RNA disruption is associated with response to multiple classes of chemotherapy drugs in tumor cell lines

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.

Iron-dependent cleavage of ribosomal RNA during oxidative stress in the yeast Saccharomyces cerevisiae

“…Site-specific cleavages of rRNA have been previously observed in different organisms that experience various types of stress (12)(13)(14)(45)(46)(47). Although these cleavages were ascribed to cellular nucleases, in most cases the enzymes responsible for initiating rRNA strand breaks were not conclusively identified.…”

Doxorubicin chemotherapy affects the intracellular and interstitial free amino acid pools in skeletal muscle

“…Recent research by Narandrula et al . [ 34 ] have shown that DOX capably induced rRNA distribution in cultured cells, suggesting that the formation of reactive oxygen species (ROS) and production of tumor necrosis factor alpha (TNFα) are likely factors responsible for the degradation of rRNA. Additionally, White and colleagues [ 35 ] have reported that DOX affects the phosphorylation of elongation factor 2 (EF-2), a key enzyme in the modulation of protein elongation during synthesis, and capably affects the rate of translation [ 36 ].…”

The RNA disruption assay is superior to conventional drug sensitivity assays in detecting cytotoxic drugs

“…Our research group developed a drug sensitivity assay called the RNA disruption assay (RDA) 12 , which monitors a biological process associated with tumour cell death, rather than parameters of active proliferating cells 13 . The process, which is triggered by a wide variety of chemotherapy agents, is characterized by the degradation of ribosomal RNAs (rRNAs) into long-lived high-molecular-weight fragments 12,13 . As the resulting RNA profile is quite distinct from that generated during autolytic RNA degradation, the phenomenon was termed 'RNA disruption' 12 .…”