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Photodynamic Tumor Therapy: Mitochondrial Benzodiazepine Receptors as a Therapeutic Target

Abstract

Background

Photodynamic therapy employs photosensitive agents such as porphyrins to treat a variety of tumors accessible to light-emitting probes. This approach capitalizes on the selective retention of porphyrins by cancer cells. Cancer cells also have elevated levels of mitochondrial benzodiazepine receptors which bind porphyrins with high affinity.

Methods

Cultured cancer cell lines were exposed to porphyrin and porphyrin-like compounds and then irradiated with light. Cytotoxicity of this treatment was measured via clonogenic assays. Mitochondrial benzodiazepine receptor pharmacology was studied using [3H] PK11195 binding to cancer cell homogenates and isolated kidney mitochondrial membranes.

Results

We show that therapeutic potencies of porphyrins correlate closely with affinities for mitochondrial benzodiazepine receptors. Sensitivities of tumor cell lines to photodynamic therapy parallel their densities of these receptors.

Conclusion

We propose that porphyrin photodynamic therapy is mediated by mitochondrial benzodiazepine receptors.

Introduction

Photodynamic therapy (PDT) employs the dyesensitized photooxidation of biological matter to treat various conditions, especially tumors that are accessible to light probes, such as skin, bladder, vaginal, bronchial, and rectal cancers (14). Although the molecular mechanism for therapeutic actions for PDT has not been established, substantial evidence indicates a prominent role for mitochondria. Porphyrins, the class of photosensitive dyes most often employed, tend to concentrate in mitochondria (5,6). Moreover, damage to mitochondrial function is one of the earliest events in porphyrin PDT (79). We speculated that mitochondrial benzodiazepine receptors (MBR) might play a role in the mechanism of PDT. MBR are distinct from the neuronal “central” benzodiazepine receptors but can bind clinically employed benzodiazepines with high affinity. MBR are localized to mitochondria where they comprise a complex of an 18 kilodalton (kD) receptor protein, the 32 kD voltage-dependent anion carrier (VDAC), and the 30 kD adenine nucleotide carrier (1014). Porphyrins represent endogenous ligands for MBR. In tissue extracts, porphyrins are the only substances that bind with nanomolar affinity to MBR (1517). Porphyrin PDT targets mitochondrial membranes where MBR are localized (18). Incubation of isolated mitochondria with porphyrins and light results in the oxidation of sulfhydryl groups in specific membrane proteins with molecular weights similar to MBR constituents (19). Moreover, loss of ATP/ADP exchange via the adenine nucleotide carrier is a major early step in porphyrin photosensitization of isolated mitochondria (20). In the present study, we show that the relative potencies of porphyrins in eliciting photodynamic killing of tumor cells correlate closely with their affinities for MBR. Moreover, the relative sensitivities of tumor cell lines to PDT parallel their MBR levels.

Materials and Methods

Materials

Cells were obtained from American Tissue Culture Collection (Rockville, MD). [3H] PK11195 was obtained from NEN/Dupont (Boston, MA).

Porphyrins were obtained from Porphyrin Products (Logan, UT). PK11195 was a gift from Pharmuka and R05-4864 was a gift from Hoffman-LaRoche. All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO).

Cell Culture

Cells were maintained at 37°C in a humidified CO2 incubator in the following media: mouse L cells were grown in MEM plus 10% horse serum; C6 glioma cells were grown in Ham’s F-10 plus 15% horse serum and 2.5% fetal bovine serum (FBS); V79 were grown in MEM plus 10% FBS; HeLa, WiDR, and LS174T cells were grown in MEM plus nonessential amino acids and 10% FBS; and SVEC 4–10 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (low glucose) plus 10% FBS. For clonogenic survival experiments, cells were plated at appropriate densities as suspensions approximately 12 hr before drug/light exposure.

