cAMP-dependent phosphorylation of CYP2B1 as a functional switch for cyclophosphamide activation and its hormonal controlin vitro andin vivo

Abstract

An important feature of cytochrome P450 (CYP) 2B1 is its high ability to convert the prodrug cyclophosphamide (CPA) to therapeutically cytotoxic metabolites, resulting in interstrand DNA-cross-linking and cell death. We have examined whether and how the phosphorylation of CYP2B1 influences CPA metabolic activation in vitro and in vivo. We found first that only part of the total CYP2B1 pool undergoes phosphorylation. This part is fully inactivated. Second, phosphorylation of CYP2B1 in intact hepatocytes reduced by up to 75% toxification of CPA to mutagenic metabolites (totally dependent on the same preferentially CYP2B-catalyzed 4-hydroxylation of CPA as is the generation of highly cytotoxic species). Third, the phosphoacceptor-serine 128 of CYP2B1 in the consensus sequence for interaction with the protein kinase A represents an on/off switch for the activation of CPA depending on the phosphorylation conditions in the cell. Fourth, evidence is presented that the above-described events also occur in vivo. In conclusion, a successful therapy with CPA, helped by forced expression of CYP2B1 in tumor cells (as recently proposed) will, in addition, be profoundly modified by its phosphorylation status. © 2001 Wiley-Liss, Inc. Key words: cyclophosphamide; CYP2B1; phosphorylation; hormonal controlThe activity of many of the foreign compound metabolizing enzymes, e.g., CYP, is of obvious importance for both physiologic homeostasis as well as effects of drugs and environmental chemicals. 1 Much of the detoxification is performed by CYP enzymes. Many of them are inducible and have a broad and overlapping substrate specificity. 2 Interindividual differences in the expression level of CYP enzymes are known, 3 thus contributing to the variations in toxic symptoms and therapeutic response evoked by antineoplastic drugs. 4 CYP2B1 (the major phenobarbital-inducible form) has long been suggested as 1 of the most important enzymes involved in the activation of widely used antineoplastic prodrugs. It catalyzes oxidative desulfuration of thio-TEPA to TEPA as well as 1-electron reduction of Adriamycin to its semiquinone free radical [5][6][7] and, most importantly, is responsible for conversion of the oxazaphosphorines CPA and IFA to their active metabolites. 8,9 It has been shown that 3 specific rat liver CYP enzymes (CYP2B1, CYP2C6 [constitutively expressed, gender independent] and CYP2C11 [constitutively expressed, male specific]) are major catalysts of CPA activation in adult rat liver, 8,10 while a rat CYP3A enzyme additionally contributes to the activation of IFA. 9 The corresponding human enzymes CYP2B6 and CYP3A4 activate CPA and IFA in human liver, respectively. 11-13 It was demonstrated that among rat enzymes CYP2B1 (the ortholog of the human CYP2B6) is by far the most active catalyst. 8,11,12 Activation of CPA to the therapeutic active species has its initial step in the liver. 8 The CYP2B1-catalyzed 4-hydroxylation reaction forms a highly unstable metabolite, which undergoes spontaneous ␤-elimination and gives rise to 2 pro...

The Localization of Cytochrome P450s CYP1A1 and CYP1A2 into Different Lipid Microdomains Is Governed by Their N-terminal and Internal Protein Regions

“…Even though such lipid modifications have not been examined with P450, increasing evidence has shown post-translational modifications of P450, including phosphorylation, nitration, and glycosylation, suggesting that the modifications are involved in facilitating the proper catalytic function of P450 (41). For example, phosphorylation was found in many P450s, including CYP2B1, CYP2E1, and CYP3A4, and the modification resulted in modulation of enzyme activity, dual targeting of P450s to the ER and mitochondria, or degradation (42)(43)(44). Further studies are required to determine whether amino acid sequences within the identified regions are targeted for post-translational modifications that may enhance interaction of P450 with specific membrane regions.…”

The Pharmacology of the Cytochrome P450 Epoxygenase/Soluble Epoxide Hydrolase Axis in the Vasculature and Cardiovascular Disease

“…Although this sequence of events is certainly plausible, it is highly likely that additional mechanisms, such as phosphorylation, play a role in regulating P450 activity. Indeed, some P450 enzymes (CYP2B1, CYP2B2, and CYP2E1) are reported to be phosphorylated by protein kinase A (PKA), and the consequences of P450 phosphorylation range from the regulation of activity (Oesch-Bartlomowicz et al, 2001) and subcellular localization (Anandatheerthavarada et al, 1999;Korsmeyer et al, 1999) to proteasome degradation (Eliasson et al, 1992;Korsmeyer et al, 1999). Although there has been little advancement in this area over the last few years, other P450 enzymes such as the hepatic-enriched CYP3A4 and CYP2E1 are phosphorylated by PKA and protein kinase C, which accelerates their ubiquitination and endoplasmic reticulumassociated degradation (Correia et al, 2014).…”

Cyclic adenosine monophosphate involvement in low-dose cyclophosphamide-reversed immune evasion in a mouse lymphoma model

“…The cAMP signaling pathway also participates in CY metabolism in the liver. 49,50 The appropriate sequential administration of CY and cAMP analogs/PDE4B inhibitors to lymphoma patients may be an effective approach for cancer therapy.…”