Bcl-x(L) antisense oligonucleotides radiosensitise colon cancer cells

Abstract

Advanced colon cancer is a malignancy with poor response to various treatment modalities including ionising radiation (IR) and chemotherapy. Both IR and chemotherapeutic agents have been shown to act by inducing apoptosis, a type of cell death antagonised by the Bcl-x(L) gene product. Since approximately 60% of human colon cancers express Bcl-x(L), it was the aim of this study to explore the potential of Bcl-x(L) antisense oligonucleotides as a novel radiosensitisation strategy. Caco-2 colon cancer cells were treated with Bcl-x(L) antisense oligonucleotides in combination with IR or cisplatin, and Bcl-x(L) protein expression, apoptosis, cell viability and clonogenic survival were examined. Bcl-x(L) antisense oligonucleotide specifically reduced the Bcl-x(L) protein level by almost 50% in Caco-2 cells. The decreased threshold for the induction of apoptosis resulted in a 300% increase of apoptosis after IR or cisplatin treatment and led to a 60% reduction of cell proliferation beyond response rates achieved with IR. These data suggest that Bcl-x(L) is an important factor contributing to the treatment resistance of human colon cancer. Specific reduction of Bcl-x(L) protein levels by antisense oligonucleotides qualifies as a promising therapeutic strategy for colon cancer that may help overcome resistance and improve clinical outcome in this malignancy.

Figure 1

Screening of Bcl-x_L AS oligonucleotides: Western blots of Caco-2 cells 48 h (A) and 72 h (B) after treatment with four different AS oligonucleotides at a concentration of 50 μM; lane 1: saline (Sal), lane 2: ISIS 22783, lane 3: ISIS 16009, lane 4: ISIS 16011, lane 5: ISIS 15999, and lane 6: saline (Sal).

Figure 2

Bcl-x_L downregulation by Bcl-x_L AS oligonucleotides (ISIS 16009): Western blot of Caco-2 cells 48 h after a 4-h treatment with 200 nm oligonucleotides in the presence of 10 μg ml⁻¹ lipofectin; lane 1: saline (Sal), lane 2: ISIS 16009 Bcl-x_L AS oligonucleotides (AS), lane 3: 8-base mismatch oligonucleotides (MM). A representative blot of four independent experiments is presented.

Figure 3

Bcl-x_L AS oligonucleotides facilitate the induction of apoptosis in human colon cancer cells. Caco-2 cancer cells were incubated for 4 h with saline (Sal), antisense (AS), or eight-base mismatch (MM) oligonucleotides at a concentration of 200 nM in the presence of 10 μg ml⁻¹ lipofectin. After 48 h, cells were treated with increasing doses of IR (0–12 Gy). At 96 h after oligonucleotide treatment, cells were harvested and analysed by FACS for apoptosis. Columns represent mean percentages of apoptotic cell death from four independent experiments; bars=s.d.

Figure 4

Bcl-x_L AS oligonucleotides radiosensitise human colon cancer cells. Time course of Caco-2 cells incubated with saline (Sal), antisense- (AS), or eight-base mismatch (MM) oligonucleotides at a concentration of 200 nM (A) alone, (B) in combination with IR (12 Gy 48 h after oligonucleotides). (C) Dose–response experiment of Caco-2 pretreated with saline (Sal), antisense (AS), or mismatch (MM) oligonucleotides at 200 nM and exposed to increasing IR doses (0–12 Gy). Cell viability was measured 96 h after oligonucleotide treatment by WST-1 assay. Representative data from four independent experiments are presented; bars=s.d.

Figure 5

Bcl-x_L AS oligonucleotides decrease clonogenic survival of human colon cancer cells after ionising irradiation. Caco-2 cells were incubated with saline (Sal), antisense (AS), or eight-base mismatch (MM) oligonucleotides at a concentration of 200 nM in the presence of 10 μg ml⁻¹ lipofectin and irradiated 48 h later. Survival was assessed by colony-forming assay and expressed relative to solvent-treated cells. Columns represent mean percentages from three independent experiments; bars=s.d.