The small molecule compound, MCP1, inhibits Ras-Raf interaction in mammalian cells and induces apoptosis in various hematopoietic cancer cells

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Activation of the Ras signaling pathway has been identified in a wide range of human cancers, making the Ras signaling pathway an ideal target for anti-cancer drug development. Previously we have identified a small molecule compound MCP1 as a potential inhibitor of Ras/Raf interaction using the yeast two-hybrid system-based screening (Khazak et al., AACR meeting, 2002) and demonstrated that it inhibits the receptor tyrosine kinase-Ras-Raf-MEK pathway as well as transformed phenotypes of various human cancer cells with an activated ras mutation (Kato-Stankiewicz et al., PNAS, 2002). Here we present effects of MCP1 on a variety of hematopoietic cancer cells including multiple myeloma cell lines MM1.S and U266, B lymphoblast IM-9, acute promyelocytic leukemia HL-60, and acute lymphoblastic leukemia CEM cells. Cells were incubated with MCP1 or MCP122, the MCP1 derivative with much less potency in Ras-MAPK cascade inhibition, under various serum concentrations (0.1-10%) and the MTT cell viability assay was performed. MCP1 showed better reduction in cell viability at lower serum concentration. Since Raf has been suggested to suppress apoptosis, we examined if this reduction is due to apoptosis by flow cytometric analysis with AnnexinV/PI-staining. MCP1, but not MCP122, increased apoptotic cells 10-20% after 16 hours incubation in the 0.1% serum-containing media. Also, MCP1 significantly activated caspase-3 and -9 in these cells. These results suggest that MCP1 is effective against hematopoietic malignancies. Interestingly, MEK inhibitors did not induce apoptosis, suggesting that inhibition of Raf but not MEK is important in MCP1-induced apoptosis in these cells. A possible molecular mechanism of apoptosis induction by MCP1 will be discussed. An important feature of MCP1 is that it is capable of inhibiting Ras/Raf interaction. To further assess this property in mammalian cells, we have recently established a novel assay system using a split renilla luciferase fusion protein-based bioluminescence assay. Two luciferase fusion proteins: the Nt-luciferase fused to Ras (NLuc-Ras) and the Ras-binding domain (RBD, 51-131 a.a.) of Raf fused to the Ct-luciferase (RafRBD-CLuc) were constructed. Coexpression of the NLuc fusion construct with constitutively active Ras(L61) and RafRBD-CLuc in COS7 cells induced several thousand-fold luciferase activity. In contrast, the dominant-negative Ras(N17)-fusion construct had no effect, as it does not interact with Raf. We demonstrated that MCP1 but not MCP122, inhibits luciferase activity induced by Ras/Raf interaction in living cells in a dose-dependent manner at similar concentrations that inhibits Ras-MAPK cascade. This approach will provide a valuable tool to observe effects of MCP1 on Ras/Raf interaction in Xenograft system under physiological conditions.