MBR Assays

Rat kidney mitochondria were prepared as described (16). Cell homogenates used for binding studies were prepared by removing culture media, rinsing with phosphate buffered saline (PBS), and homogenizing in 10 mM HEPES (pH, 7.4) using a Dounce B homogenizer with 10 to 15 strokes at 4°C. Protein was determined by Coomassie Blue protein assay (BioRad, Hercules, CA) and adjusted to a final value of 0.5 mg/ml in the binding assay. IC50 and Ki values for porphyrins and analogs were determined by displacement of [3H] PK11195 binding to cell homogenates and isolated rat kidney mitochondria, respectively, as described earlier (16). MBR densities of the various cell lines were determined by performing Scatchard analyses on [3H] PK11195 saturation isotherms for each cell line as reported earlier (16). For [3H] PK11195 binding studies in live cells, confluent cultures in 60-mm dishes were used. After medium removal and rinsing with PBS, live cell monolayers were incubated in 2 ml PBS containing 2 nM [3H] PK11195 with and without 10 µM PK11195 or varying concentrations of Photofrin II at 37°C for 1 hr. Cells were scraped from the dishes, collected over GF/C glass fiber filters, and washed three times with ice-cold PBS. The filters were counted for radioactivity in scintillation cocktail.

Phototoxicity Studies

All cultured cells were plated at a density of 300 to 10,000 per dish. Cells were exposed to varying concentrations of porphyrins and analogs in DMEM for 4 hr. After a brief wash with PBS, cells were irradiated for 60 sec with a broad spectrum light from a Kratos lamp at 60 cm distance and cultured in DMEM. Clonogenic assessment for cell survival was performed after 7 to 14 days of cell growth. D37 values represent concentrations that reduce survival to 37% of control.

Results

To explore a role for MBR in porphyrin phototoxicity we first determined whether V79 cell homogenates display high-affinity [3H] PK11195 binding with an MBR pharmacological profile. [3H] PK11195 bound to a single site on V79 homogenates with a Kd of 1.2 nM and Bmax of 3.9 pmol/mg protein. Binding of 1 nM [3H] PK11195 to V79 homogenates was potently displaced by unlabeled PK11195, R05-4864, and protoporphyrin IX, which are all high-affinity ligands for MBR. Diazepam, a mixed MBR and central benzodiazepine receptor (CBR) ligand. showed intermediate inhibitory potency, whereas the CBR-specific ligands, clonazepam and RO15-1788, were without effect (Fig. 1).

Fig. 1
figure 1

Pharmacologie profile of [3H] PK11195 binding to V79 cell homogenates. Binding studies performed as described in Materials and Methods demonstrate the presence of MBR in V79 cells. Key for symbols: PK11195 (filled circles), R05-4864 (open circles), protoporphyrin (PP) IX (filled triangles), diazepam (open triangles), clonazepam (filled squares), R015-1788 (open squares).

[3H] PK11195 binding to V79 homogenates was also potently inhibited by Photofrin II, a clinically employed tumor photosensitizing dye (largely composed of di-hematoporphyrin ether), but not by three other nonporphyrin phototoxic dyes—rhodamine 6G (21), rhodamine 123 (22), and merocyanine (23) (Fig. 2). Photofrin II also inhibited [3H] PK11195 binding to live V79 cells (Fig. 2). Similar Ki values for Photofrin II were found for rat kidney mitochondria (Table 1) and other cell lines (data not shown). In a series of 27 porphyrin derivatives, relative potencies in eliciting photodynamic cell toxicity in the V79 cells correlated very closely with their affinities for MBR (p < 0.001).

Fig. 2
figure 2

Inhibition of [3H] PK11195 binding to V79 cell homogenates and live cells (Photofrin only) by photosensitive dyes. Binding studies performed as described in Materials and Methods demonstrate the selective interaction of the porphyrin phototoxic dye, Photofrin II, with MBR in homogenized and live V79 cells. Key for symbols: rhodamine 6G (open circles), rhodamine 123 (filled circles), merocyanine (open triangles), Photofrin II with V79 homogenates (filled squares), Photofrin II with live V79 cells (open squares).

Table 1 Relationship of porphyrin phototoxicity and MBR affinity

If PDT involves MBR, then tumor cell lines with higher densities of MBR should be more sensitive. We evaluated the sensitivity to porphyrin photodynamic toxicity in 8 cell lines (Table 2). The relative sensitivity closely paralleled MBR density in these cell lines.

