Background
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 regulatory protein with numerous biochemical activities, and it has been classified as a member of superfamily III helicase and can unwind dsDNA and RNA [
4,
5]. All other proteins are supplied by host cells. In replication, replication protein A (RPA) mediates unwinding of SV40 origin-containing DNA in the presence of SV40 T-Ag and the DNA polymerase α-primase complex (pol α-primase) [
6,
7], which is necessary for the initiation of SV40 DNA replication [
8,
9].
Psammaplin A is a symmetrical bromotyrosine-derived disulfide dimer that was originally isolated in 1987 from the
Psammaplysilla sponge [
10]. Early studies revealed that psammaplin A had general antibacterial and antitumor properties. In 1999, it was found that psammaplin A exhibited significant
in vitro antibacterial activity against both
Staphylococcus aureus (SA) and methicillin-resistant
Staphylococcus aureus (MRSA), which was inferred to be the result of induced bacterial DNA synthesis arrest by psammaplin A through inhibiting DNA gyrase [
11]. Given the increasingly rapid emergence of multi-drug resistant bacterial strains and the corresponding threat to public health, there is significant interest in the development of structurally novel antibacterial agents such as psammaplin A. Additionally, psammaplin A has been reported to exhibit certain inhibition of a number of enzymes including topoisomerase II (topo II) [
12], farnesyl protein transferase [
13], leucine aminopeptidase [
13], and latest reported chitinase [
14]. Among these enzymes, topo II, as one required protein for eukaryotic DNA replication, as well as bacterial DNA gyrase belongs to the topoisomerase family of enzymes responsible for the remolding of DNA topology. Since psammaplin A can inhibit bacterial DNA synthesis through DNA gyrase inhibition, and much of the basic enzymology of the eukaryotic replication fork has close homologies with its prokaryotic counterpart, we wonder whether psammaplin A also can induce eukaryotic DNA replication arrest or not.
We have reported that psammaplin A displayed significant cytotoxicity against human lung (A549), ovarian (SK-OV-3), skin (SK-MEL-2), CNS (XF498), and colon (HCT15) cancer cell lines [
15]. In this paper, psammaplin A was found to have dose-dependent cytotoxicity on macrophage cell line. In order to clarify the possible mechanism of the cytotoxicity and also verify our conjecture of its possible action on DNA replication, the effect of psammaplin A on eukaryotic DNA replication was examined by using
in vitro SV40 DNA replication system. According to our result that psammaplin A can induce eukaryotic DNA replication arrest through inhibiting some important replication proteins, we suggest that psammaplin A-induced cytotoxicity may correlate with its inhibition on DNA replication, and one of the main target molecules could be DNA polymerase α-primase.
Discussion
Psammaplin A has exhibited inhibition on general bacterium, some actinomycetes and fungi, and it also has cytotoxicity toward several cancer cell lines. In our research, we found that psammaplin A deliver significant cytotoxic activity against macrophage cell line RAW264.7. Which process is mostly affected by psammaplin A in cell cycle? What is the mechanism of the inhibition? Enlightened by the inhibitory effects of psammaplin A on bacterial DNA synthesis, bacterial DNA gyrase and eukaryotic topo I, we investigated the effect of psammaplin A on DNA replication using SV40 replication in vitro system attempting to find out the target process and molecules of psammaplin A and have a glimpse on cell cycle regulation.
