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

Abstract

Background: Cellular 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.

Methods: Ovarian (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.

Results: All 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.

Conclusions: Supporting 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.

Figures

Fig. 1

Dose and time-dependent ribosomal RNA disruption in response to taxanes. A2780 and CaOV3 cells were exposed to increasing concentrations of either docetaxel (DXL) or paclitaxel (TAX) for times ranging from 24 to 72 h. Total RNA was isolated from cells following drug exposure and RNA quality was analyzed by capillary electrophoresis. Electropherograms showing RNA mobility were converted to gel images using the Bioanalyzer software. a Gel image of RNA from A2780 cells treated with paclitaxel. b RDI analysis of RNA isolated from paclitaxel treated A2780 cells. c Gel image of RNA from A2780 cells treated with docetaxel. d RDI analysis of RNA isolated from docetaxel treated A2780 cells. e Gel image of RNA isolated from CaOV3 cells treated with docetaxel. f RDI analysis of RNA isolated from CaOV3 cells treated with docetaxel

Fig. 2

Dose dependence of carboplatin-induced RNA disruption in A2780 and CaOV3 cells. A2780 and CaOV3 cells were treated with increasing concentrations of carboplatin (CBN) for 72 h, the length of time required to detect the RNA disruption response to carboplatin. Total RNA was isolated from cells following drug exposure and RNA quality was analyzed by capillary electrophoresis. a Gel image of RNA from A2780 cells treated with carboplatin. b RDI analysis of RNA isolated from carboplatin treated A2780 cells. c Gel image of RNA from CaOV3 cells treated with carboplatin. d RDI analysis of RNA isolated from carboplatin treated CaOV3 cells

Fig. 3

Multiple chemotherapy agents induce RNA disruption in breast and ovarian cancer cell lines. Multiple breast and ovarian cancer cell lines were exposed to various chemotherapy agents and total RNA was isolated from the cells following drug exposure. a Gel image of total RNA isolated from A2780 cells treated with multiple chemotherapy agents. All treatments were for 72 h, except carboplatin, which was treated for 120 h. b Gel image of RNA isolated from MDA-MB-231, a breast cancer cell line, following treatment with multiple chemotherapy agents. All treatments were for 72 h, except for the control (0 μM) and cisplatin which were treated for 96 h. c Gel image of RNA isolated from various breast (MCF-7, MB231, SKBR3, BT474) and ovarian cancer (A2780, CaOV3) cells following 72 h docetaxel treatment, except for MDA-MB-231-0 μM, from which RNA was isolated after 96 h. Abbreviations: TAX-paclitaxel, DXL-docetaxel, CBN-carboplatin, CIS-cisplatin, ETOP-etoposide, VIN-vincristine, IRN-irinotecan, DOX-doxorubicin

Fig. 4

Northern blot analysis of RNA isolated from A2780 cells treated with docetaxel. A2780 cells were treated with 0.2 μM docetaxel for 48 h and total RNA was isolated. RNA was resolved by agarose gel electrophoresis and transferred to PVDF membranes for hybridization with the 32P-end-labeled oligonucleotide probe, 28S-5. a The panel on the left shows the agarose gel and the panel on the right shows the Northern blot of the gel. RNA bands are indicated by arrows with the size of the band in nucleotides alongside. b A schematic diagram of the 28S rRNA sequence showing conserved and variable regions, based on the structure of the 28S rRNA as defined by Gorski et al. (1987) [53] and Wakeman et al. [28]. Location of the probes in the 28S rRNA sequence is shown above the diagram using arrows and the location of the cleavage sites and resulting bands are shown below the diagram

Fig. 5

Lack of RNA disruption response in drug resistant cells. A2780 and A2780DXL (resistant to docetaxel) cells were treated with 0, 0.005 and 0.2 μM docetaxel (DXL) for 48 and 72 h. RNA isolated from the cells was analyzed by capillary gel electrophoresis. A2780 and A2780CBN (resistant to carboplatin) cells were treated with 0 and 10 μM carboplatin (CBN) for 72 h. To test for cross-resistance, A2780DXL cells were treated with 0 and 5 μM carboplatin while A2780CBN cells were treated with 0 and 0.2 μM docetaxel. RNA isolated from the cells was analyzed by capillary gel electrophoresis. a RDI analysis of RNA isolated from A2780 and A2780DXL cells treated with docetaxel. b RDI analysis of RNA isolated from A2780 and A2780CBN cells treated with carboplatin. c RDI analysis of A2780DXL cells treated with 0 and 5 μM carboplatin. d RDI analysis of RNA isolated from A2780CBN cells treated with 0 and 0.2 μM docetaxel

Fig. 6

Early and late markers of apoptosis in A2780 cells treated with 0.2 μM docetaxel.A2780 cells were treated with 0.2 μM docetaxel (DXL) for 8, 24, 48 and 72 h. a Cells were stained with annexin V-FITC and propidium iodide and analyzed by flow cytometry. Scatter plots of stained cells at each time point are shown. b A2780 cells were stained with propidium iodide only and analyzed by flow cytometry. Histograms of stained cells are shown for each time point. c DNA laddering in A2780 cells treated with 0 and 0.2 μM docetaxel for times ranging from 2 to 72 h. Jurkat cells were treated with 5 and 30 μM etoposide as a positive control. DNA was isolated from cells at each time point and analyzed by agarose gel electrophoresis. A representative ethidium bromide stained gel is shown

Fig. 7

Recovery and proliferation of A2780 cells following docetaxel treatment. In order to assess whether the A2780 cells treated with docetaxel (DXL) that show RNA degradation are able to recover and proliferate, cells were treated with 0, 0.005 or 0.2 μM docetaxel for 24, 48 and 72 h. Following the treatment end point, cells were collected and replated in fresh drug-free medium, and their proliferation was assessed following recovery for 24, 48, 72 and 96 h of replating. a Recovery of cells after 24 h treatment. b Recovery after 48 h drug treatment. c Recovery following 72 h drug treatment

Fig. 8

Caspase-3 activation is associated with RNA disruption after docetaxel treatment in A2780 cells. A2780 cells were treated with 0.2 μM docetaxel (DXL) for 24, 48 or 72 h. a Caspase-3 activity, assayed using a DEVD substrate, in lysates from A2780 cells. b Effect of a caspase-3 inhibitor on caspase-3 activation in lysates from docetaxel treated A2780 cells. c Gel image of RNA disruption in A2780 cells treated with docetaxel, in the presence (+) or absence (−) of caspase-3 inhibitor (Q-DEVD-Oph). d RDI analysis of RNA from A2780 cells treated with docetaxel for 72 h, with and without the caspase-3 inhibitor