Table 2 Relationship of MBR density and PDT susceptibility in cell lines

Discussion

Our most striking findings are two strong correlations that together implicate MBR as playing a major role in PDT. First, we observed a close correlation between the potencies of porphyrins to catalyze cellular phototoxicity and their affinity for MBR, establishing a structure-activity profile for porphyrins and porphyrin-like compounds to mediate phototoxicity. Second, we observed that MBR density in tumors parallels their susceptibility to porphyrin-catalyzed phototoxicity. Such data suggest that mitochondrial damage is the proximal event that leads to observed cell death. This conclusion is supported by recent studies by Munday et al. (24), who show that mammalian cells deficient in mitochondria are refractory to porphyrin-catalyzed phototoxicity.

How might MBR mediate PDT? MBR comprises a complex of proteins apparently located at junctional sites of outer and inner membrane of mitochondria (11,12). A molecular complex with such a localization could influence transactions between cytosolic and mitochondrial compartments. For example, hexokinase partitions from cytosol onto the outer mitochondrial surface where it binds to VDAC (25,26). Interactions of VDAC with the inner membrane adenine nucleotide carrier may afford a path for exchange of mitochondrial ATP with ADP generated by hexokinase (26). This would provide direct ATP supply to hexokinase and yield improved mitochondrial coupling through kinase-generated ADP. Tumor cells display especially high levels of hexokinase attached to mitochondria (2527), which might account for the high rate of “aerobic glycolysis” seen in many neoplastic cells. Porphyrin photooxidation of MBR-associated VDAC or adenine nucleotide carrier could thus profoundly affect tumor bioenergetics.

Recent findings have implicated MBR in the transport of porphyrins in and out of mitochondria (28). Porphyrins used in photodynamic therapy may thus enter cells and accumulate on mitochondrial porphyrin transport sites. Porphyrins with the highest affinity for MBR would be expected to accumulate the most in this manner. This may explain the suggested role of MBR in delta-aminolevulinic acid (ALA)-mediated phototoxicity in pancreatoma cells (29), as ALA phototoxicity requires conversion to protoporphyrin IX prior to its phototoxic effects.

The ability of MBR to modulate ion conductances (30) and cholesterol movement across mitochondrial membranes (31) implies a substantial role for MBR in regulating mitochondrial compartmentation. Thus, the adenine nucleotide carrier is believed to mediate the nonspecific increase in mitochondrial inner membrane permeability observed after Ca2+ overloading and oxidative injury to mitochondria (32). This protein is labeled by the alkylating benzodiazepine [3H] AHN086 (11) which causes a large release of calcium from isolated mitochondria (33). We hypothesize that porphyrins, when bound to MBR and exposed to light, generate radical oxygen species that oxidize the adenine nucleotide carrier to increase mitochondrial permeability. This would account for the rapid loss of adenine nucleotide exchange and Ca2+ release from mitochondria following porphyrin PDT (9,20,34) and associated cytotoxicity. The oxidative sensitivity displayed by the adenine nucleotide carrier (35) supports our hypothesis and indeed may implicate the MBR complex in diverse pathological and physiological phenomena.

Our findings have clear clinical relevance. Some benzodiazepines and related agents have extremely high affinity for MBR in the low nanomolar range and, like porphyrins (36), radiolabeled forms of these agents have been employed to localize neoplasms in vivo (37). Photosensitive compounds with high affinity for MBR may afford more specific PDT agents with fewer side effects. Such compounds may also be readily screened for using binding studies with [3H] PK11195 and isolated mitochondria. Interestingly, MBR levels are increased in several types of tumors (38,39,40).

References

  1. Spikes JD, Jori L. (1987) Photodynamic therapy of tumors and other diseases using porphyrins. Lasers Med. Sd. 2:3–15.

    Article  Google Scholar 

  2. Moan J. (1988) Porphyrin photosensitization and phototherapy. Photochem. Photobiol. 6:681–690.

    Google Scholar 

  3. Brown SB, Kessel D. (1990) Cancer and porphyrin photochemotherapy. Mol. Aspects Med. 11:99–111.

    Article  Google Scholar 

  4. Pass HI. (1993) Photodynamic therapy in oncology: mechanisms and clinical use. J. Natl. Cancer Inst. 85:443–456.

    Article  CAS  PubMed  Google Scholar 

  5. Berns SB, Dahlman A, Johnson FM, et al. (1982) In vitro cellular effects of hematoporphyrin derivative. Cancer Res. 42: 2325–2329.

