treatment of WLY-1. The reporter assay showed that NF-kB transcriptional activity was dose-dependently decreased by WLY-1 treatment. Western blot analysis confirmed that the phospho-Akt decreased and IkB accumulated after the treatment of WLY-1. We concluded that WLY-1 can induce cell cycle G2M arrest and apoptosis through activating MAPK pathway as well as inhibiting NF-kB signaling pathway, and thus, is a candidate of anti-cancer agents for cancer therapy.

99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA