Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

Abstract

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multicentrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent γ-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered γ-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis. Video LinkThe video component of this article can be found at

PARP targeting counteracts gliomagenesis through induction of mitotic catastrophe and aggravation of deficiency in homologous recombination in PTEN-mutant glioma

“…As a first approach to analyze the potential of PARP inhibition (PARPi) as monotherapy against GBM we evaluated self-renewal capability, which is a marker of stemness in GSCs, using neurospheres formation assay in primary patient-derived PTEN-proficient GSCs TG1 [ 21 ] with two different PARP inhibitors: PJ34 (IC 20 nM) and olaparib (IC 5 nM). PJ34 targets mainly PARPs synthesizing proteins but some off-target effects have also been reported, suggesting the effect of PJ34 on cancer cells may not be attributed exclusively to PARP inhibition [ 22 , 23 ]. For that reason we also used the clinically relevant PARPi olaparib.…”

The Modified Phenanthridine PJ34 Unveils an Exclusive Cell-Death Mechanism in Human Cancer Cells

“…It was observed that apart from PARP inhibition, some of these molecules target a variety of kinases implicated in signal transduction pathways in both healthy and malignant cells [27]. Unexpectedly, this research also disclosed that a group of phenanthrene derivatives acting as PARP inhibitors, exclusively kill human cancer cells without affecting benign cells [28][29][30][31][32]. Unlike other PARP inhibitors, these small molecules exclusively eradicated a variety of human cancer cells without affecting proliferating and non-proliferating healthy somatic cells.…”

“…After years of research based on PJ34-induced PARP inhibition in a variety of cell types under pathological conditions [1,2,8], additional activities of PJ34 have been disclosed. It was observed that PJ34 causes an irreversible cell growth arrest in cancer cells, that it interferes with angiogenesis, and, most interestingly, that PJ34 exclusively eradicates human cancer cells [29,30,39,40].…”

“…In view of the decreased expression of PARP1 and NFkappaB in several cancer cell types eradicated by PJ34, it has been suggested that eradication by PJ34 can be attributed to the attenuation of PARP1-dependent NF-kappaB activity that promotes proliferation [45]. However, the suggested mechanisms of PARP1-dependent activity of PJ34 in cancer cells are inconsistent with its exclusive PARP1 independent cytotoxic activity in human cancer cells at a higher concentration range than that causing PARP1 inhibition [29][30][31]35]. Its exclusive cytotoxic activity in human cancer cells was not shared by other potent PARP inhibitors [29][30][31]61].…”

“…On the other hand, their exclusive cytotoxic activity in human cancer cells resembles the cytotoxic activity of other phenanthridines [36][37][38]. The modified phenanthridine PJ34, one of the molecules in this group, was the most potent in a variety of human cancer cells, including cells that are resistant to currently offered therapies [28][29][30][31][32]. Its specific cytotoxic activity in human cancer cells is summarized in this overview.…”