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

Abstract

conventional drug sensitivity assays used to screen prospective anti-cancer agents for cytotoxicity monitor biological processes associated with active growth and proliferation, used as proxies of cell viability. However, these assays are unable to distinguish between growth-arrested (but otherwise viable) cells and non-viable/dead cells. As a result, compounds selected based on the results of these assays may only be cytostatic, halting or slowing tumour progression temporarily, without tumour eradication. Because agents capable of killing tumour cells (cytotoxic drugs) are likely the most promising in the clinic, there is a need for drug sensitivity assays that reliably identify cytotoxic compounds that induce cell death. We recently developed a drug sensitivity assay, called the RnA disruption assay (RDA), which measures a phenomenon associated with tumour cell death. in this study, we sought to compare our assay's performance to that of current commonly used drug sensitivity assays (i.e, the clonogenic, the cell counting kit-8 and the Trypan blue exclusion assays). We found that RnA disruption occurred almost exclusively when total cell numbers decreased (cytotoxic concentrations), with little to no signal detected until cells had lost viability. in contrast, conventional assays detected a decrease in their respective drug sensitivity parameters despite cells retaining their viability, as assessed using a recovery assay. We also found that the RDA can differentiate between drug-sensitive and-resistant cells, and that it can identify agents capable of circumventing drug resistance. taken together, our study suggests that the RDA is a superior drug discovery tool, providing a unique assessment of cell death.

RNA disruption is a widespread phenomenon associated with stress-induced cell death in tumour cells

“…These different levels of RNA disruption may reflect the relative sensitivity of the cell lines to specific chemotherapy agents and stressors. Consistent with this view, we have shown a clear association between the sensitivity of specific cell lines to known chemotherapy agents (as measured using clonogenic assays) and the ability of such agents to induce RNA disruption 25 , 28 . Additional factors likely play a role in the degree of RNA disruption induced by specific chemotherapy agents or stressors, including the drug’s mechanism of action and its eventual effects on ribosomes.…”

“…Given the strong relationship between RNA disruption and cell death outlined above, the RDA is particularly apt at detecting genuine changes in cell viability. This is in contrast to many classical drug sensitivity assays which are unable to discriminate between cell cycle arrest and cell death, and whose results can be misinterpreted to mean a loss in viability 25 . Although subG1 DNA content analysis of tumour cells also offers an accurate means of quantifying cell death, this approach is not feasible when working with tumours from humans or animals, as cells within tumours are held together through adhesion to each other and to the extracellular matrix.…”

“…RNA disruption is associated with the onset of cell death. While our previous studies have shown that induction of RNA disruption by doxorubicin and cycloheximide is associated with cytotoxicity 25 , they did not assess whether RNA disruption is associated with the onset of cell death (as determined by meeting all three phenotypes commonly associated with cell death: reductions in cell numbers/cell destruction, cessation of cell replication, and the generation of cells with a subG1 DNA content). To begin this assessment, we examined a variety of chemotherapy agents for their effect on the number of cells in culture.…”

“…The effect of chemotherapy agents and other stressors on cell numbers and on cellular replicative capacity in nutrient-rich medium post-treatment was measured as described in our prior publication 25 . Briefly, in the former assay, floating and adherent cells present in cell culture flasks were counted prior to and at various time points after the addition of drug or application of the stressor.…”

“…Moreover, RNA disruption was shown to predict pCR as early as one cycle of chemotherapy in patients with HER2 + breast cancer 23 , 24 . Our most recent study has revealed that chemotherapy-induced RNA disruption in cultured A2780 ovarian tumour cells is associated with a reduction in cell numbers (cytotoxicity) and appears to be superior to the Cell Counting Kit-8 (CCK8), dye exclusion, recovery, and clonogenic assays at identifying cytotoxic drugs that promote cell destruction 25 . Nevertheless, several important questions relating to RNA disruption remain unanswered, including how widespread is the disruption phenomenon among cell types, how specific are the triggers for RNA disruption, and how tightly is it associated with cell death.…”