In our study, we found that psammaplin A inhibited SV40 DNA replication
in vitro. In order to clarify the inhibition mechanism, further work were carried out. In SV40 DNA replication, three factors, SV40 T-Ag, RPA, and pol α-primase complex, are essential for initiation process. In the presence of topo I, SV40 T-Ag will continue to unwind the DNA to form a highly unwound DNA [
21]. DNA synthesis with three factors and topoisomerase can be quite extensive [
22]. We have suggested that psammaplin A might interfere with some molecules that are required to establish replication forks during the initiation reaction. To address this possibility, we asked whether psammaplin A inhibits topo I, RPA's ssDNA binding activity, and pol α-primase activity. In addition, the topo I is now considered to be important cancer chemotherapeutic target. In mammalian cells, actions of antitopoisomerase drugs on replication, transcription, and other processes ultimately activate pathways of programmed cell death [
23]. Psammaplin A inhibited the DNA relaxation activity of topo I and the ssDNA binding activity of RPA in a dose-dependent manner, and up to 500 μM, psammaplin A can inhibit both the activities of topo I and RPA completely. On the other hand, psammaplin A significantly reduced pol α-primase activity at 40 μM. The above results indicate that major inhibition of SV40 DNA replication by psammaplin A may be due to the inhibition of pol α-primase activity. Here, we cannot rule out the possibility that psammaplin A inhibit the activity of SV40 T-Ag, because it is essential for initiation process.
It is puzzling that the DNA pol α-primase, RPA and topo I were readily inhibited by low concentration of psammaplin A whereas the SV40 DNA replication assay still showed about more than 80% DNA replication in the presence of 125 μM psammaplin A. In our opinion, at least three points could account for this discrepancy. First, the cell extract we have used to support in vitro DNA replication system includes a large number of proteins, while in topo I assay, RPA binding assay and pol α-primase assay, purified proteins were used. Many of the proteins in the crude extracts can affect each other by physical or functional interactions. For example, in the process of DNA replication initiation, RPA interacts with T-Ag and DNA pol α-primase, and it is believed that RPA can both stabilize the unwound DNA and stimulate DNA pol α. The universal protein-protein interactions in crude extracts make its working environment quite different from that of purified protein assay system. Second, even for the same protein, for example, pol α-primase, the concentration in the cell extract and in the purified pol α-primase assay is not comparable before any quantification of the replication proteins in the cell extract. Third, due to the active disulfide moiety in the structure of psammaplin A, it is possible that psammaplin A could interact with some particular cellular targets in the crude extract, which may lead to covalent modification of the biological targets and psammaplin A itself. Therefore, the free available concentration of psammaplin A in the crude extract might be different from that in purified protein assay system.
Different from the effect of psammaplin A on SV40 DNA in vitro replication, the significant inhibition of psammaplin A on the viability of macrophage RAW264.7 cells occurred at a relatively low concentration. There is the possibility that DNA replication might not be the single or primary event that affected by psammaplin A. It should be evident that the event of DNA replication in a living cell is more complicated than that in the crude extract system because of many existing cell cycle signals. So, it is still vague whether the ability of psammaplin A to inhibit cellular viability is correlated with its ability to inhibit DNA replication. In order to make it clear, it is necessary to check the effects of psammaplin A on other macromolecular synthesis (RNA synthesis and protein synthesis). Although the results in this paper do not clearly define the mechanism of cytotoxicity of psammaplin A, they have convincingly shown that psammaplin A possesses the abilities to inhibit DNA replication and some important replication proteins.
Because of the disulfide bridge linking two identical subunits in the structure of psammaplin A, in this study, we also paid attention to the potential reduction effect of DTT present in the topo I assay. Different from the report of Nicolaou [
24], we didn't catch any difference between the reactions in the presence and absence of DTT. There exist two possibilities. One is that in our reaction system, DTT couldn't reduce psammaplin A to the corresponding free thiol. Comparing the
in vitro assay conditions in this study, we can find that all these assays were performed in similar conditions (for example: pH 7.5~7.7, 0.5 mM DTT and Mg
2+ ion environment). Given the mildness and tolerance of these reaction mixtures, we guess that psammaplin A is stable in the assays. Of course there is the second possibility that psammaplin A was reduced in the reaction, but the reduction product had nearly the same inhibition effect on topo I as the original compound. Further investigation aimed at this question need to be carried out.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
YHJ and DKK conceived and designed the experiments and wrote the manuscript. YHJ performed all of the experiments. JHJ provided psammaplin A sample. EYA, SHR, JSP, HJY, SY, BJL and DSL participated in the conception, supervision, coordination and guidance of the study and manuscript preparation. All authors read and approved the final manuscript.