A mechanism for the proapoptotic activity of ursodeoxycholic acid: effects on Bcl-2 conformation

Abstract

Ursodeoxycholic acid (UDCA), a relatively nontoxic bile acid, enhanced the apoptotic response of tumor cells to both photosensitizers that cause photodamage to Bcl-2 and to the nonpeptidic Bcl-2/Bcl-x L antagonist HA14-1. The latter agent binds to the surface pocket formed by the BH1, BH2 and BH3 domains of Bcl-2 and Bcl-x L . Fluorescence polarization studies indicated that affinity of HA14-1 for Bcl-2 was enhanced in the presence of UDCA. Moreover, Bcl-2 photodamage was promoted by UDCA using a photosensitizing agent with affinity for the endoplasmic reticulum, a site of Bcl-2 localization. Fluorescence resonance energy transfer (FRET) studies revealed that the proximity of Bcl-2 to a hydrophobic photosensitizing agent embedded in liposomes was enhanced by UDCA. Since photodamage will occur only if a protein is in close contact with a photosensitizing agent, we propose that these findings support the hypothesis that UDCA causes a conformational change in Bcl-2, promoting HA14-1 binding and enhancing affinity for certain membrane-bound photosensitizers. Cell Death and Differentiation (2004) 11, 906-914. doi:10.1038/sj.cdd.4401433 Keywords: apoptosis; Bcl-2; CPO; NPe6; SnET2; photodynamic therapy (PDT)Abbreviations: CPO, 9-capronyloxy-tetrakis(methoxyethyl) porphycene; DCA, deoxycholic acid; DEVD-R110, asp-glu-valasp-rhodamine 110 (fluorogenic caspase-3 substrate); ER, endoplasmic reticulum; flu-Bak, 5-carboxyfluorecein coupled to the N terminus of a peptide GQVGRQLAIIGDDINR derived from the BH3 domain of Bak; HA14-1, ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate; HO342, Höchst dye HO33342; mTHPC, meta-(tetrahydroxyphenyl) chlorin; NPe6, N-aspartyl chlorin e6; P, fluorescence polarization value; PDT, photodynamic therapy; SnET2, tin etiopurpurin; UDCA, ursodeoxycholic acid.

Death pathways associated with photodynamic therapy

“…This occurred in both L1210 mouse leukemia and 1c1c7 mouse hepatoma cells, using either SnET2 or CPO as the photosensitizing agent. The mechanism derived from a UDCA-induced conformational change in the Bcl-2 protein promoting affinity for the photosensitizer and thereby increasing Bcl-2 photodamage [11]. UDCA also enhanced binding to Bcl-2 by the small-molecule non-peptidic Bcl-2 antagonist HA14-1, initially described by Huang's group [12].…”

“…A persisting question relating to PDT targets was the apparent resistance of the Bcl-2 analog Bcl-x L to photodamage in L1210 murine leukemia cells in suspension culture [11]. In contrast, Oleinick's group had found that Bcl-x L was as sensitive as Bcl-2 to photodamage in adhering cells [14].…”

Protection of Bcl-2 by salubrinal

“…This substrate releases the fluorescent dye Rhodamine 110 upon enzymatic hydrolysis. The increase in fluorescence as a function of time was measured with a Fluoreskan fluorescence plate reader using 485 nm excitation and 510 nm emission.Western blots for Bcl-2, phosphorylated and non-phosphorylated eIF2α, and LC-3Cells were lysed and extracts prepared for Western blot analysis [9]. Phosphatase inhibitors (5 mM NaF, 10 mM Na 4 P 2 O 7 , and 5 mM Na 3 VO 4 ) were present during extraction procedures.…”

“…2E). These results are consistent with the proposal that salubrinal protects Bcl-2 from photodamage.A possible mechanism for the latter effect was provided by FRET analysis, using a procedure we had previously adopted for assessing the affinity of CPO for the Bcl-2 protein in a lysosomal environment [9]. Excitation of CPO at 400 nm leads to fluorescence at 642 nm, but excitation at 280 nm does not result in any detectable fluorescence emission (Fig.…”

“…3A). Upon excitation at 280 nm, the aromatic amino acids in the Bcl-2 protein fluorescence in the vicinity of 400 nm [9]. When both Bcl-2 and CPO were simultaneously present in the liposomes, the two products bind in sufficient proximity so that excitation at 280 nm yields CPO fluorescence at 642 nm (Fig.…”