Mechanisms by which the bile acid UDCA enhances the efficacy of Bcl-2 targeting photodynamic therapy

The study was designed to explore the mechanism whereby the bile acid ursodeoxycholic acid (UDCA) promotes the efficacy of photodynamic therapy (PDT) directed against the anti-apoptotic protein Bcl-2. The hypothesis being examined was that UDCA either promoted Bcl-2 photodamage or enhanced interactions between the pro-apoptotic protein Bax and the mitochondrial membrane subsequent to Bcl-2 inactivation. Using photosensitizers with known targets, we established that UDCA only promoted phototoxicity of agents that targeted the endoplasmic reticulum (ER), resulting in loss of the Bcl-2 signal on western blots. These results suggested that Bcl-2, localized in the ER, was the pertinent PDT target. UDCA also promoted the pro-apoptotic effects of HA14-1, a new drug that inactivates anti-apoptotic Bcl-2 family members by binding to the hydrophobic cleft of these proteins. Fluorescence polarization (P) studies had previously been used to identify HA14-1, by using a fluorescent peptide (flu-BAK) that mimicked the BH3 portion of the Bak protein. Binding of flu-BAK to Bcl-2, leads to an increase in P; subsequent addition of HA14-1 caused P to decrease as flu-BAK was displaced from this binding site. UDCA enhanced this displacement, consistent with an enhanced Bcl-2:HA14-1 affinity. Fluorescence resonance energy transfer (FRET) was used to determine whether the affinity of Bcl-2 for an ER photosensitizer was also promoted by UDCA. Liposomes were prepared containing recombinant Bcl-2 and the ER-sensitizer termed CPO. Under FRET conditions, there was a detectable fluorescence signal arising from CPO when the liposomes were excited with 280 nm light, a wavelength that excites Bcl-2 but not CPO fluorescence. Addition of UDCA resulted in a doubling of the FRET signal, indicating that CPO:Bcl-2 proximity was enhanced. Bax:mitochondrial interactions were promoted by UDCA, but this resulted from an increase in Bcl-2 photodamage rather than from increased affinity of Bax for the mitochondrial membrane. We conclude that the mode of action of UDCA involves a conformational change in Bcl-2 resulting in enhanced affinity for certain photosensitizing agents, leading to an increased sensitization of Bcl-2 to photo-inactivation.