Bcl-xL antisense oligonucleotides radiosensitise colon cancer cells

Colorectal carcinoma is the second leading cause of cancer death in Western countries with an incidence rate of 1 : 3000 (Midgley and Kerr, 1999; Greenlee et al, 2000). Surgical resection is the first choice of therapy for localised tumours, but at least 40% of patients with colorectal cancer will develop local recurrence or metastases during the course of the disease. For patients with advanced colorectal cancer, adjuvant chemotherapy and/or ionising radiation (IR) offer a small but significant survival advantage (Midgley and Kerr, 1999; Wils et al, 2001). While in the US postoperative (chemo)radiotherapy is considered the adjuvant treatment of choice, most European investigators have advocated for preoperative intensive short-course irradiation instead (Wils et al, 2001). Nevertheless, irrespective of the therapeutic strategy selected, advanced colorectal cancer remains a prime example for poor response to adjuvant treatment due to low sensitivity to both IR and chemotherapy. The mechanisms responsible for the resistance of this malignancy to IR or chemotherapeutic drugs are not yet fully understood. Apoptosis is currently a subject of intense research, and there is growing evidence that tumour cells, at least in part, die by apoptosis in response to IR or cytotoxic treatments (Desoize, 1994; Huang et al, 1997; Coultas and Strasser, 2000; Evan and Vousden, 2001; Reed, 2001). The members of the Bcl-2 multigene family are a pivotal set of apoptotic regulators that consist of partially interacting proteins highly conserved from nematodes to mammals (Kroemer, 1997; Antonsson and Martinou, 2000; Tsujimoto and Shimizu, 2000). Among the various Bcl-like proteins, the effects and functions of Bcl-x in controlling apoptosis induced by IR or chemotherapy have been studied recently. The Bcl-x gene is a Bcl-2 homologue and plays an important role in the regulation of programmed cell death in a variety of tissues (Xerri et al, 1998; Tsujimoto and Shimizu, 2000). Bcl-x is alternatively spliced into two mRNAs. The protein product of the larger Bcl-x mRNA (Bcl-xL) functions as a repressor of programmed cell death (Kroemer, 1997), whereas the smaller splicing product Bcl-xS, encodes a protein capable of accelerating cell death (Antonsson and Martinou, 2000; Tsujimoto and Shimizu, 2000). While it becomes increasingly clear that the two close relatives Bcl-2 and Bcl-xL show different cellular expression patterns and may complement each other's antiapoptotic function, the exact mechanisms of action remain unclear (Kroemer and Reed, 2000; Robertson and Orrenius, 2000). The antiapoptotic effects of Bcl-xL against IR- and chemotherapy-induced apoptosis have been demonstrated in various human cancer cell lines (Huang et al, 1997; Amarante-Mendes et al, 1998; Nagane et al, 1998; Srinivasan et al, 1998). The most pronounced effects were observed in cells containing the highest levels of Bcl-xL expression. Antisense (AS) oligonucleotides are modified single-strand stretches of nucleotides capable of inhibiting protein expression by complexing with the complementary target mRNA preventing translation. Antisense oligonucleotides hold great promise as agents for specific manipulation of gene expression and have been used to inhibit gene expression both in vitro and in vivo (Kitada et al, 1994; Keith et al, 1995). Bcl-xL downregulation by AS oligonucleotides has been observed in different types of cancer cells leading to an increase in susceptibility to apoptotic stimuli (Amarante-Mendes et al, 1998; Lebedeva et al, 2000). Recently, it was shown that Bcl-xL AS oligonucleotides are capable of sensitising colon cancer cells in vitro to 5-fluorouracil (Nita et al, 2000). Furthermore, bcl-2/bcl-xL bispecific oligonucleotides significantly reduced Bcl-xL expression that leads to increased apoptosis and delayed tumour growth in a xenotransplantation model for colon cancer (Gautschi et al, 2001). Taylor et al (1999) demonstrated specific downregulation of Bcl-xL by AS oligonucleotides (ISIS 16009) in keratinocytes and epithelial cells and sensitisation to UV-B radiation- and cisplatin-induced apoptosis. However, the effect of Bcl-xL AS oligonucleotides on radiosensitivity of colon cancer has not yet been explored. Given the overexpression of Bcl-xL protein in more than 60% of human colon cancers (Krajewska et al, 1996; Maurer et al, 1998) and its positive correlation with poor prognosis (Biroccio et al, 2001), we hypothesised that downregulation by Bcl-xL by AS oligonucleotides may sensitise colon cancer cells to IR or cisplatin.