Our results suggest that the exclusive eradication of human cancer cells with extra centrosomes by the phenanthridine derivative PJ-34 is attributable to its extra-centrosomes de-clustering activity in mitosis (Figures 2, 4, 5, 6, Additional File 1 and Additional File 2).
According to our observations, PJ-34 did not impair centrosomes in both human normal and cancer cells, nor did it interfere with the formation of centrosomes in the interphase of multi-centrosomal cancer cells or impaired the bi-polar assembly of centrosomes in mitosis of normal proliferating cells with two centrosomes (Figures 2a, 8, Additional File 1 and Additional File 2). These features may underlie its outstanding capability to exclusively eradicate human cancer cells while keeping normal proliferating cells un-affected (Figures 5 and 7).
Theoretically, in cancer cells with high occurrence of extra-centrosomes (>50%) when mitosis is accompanied by eradication of multi-centrosomal cells, an exponential reduction of the fraction of bi-centrosomal cancer cells is expected. In this case, the higher their proliferation rate the more rapidly will these cancer cells be eradicated by extra-centrosomes de-clustering agents.
The indicated interference of the tested phenanthrene derivatives with the bipolar clustering of supernumerary centrosomes was not shared by non-phenanthrene derivatives acting as potent PARP inhibitors as well (Figure 3). This questions an exclusive role of PARP inhibition in extra centrosomes de-clustering. Nevertheless, these results could also reflect a possible, yet un-identified selective inhibition of PARP isoforms by phenanthridines.
Results obtained from genome-wide RNAi screen in multi-centrosomal cells, including embryonic Drosophila S2 cells and human malignant cells, identified genes required to suppress multipolar mitosis. Proteins involved in the organization and regulation of the cytoskeleton, including Kinesin HSET/Ncd (which is not required for polar organization in normal somatic cells) and the four chromosomal passenger complex (CPC) components, Aurora-B, INCENP, Survivin, and Borealin were identified among the main proteins required for bifocal clustering of extra-centrosomes in these cells [12, 17]. Evidence for the necessity of tankyrase-I and a putative human PARP-16 homolog in centrosomes clustering has been also provided [17, 20]. PolyADP-ribosylation of tankyrase-1 may contribute to spindle bipolarity by providing a static matrix, anchoring microtubule-associated motor proteins and spindle proteins [17, 20]. A specific role for PARP-16 in mitosis has not been identified yet .
PARP proteins are present in centrosomes [21–24], and both Aurora-B and INCENP are targets for polyADP-ribosylation [22, 24]. In addition, supernumerary centrosomes were found in PARP-1 deficient cells . Thus, in view of the exclusive interference of PJ-34 with extra centrosomes de-clustering in mitosis, a possible synergism between PARP activity and other mechanisms underlying extra-centrosomes clustering should be further investigated.
The indicated cytotoxic activity of the phenanthrene derivatives in human cancer cells with high occurrence of extra-centrosomes links for the first time between two well known, but poorly understood phenomena; Some anti-tumor effects of natural phenanthridines were first described in the early fifties [26–28], and a higher cytotoxicity of some phenanthridines in cancer cells as compared to normal cells has been reported before [29, 30], although the underlying mechanisms have never been understood. In addition, cell-cycle arrest resulting from failure of centrosomes to accomplish bipolar clustering in multi-centrosomal cells has been acknowledged for nearly a century [6, 31, 32]. Nevertheless, the causal relationship between multiple centrosomes and malignancy has only recently been clarified [32–35].
Mitotic-spindle microtubules are among the most effective targets for anti-cancer therapy [5, 10, 17, 36–39]. Therefore efforts are invested in trying to find mechanisms exclusively targeting mitosis in cancer cells [40–42].
Unlike the exclusive interference of PJ-34 with extra centrosomes bi-polar clustering in mitosis (Figures 2, 3, 4, 5, 6, Additional File 1 and Additional File 2), the currently known small molecules interfering with mitosis in cancer cells target microtubule polymerization (vinca alkaloids, taxanes, colchicines, and also griseofulvin). These compounds therefore impair normal cells as well, and cause severe side effects [36–39].
The discovered potency of PJ-34 in preventing bipolar clustering of extra centrosomes in mitosis may pave the way for a new efficient therapy lacking intolerable side effects, which is based on compounds that exclusively target the unique dependence of many human cancer cells on their supernumerary centrosomes clustering for their survival.
Since many human cancer cells have high occurrence of multicentrosomal cells, the exclusive cytotoxic activity of PJ-34 in multi-centrosomal human cancer cells holds promise as a therapeutic tool for a selective chemotherapy in a wide range of human cancers, beyond the currently used selective therapeutic tools targeting specific genes/proteins that affect few cancer cell types [43–45].