    PubMed  CAS  Google Scholar 

  6. Roberts WG, Berns MW. (1989) In vitro photosensitization I. Cellular uptake and subcellular localization of mono-L-aspartyl chlorine6, chloro-aluminum sulfonated phthalocyanine, and Photofrin n. Lasers Surg. Med. 9:90–101.

    Article  CAS  PubMed  Google Scholar 

  7. Perlin DS, Murant RS, Gibson SL, Hilf R. (1985) Effects of photosensitization by hematoporphyrin derivative on mitochondrial adenosine triphosphate-mediated proton transport and membrane integrity of R3230AC mammary adenocarcinoma. Cancer Res. 45:653–658.

    PubMed  CAS  Google Scholar 

  8. Hilf R, Murant RS, Narayanan V, Gibson SL. (1986) Relationship of mitochondrial function and cellular adenosine triphosphate levels to hematoporphyrin derivative-induced photosensitization in R3230AC mammary tumors. Cancer Res. 46:211–217.

    PubMed  CAS  Google Scholar 

  9. Salet C. (1986) Hematoporphyrin and hematoporphyrin-derivative photosensitization of mitochondria. Biochemie 68:865–866.

    Article  CAS  Google Scholar 

  10. Krueger KE. (1991) Peripheral type benzodiazepine receptors: A second site of action for benzodiazepines. Neuropharmacology 4:1417–1423.

    Google Scholar 

  11. Verma A, Snyder SH. (1989) Peripheral type benzodiazepine receptors. Annu. Rev. Pharmacol. Toxicol. 29:307–322.

    Article  CAS  PubMed  Google Scholar 

  12. McEnery MW, Snowman AM, Trifiletti RR, Snyder SH. (1992) Isolation of the mitochondrial benzodiazepine receptor: Association with the voltage-dependent anion channel and the adenine nucleotide carrier. Proc. Natl. Acad. Sci. U.S.A. 89: 3170–3174.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Krueger KE, Mukhin AG, Antkiewicz-Michaluk L, et al. (1990) Purification, cloning and expression of a peripheral type benzodiazepine receptor. Adv. Biochem. 46:1–13.

    CAS  Google Scholar 

  14. Doble A, Burgevin MC, Meager J, et al. (1987) Partial purification and pharmacology of peripheral type benzodiazepine receptors. J. Recept. Res. 7:55–70.

    Article  CAS  PubMed  Google Scholar 

  15. Verma A, Nye JS, Snyder SH. (1987) Porphyrins are endogenous ligands for the mitochondrial (peripheral type) benzodiazepine receptor. Proc. Natl. Acad. Sci. U.S.A. 84: 2256–2260.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Verma A, Snyder SH. (1988) Characterization of porphyrin interactions with peripheral-type benzodiazepine receptors. Mol. Pharmacol. 34:800–805.

    PubMed  CAS  Google Scholar 

  17. Cantoni L, Rizzardini M, Skorupska M, et al. (1992) Hepatic protoporphyria is associated with a decrease in ligand binding for the mitochondrial benzodiazepine receptors in the liver. Biochem. Pharmacol. 44: 1159–1164.

    Article  CAS  PubMed  Google Scholar 

  18. Hilf R, Warne NW, Smail DB, Gibson SL. (1984) Photodynamic inactivation of selected intracellular enzymes by hematoporphyrin derivatives and their relationship to tumor cell viability in vitro. Cancer Lett. 24: 165–172.

    Article  CAS  PubMed  Google Scholar 

  19. Yamamoto K, Kawanishi S. (1991) Oxidation of specific S4 protein of mitochondria by photodynamic action of hematoporphyrin. Biochem. Pharmacol. 42:1087–1092.

    Article  CAS  PubMed  Google Scholar 

  20. Atlante A, Passarella S, Quagliariello E, Moreno G, Salet C. (1989) Hematoporphyrin derivative (Photofrin II) photosensitization of isolated mitochondria: Inhibition of ADP/ATP translocator. J. Photochem. Photobiol. 4:35–46.

    Article  CAS  Google Scholar 

  21. Saetzler RK, Jallo J, Lehr HA, Phillips CM, Vasthare U, Arfors KE, Tuma RF. (1997) Intravital fluorescence microscopy: Impact of light-induced phototoxicity on adhesion of fluorescently labeled leukocytes. J. Histochem. Cytochem. 45:505–513.

