Potentiation of Photodynamic Therapy by Ursodeoxycholic Acid
By Michelle Castelli and David Kessel, PhD
Photodynamic therapy (PDT) is a procedure used to bring about selective photodamage to neoplastic tissues. PDT involves the use of photosensitizing agents that tend to localize somewhat selectively in neoplastic tissues, tumor vasculature, and other sites that are spared from toxicity, since irradiation is needed to "activate" these agents. The determinants of localization are not yet clear. PDT also has been used for treatment of vascular diseases, e.g., atherosclerotic plaque and macular degeneration. A general review of the field was recently published.1 Several photosensitizing agents have been identified with the sufficient selectivity for pathologic vs. normal tissues to be clinically useful. Irradiation of cells treated with one class of sensitizers results in a rapid loss of the mitochondrial membrane potential (Dym). This is accompanied by the translocation of cytochrome c to the cytosol, followed by the prompt initiation of an apoptotic response.2 The resulting mode of cell death is mediated by the pathway leading to caspase 3 activation initially identified by Wang’s group.3 We reported that an important target for this class of photosensitizing agents is the anti-apoptotic protein bcl-2.4 A recent report indicated that the efficacy of this group of photosensitizers could be promoted by a bile acid in current clinical use for other indications.5 Another group of photosensitizing agents targets lysosomes for photodamage.6 Preliminary studies indicate that the photodynamic properties of these agents are not promoted by bile acids.
Background
The bile acid UDCA (ursodeoxycholic acid) commonly is used for the solubilization of gallstones and in the treatment of biliary cirrhosis.7 UDCA has been shown to protect hepatocytes, hepatoma, osteogenic sarcoma, and HeLa cells from apoptosis induced by a variety of stimuli, including okadaic acid, hydrogen peroxide, ethanol, and deoxycholic acid.8 In the latter case, UDCA prevented both loss of Dym and release of cytochrome c from mitochondria into the cytosol.9,10 These results suggest that UDCA can protect mitochondria from adverse effects that may lead to the opening of the mitochondrial pore and the release of cytochrome c, a trigger for the apoptotic program.
Deoxycholate is a more hydrophobic analog of UDCA. The ability of deoxycholate to elicit an apoptotic response10 could be a result of the amphipathic properties of this agent, since exposure of cells to detergents such as Triton X-100 also results in apoptotic death.11 If the release of cytochrome c is mediated by chaotropic interactions with the mitochondrial membrane, the protective effect of UDCA could be derived from competition with more hydrophobic agents for mitochondrial binding sites. Such a competition could not, however, explain the protection from hydrogen peroxide and ethanol.
Based on the ability of UDCA to protect mitochondria from stimuli that lead to an apoptotic response, we considered it highly probable that this agent also would protect cells from apoptosis induced by photodynamic therapy.
Experimental Approach
To evaluate the potential for UDCA-induced changes in PDT phototoxicity, we carried out studies using two different murine neoplastic cell lines, the L1210 lymphoblastic leukemia, and the 1c1c7 hepatoma.5 In contrast to data previously reported, we found that UDCA enhanced the cytotoxic response to PDT. At levels as low as 20 µM, a substantial increase in PDT efficacy by UDCA was observed when cells were sensitized with agents that target bcl-2 for photodamage.6 Treatment with UDCA enhanced caspase-3 activation, appearance of apoptotic morphology, and loss of cell viability after irradiation. UDCA promoted loss of Dym and release of cytochrome c into the cytosol after photodamage. Controls were carried out to demonstrate that UDCA alone, at levels as high as 100 µM, did not result in cytochrome c release into the cytosol or any loss of cell viability.
It is known that the administration of UDCA in man results in a substantial conversion of UDCA to the glycine and taurine conjugates GUDCA and TUDCA, respectively.12 We considered it important to evaluate these potential detoxification products for activity. Neither GUDCA nor TUDCA was cytostatic or cytotoxic to L1210 cells, but both conjugates were as active as UDCA in potentiating the cytotoxic effects of PDT.5 Such a result is interpreted to mean that UDCA catabolism will not decrease the effectiveness of the product.
The promotion of PDT efficacy could be explained if UDCA acted to lower the threshold for photodamage to bcl-2. This could occur if an interaction between UDCA and bcl-2 resulted in a conformational change such that certain regions of the protein were better exposed to the photosensitizers. A second possibility is that UDCA alters the mitochondrial pore, resulting in an enhanced interaction with the pro-apoptotic protein bax. It has been established that bax is not affected by PDT under conditions where bcl-2 is sufficiently altered so as not to be detectable on Western blot.4 It also is possible that the interaction between UDCA and the mitochondrial membrane results in the promotion of sensitizer binding. In this case, direct photodamage to the membrane could result in release of cytochrome c without any intermediate steps.
Conclusions
Photodynamic therapy currently is being investigated as a means for selective tumor eradication.1 It has been demonstrated that UDCA can promote the phototoxic response to PDT when used in conjunction with photosensitizing agents that initially catalyze alterations in the bcl-2 molecule. A variety of clinically-useful agents fall into this class, including Photofrin, protoporphyrin derived from administration of 5-aminolevulinic acid,13 m-tetrahydroxyphenyl-chlorin (mTHPC), and the etiopurpurin SnET2.1 These agents have either received FDA approval for photodynamic therapy or are in clinical trials.
Several other procedures have been suggested for enhancing the efficacy of PDT, including fractionated light dose14 and hyper-oxygenation of tissues.15 The use of UDCA may be a simpler approach to this same end. Since UDCA has a long history of clinical safety,7 addition of this agent to a clinical protocol might present minimal challenge with regard to potential adverse reactions, although the effect on selectivity remains to be established. It is noteworthy that the taurine and glycine conjugates of UDCA also enhance PDT efficacy, since metabolism of UDCA to these conjugates readily occurs in man.16 Further studies with animal models are underway to determine whether UDCA pharmacokinetics will be suitable for promotion of PDT responses.
Photodynamic therapy is known to cause loss of viability of malignant cells by several mechanisms.1 These include direct cell kill, vascular shutdown, and the evoking an enhanced immunologic response. Direct cell kill can eradicate, at best, two tumor logs, meaning that seven doublings will restore the initial tumor mass. Vascular shutdown likely provides the additional cell kill that accounts for the tumor eradication commonly seen after PDT. Immunologic phenomena are now being examined as an additional factor in PDT efficacy. At this stage in the investigation, it is apparent that one effect of UDCA is the enhancement of the direct tumor cell kill after PDT. Effects on vascular shutdown and immune effects remain to be explored. (Dr. Kessel is a Professor of Pharmacology and Medicine, and Ms. Castelli is a graduate student in the Cancer Biology Program, Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI.)
References
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