Cyclophosphamide induces caspase 9-dependent apoptosis in 9L tumor cells

Cyclophosphamide (CPA), a widely used oxazaphosphorine anti-cancer prodrug, is inactive until it is metabolized by cytochrome P450 to yield phosphoram ide mustard and acrolein, which alkylate DNA and proteins, respectively. Tu mor cells transduced with the human cytochrome P450 gene CYP2B6 are greatly sensitized to CPA, however, the pathway of CPA-induced cell death is unkno wn. The present study investigates the cytotoxic events induced by CPA in 9 L gliosarcoma cells retrovirally transduced with CYP2B6, or induced in wild -type 9L cells treated with mafosfamide (MFA) or 4-hydroperoxyifosfamide (4 OOH-IFA), chemically activated forms of CPA and its isomer ifosfamide. CPA and MFA were both shown to effect tumor cell death by stimulating apoptosis , as evidenced by the induction of plasma membrane blebbing, DNA fragmentat ion, and cleavage of the caspase 3 and caspase 7 substrate poly(ADP-ribose) polymerase (PARP) in drug-treated cells. Caspase 9 was identified as the r egulatory upstream caspase activated in 9L cells treated with CPA, MFA, or 4OOH-IFA, implicating the mitochondrial apoptotic pathway in oxazaphosphori ne-induced tumor cell death. Correspondingly, expression of the mitochondri al proapoptotic factor Bax enhanced caspase 9 activation, plasma membrane b lebbing, and drug-induced cytotoxicity. Conversely, overexpression of the m itochondrial antiapoptotic factor Bcl-2 blocked caspase 9 activation, leadi ng to an inhibition of drug-induced plasma membrane permeability and blebbi ng, terminal deoxynucleotidyl transferase dUTP nick-end labeling positivity , PARP cleavage, Annexin V positivity, and drug-induced cell death. Althoug h Bcl-2 thus blocked the cytotoxic effects of activated CPA, it did not inh ibit the drug's cytostatic effects. CPA induced S-phase cell cycle arrest f ollowed by conversion to an apoptotic pre-G, state in wild-type 9L cells; b y contrast, Bcl-2-expressing 9L cells accumulated in G(2)/M in response to CPA treatment. Intratumoral expression of Bcl-2 and related family members, including both apoptotic and antiapoptotic factors, is thus an important d eterminant of the responsiveness of tumor cells to CPA and ifosfamide, both in the context of conventional chemotherapy and in patients sensitized to these oxazaphosphorine drugs by the use of cytochrome P450-based gene thera py.