Inducible Nitric Oxide Synthase Neutralizes Carbamoylating Potential of 1 , 3-Bis ( 2-chloroethyl )-1-nitrosourea in C 6 Glioma Cells

Expression of iNOS in glioma and other tumors has been extensively documented but the effects of NO derived from iNOS on tumor-killing mechanisms of chemotherapy drugs remain to be fully defined. We note that increased NO synthesis by cytokine exposure or iNOS overexpression neutralized the cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), but not cisplatin, in rat C6 glioma cells. Suppression of BCNU cytotoxicity associated with iNOS overexpression could be abolished by pharmacological inhibition of NOS or coexpression of an antisense RNA against iNOS. Both BCNU and CCNU are chloroethylnitrosoureas that kill tumor cells via carbamoylating and alkylating actions. Further studies using compounds that each carry these different activities indicate that iNOS neutralized carbamoylating, but not alkylating, action of chloroethylnitrosoureas. Temozolomide, a novel chemotherapy drug recently available for treating brain tumors, carries only alkylating, but not carbamoylating, action. Overexpression of iNOS in C6 cells failed to neutralize temozolomide cytotoxicity. Results from the present study demonstrate the ability of iNOS-derived NO to confer chemoresistance against the carbamoylating potential of chloroethylnitrosoureas in vitro. Further investigation is needed to test whether iNOS expression, frequently noted in malignant brain tumors, also enhances chemoresistance against chloroethylnitrosoureas in vivo. Glioblastoma multiforme (GBM) is the most common type of primary brain tumor accounting for more than 40% of neoplasm in the central nervous system (Kleihues et al., 1995). It carries poor prognosis despite multimodality approaches consisting of surgical resection, radiotherapy, and chemotherapy (Fine, 1994). The life expectancy of patients with GBM is usually less than 1 year from the time of diagnosis. To date, the 5-year survival remains at 1% (Davis et al., 1998). Surgery alone gave a median survival length of 14 weeks. Radiotherapy following surgical resection extended this figure to 36 weeks. Combination of surgery, radiotherapy, and chemotherapy prolonged life expectancy to approximately 14 months (Rajkumar et al., 1999). BCNU is the most commonly used adjunct chemotherapy for GBM because of its lipophilic character. Unfortunately, BCNU did not substantially prolong median survival, even though the proportion of patients living more than 18 months increased from 5 to 15% with chemotherapy (Walker et al., 1980; Green et al., 1983; Fine et al., 1993). A recent analysis of results from two Brain Tumor Study Group protocols reaffirms a modest effect of adjunct chemotherapy in increasing longterm survival (DeAngelis et al., 1998). GBM develops chemoresistance against BCNU. Several mechanisms have been proposed that may account for BCNU chemoresistance in GBM. These include an increase in the synthesis of the reduced form of glutathione (Ali-Osman et al., 1990), DNA mismatch repair and O6-alkylguanine-DNA alkyltransferase expression (Friedman et al., 1998). Interventions that enhance chemosensitivity of glioma cells to BCNU may improve