Cytotoxicity of psammaplin A from a two-sponge association may correlate with the inhibition of DNA replication

Cell viability was determined by Cell Counting Kit-8 (CCK-8) to count living RAW264.7 cells by combining 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8) and 1-methoxy-phenazine methosulfate (1-methoxy-PMS). The effect of psammaplin A on DNA replication was carried out in SV40 DNA replication system in vitro. The activities of topoisomerase I and polymerase α-primase were measured by the relaxation of superhelical plasmid DNA and the incorporation of [3H]dTTP to the template respectively. The ssDNA binding activity of RPA was assessed by Gel Mobility Shift Assay (GMSA).We have found that psammaplin A delivers significant cytotoxic activity against the RAW264.7 cell line. It was also found that psammaplin A could substantially inhibit SV40 DNA replication in vitro, in which polymerase α-primase is one of its main targets.Taken together, we suggest that psammaplin A-induced cytotoxicity may correlate with its inhibition on DNA replication. Psammaplin A has the potential to be developed as an anticancer drug.DNA replication in eukaryotic cells is a tightly regulated process [1]. The regulation of DNA replication is central to understanding the regulation of cell cycle and virus proliferation, events that have a direct impact on our understanding human disease. One critical component of cell cycle regulation is the initiation of DNA replication. The timing of initiation is precisely controlled and is sensitive to both environmental and cellular factors. If DNA replication is blocked by inhibitors or the template is damaged by radiation or other factors, signals are generated that can induce cell cycle arrest or apoptosis [2,3].Much of what is currently known about the mechanism of DNA replication in eukaryotic cells has come from studying SV40 and related viruses. SV40 virus can use the host replication machinery for its own DNA replication together with the virally encoded SV40 T-antigen. SV40 T-Ag is a multifunctional regulato