Abstract
BackgroundCellular stressors and apoptosis-inducing agents have been shown to induce ribosomal RNA (rRNA) degradation in eukaryotic cells. Recently, RNA degradation in vivo was observed in patients with locally advanced breast cancer, where mid-treatment tumor RNA degradation was associated with complete tumor destruction and enhanced patient survival. However, it is not clear how widespread chemotherapy induced “RNA disruption” is, the extent to which it is associated with drug response or what the underlying mechanisms are.MethodsOvarian (A2780, CaOV3) and breast (MDA-MB-231, MCF-7, BT474, SKBR3) cancer cell lines were treated with several cytotoxic chemotherapy drugs and total RNA was isolated. RNA was also prepared from docetaxel resistant A2780DXL and carboplatin resistant A2780CBN cells following drug exposure. Disruption of RNA was analyzed by capillary electrophoresis. Northern blotting was performed using probes complementary to the 28S and 18S rRNA to determine the origins of degradation bands. Apoptosis activation was assessed by flow cytometric monitoring of annexin-V and propidium iodide (PI) binding to cells and by measuring caspase-3 activation. The link between apoptosis and RNA degradation (disruption) was investigated using a caspase-3 inhibitor.ResultsAll chemotherapy drugs tested were capable of inducing similar RNA disruption patterns. Docetaxel treatment of the resistant A2780DXL cells and carboplatin treatment of the A2780CBN cells did not result in RNA disruption. Northern blotting indicated that two RNA disruption bands were derived from the 3’-end of the 28S rRNA. Annexin-V and PI staining of docetaxel treated cells, along with assessment of caspase-3 activation, showed concurrent initiation of apoptosis and RNA disruption, while inhibition of caspase-3 activity significantly reduced RNA disruption.ConclusionsSupporting the in vivo evidence, our results demonstrate that RNA disruption is induced by multiple chemotherapy agents in cell lines from different tissues and is associated with drug response. Although present, the link between apoptosis and RNA disruption is not completely understood. Evaluation of RNA disruption is thus proposed as a novel and effective biomarker to assess response to chemotherapy drugs in vitro and in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2197-1) contains supplementary material, which is available to authorized users.
Highlights
Cellular stressors and apoptosis-inducing agents have been shown to induce ribosomal ribonucleic acid (RNA) degradation in eukaryotic cells
Since the RNA integrity number (RIN) algorithm quantifies the formation of low molecular weight autolytic RNA degradation products [19] and since abnormal RNA banding patterns during RNA disruption results in the assignment of “n/a” values for RIN by the Agilent Bioanalyzer, we recently developed the RNA disruption assay (RDA), which quantifies RNA disruption in tumors and tumor cells as an RNA disruption index (RDI), and is a ratio of abnormal to normal ribosomal RNA (rRNA) [20]
RNA disruption was evident in the taxane-treated cells by the presence of abnormal bands on the electropherogram, distinct from the normal RNA banding pattern seen in untreated cells (Fig. 1a and c)
Summary
Cellular stressors and apoptosis-inducing agents have been shown to induce ribosomal RNA (rRNA) degradation in eukaryotic cells. RNA degradation in vivo was observed in patients with locally advanced breast cancer, where mid-treatment tumor RNA degradation was associated with complete tumor destruction and enhanced patient survival. It is not clear how widespread chemotherapy induced “RNA disruption” is, the extent to which it is associated with drug response or what the underlying mechanisms are. Mroczek and Kufel [16] have used the term “stress-induced rRNA fragmentation” to describe the phenomenon by which various cellular stressors activate programmed cell death pathways in yeast, some of which are associated with rRNA degradation. The apparent connection between apoptosis and rRNA degradation is not compulsory, as it was shown that rRNA cleavage could occur in the absence of caspase- and BCL-2 dependent apoptosis [17]
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