The efficacy of
photodynamic therapy (
PDT) for epithelial
cancers is increased when
PDT is combined with
calcitriol (Vit D), a form of differentiation
therapy (DT). Here, we describe an underlying mechanism for this effect. Differentiation-promoting agents are known to upregulate
CCAAT/enhancer-binding proteins (C/EBP), powerful regulators of cellular differentiation. In subcutaneous A431
tumors in mice, pretreatment with Vit D induced the expression of C/EBPβ
isoforms, and of
coproporphyrinogen oxidase (CPO), a
heme pathway
enzyme responsible for the conversion of 5-aminolevulinic
acid (ALA) into
protoporphyrin IX (
PpIX), the principal light-absorbing molecule during
PDT. To further investigate this apparent link between C/EBPs and CPO, two cell lines (MEL and LNCaP) were exposed to differentiating agents, and levels of
PpIX, C/EBPs, and CPO were measured. Differentiating agents, or transfection of C/EBP expression vectors, increased C/EBP and CPO levels in parallel. Focusing on approximately 1,300 bp of upstream CPO gene promoter, we tested the ability of recombinant C/EBPα, C/EBPβ, C/EBPδ, and C/EBPζ to bind to CPO gene sequences [electrophoretic mobility shift assay (EMSA) assays] and to affect transcriptional activity (
luciferase assays). Multiple C/EBP consensus binding sites were identified (15 for mouse, 18 for human). Individual probes representing each site bound to C/EBPs with characteristic affinities (strong, moderate, or weak), but when sites were inactivated in the context of the native promoter, transcriptional activity was reduced nearly equally for strong or weak sites. Cooperative interactions between regularly spaced C/EBP sites seem critical for CPO transcriptional regulation by differentiation
therapy. These results provide a mechanistic rationale for DT/
PDT combination
therapy for
cancer.