Abstract
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.
Highlights
Photodynamic Therapy (PDT) is a relatively new anti-cancer technique in which a photolabile molecule is allowed to selectively accumulate in cancer cells, followed by illumination with high-intensity light to activate the photosensitizer and kill the cells [1, 2]
Differentiation therapy is associated with upregulation of CCAAT-enhancer binding proteins (C/EBPs), coproporphyrinogen oxidase (CPO) gene expression, and protoporphyrin IX (PpIX) production in tumors in vivo
We had previously shown that when subcutaneous A431 squamous tumors in mice (Fig. 1A) are preconditioned for 3 days with calcitriol (Vit D) and given aminolevulinic acid (ALA) for 4 hr, the PpIX photosensitizer accumulates to higher levels than in the absence of Vit D, causing more tumor cell death upon exposure to light [10]
Summary
Photodynamic Therapy (PDT) is a relatively new anti-cancer technique in which a photolabile molecule is allowed to selectively accumulate in cancer cells, followed by illumination with high-intensity light to activate the photosensitizer and kill the cells [1, 2]. One useful approach is to administer 5-aminolevulinic acid (ALA), a pro-drug selectively taken up by cancer cells and metabolically converted into intracellular porphyrins [3]. Porphyrins, mainly protoporphyrin IX (PpIX), are efficient absorbers of visible light. PpIX is synthesized from ALA by the sequential action of eight enzymes in the heme-synthetic pathway [4], including coproporphyrinogen oxidase (CPO, sixth enzyme in the cascade). CPO lies upstream of PpIX, and is of particular interest because CPO is rate-limiting for PpIX production under typical PDT conditions [5]
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