Abstract
An increased rate of cellular proliferation is a hallmark of cancer and may be accompanied by an increase in ribosome biogenesis and dysregulation in rRNA synthesis. In this regard, CX-5461 has been developed as a novel RNA polymerase I inhibitor and is currently in Phase I/II clinical trials for solid and hematological malignancies. In the present study, interactions between CX-5461 and single-dose X-ray exposure were assessed using isobologram analysis using MTS assay and drug-induced cell death was assessed using flow cytometric, confocal microscopy and Western blot analysis. Combination treatments involving CX-5461 and single-dose X-ray exposure highlighted increased effectiveness compared to individual treatment alone in the CaSki cervical cancer line, with marked synergistic interaction occurring within the low-drug (50 nM) and low-dose radiation range (2–6 Gy). Cell lines challenged with CX-5461 demonstrated the presence of DNA damage, induction of apoptosis, autophagy and senescence alongside high percentages of G2/M cell cycle arrest. In addition, we report preferential sensitivity of ovarian cancer cells with BRCA2 mutation to this novel agent. Taken together, CX-5461 displayed a broad spectrum of activity in a panel of solid cancer cell lines with IC50 values ranging from 35 nM to >1 µM. The work described herein identifies the synergistic effects of CX-5461 in combination with X-rays in solid cancers and may also aid in the design of clinical trials involving this novel agent.
Highlights
The nucleolus is a prominent subcellular organelle involved in a wide range of cellular processes [1,2]
Using the MTS assay with continuous exposure of CX-5461, a panel of 14 solid tumour cell lines investigated (Figure 1A) gave IC50 values within the nanomolar range (Table 1), The sensitivity of these solid cancer cell lines was not subject to p53 status, as shown in Table 1; CX-5461 did seem to be more selective towards PEO1 cell line harboring a BRCA2 mutation compared to its
The selectivity of CX-5461 towards RNA POL I transcription was confirmed using qPCR in CaSki cell line, where the transcription of 45s pre-rRNA was found to decrease in a dose-dependent manner over the course of 72 h (p-value < 0.0001) (Figure 1D)
Summary
The nucleolus is a prominent subcellular organelle involved in a wide range of cellular processes [1,2]. The primary function of the nucleolus is to transcribe and process the 45s pre rRNA transcripts which are located along tandem repeats within nucleolar organizer regions (NORs) located within the short arms of acrocentric chromosomes. The 45s pre rRNA transcripts are processed into the small and large ribosomal subunits within the nucleolus, which are exported into the cytoplasm and assembled with RNAs and riboproteins to form fully assembled ribosomes. RNA polymerase I is exclusively dedicated to the transcription of 45s rRNA genes and has recently become the centre of attention for a number of selective agents aimed at inhibiting the transcription of rRNA for cancer intervention [3,4]. Several approved anticancer agents have been shown to inhibit rRNA synthesis but these drugs mechanistically do not target the RNA Polymerase I machinery such as cisplatin, 5-FU and actinomycin Cancers 2019, 11, 1429 with adverse prognosis in cancer [5] and the increase in the number of nucleoli, as well as their size within individual nuclei, has been established as a marker for tumour aggressiveness [6].
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