    Article  CAS  PubMed  Google Scholar 

  22. Shea CR, Sherwood ME, Flotte TJ, Chen N, Scholz M, Hasan T. (1990) Rhodamine 123 phototoxicity in laser-irradiated MGH-U1 human carcinoma cells studied in vitro by electron microscopy and confocal laser scanning microscopy. Cancer Res. 50:4167–4172.

    PubMed  CAS  Google Scholar 

  23. Gulliya KS, Matthews JL, Fay JK, Dowben RM. (1988) Increased survival of normal cells during laser photodynamic therapy: implications for ex vivo autologous bone marrow purging. Life Sci. 42:2651–2656.

    Article  CAS  PubMed  Google Scholar 

  24. Munday AD, Sriratana A, Hill JS, Kahl SB, Nagley P. (1996) Mitochondria are the functional intracellular target for a photosensitizing boronated porphyrin. Biochem. Biophys. Acta 1411:1–4.

    Article  Google Scholar 

  25. Fiek C, Benz R, Roos N, Brdiczka D. (1982) Evidence for identity between the hexokinase-binding protein and the mitochondrial porin in the outer membrane of rat-liver mitochondria. Biochim. Biophys. Acta 688:429–440.

    Article  CAS  PubMed  Google Scholar 

  26. Nelson BD, Kabir F. (1986) The role of the mitochondrial outer membrane in energy metabolism of tumor cells. Biochemie 68:407–415.

    Article  CAS  Google Scholar 

  27. Nakashima RA, Scott LJ, Pedersen PL. (1986) The role of mitochondrial hexokinase binding in the abnormal energy metabolism of tumor cell lines. Ann. N. Y. Acad. Sci. 488:438–450.

    Article  CAS  PubMed  Google Scholar 

  28. Taketani S, Kohno H, Furukawa T, Tokunaga R. (1995) Involvement of peripheral-type benzodiazepine receptors in the intracellular transport of heme and porphyrins. J. Biochem. 117:875–880.

    Article  CAS  PubMed  Google Scholar 

  29. Ratcliffe SL, Matthews EK. (1995) Modification of the photodynamic action of delta-aminolaevulinic acid (ALA) on rat pancreatoma cells by mitochondrial benzodiazepine receptor ligands. Br. J. Cancer 71:300–305.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Kinnally KW, Zorov DB, Antonenk YN, Snyder SH, McEnery MW, Tedeschi H. (1993) Mitochondrial benzodiazepine receptor linked to inner membrane ion channels by nanomolar actions of ligands. Proc. Natl. Acad. Sci. U.S.A. 90:1374–1348.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Krueger KE, Papadopolous V. (1990) Peripheral-type benzodiazepine receptors mediate translocation of cholesterol from outer to inner mitochondrial membranes in adrenocortical cells. J. Biol. Chem. 265:15015–15022.

    PubMed  CAS  Google Scholar 

  32. LeQuoc K, LeQuoc D. (1988) Involvement of the ADP/ATP carrier in calcium-induced perturbation of the mitochondrial inner membrane permeability: Importance of the orientation of the nucleotide binding sites. Arch. Biochem. 265:249–257.

    Article  CAS  Google Scholar 

  33. Moreno SR, Brako C, Gutierrez J, Newman AH, Chiang PK. (1991) Release of Ca2+ from heart and kidney mitochondria by peripheral-type benzodiazepine receptor ligands. Int. J. Biochem. 23:207–213.

    Article  Google Scholar 

  34. Salet C, Moreno G, Vinzens F. (1983) Effects of photodynamic action on energy coupling of Ca2+ uptake in liver mitochondria. Biochem. Biophys. Res. Commun. 115:76–81.

    Article  CAS  PubMed  Google Scholar 

  35. Zwizinski CW, Hho S. (1992) Peroxidative damage to cardiac mitochondria; Identification and purification of modified adenine nucleotide translocase. Arch. Biochem. Biophys. 294:178–183.

    Article  CAS  PubMed  Google Scholar 

  36. Hill JS, Kohl SB, Kaye AH, et al. (1992) Selective tumor uptake of a boronated porphyrin in an animal model of cerebral glioma. Proc. Natl. Acad. Sci. U.S.A. 89:1785–1789.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Starosta-Rabinstein S, Ciliax BJ, Penney JB, McKeever P, Young AB. (1987) Imaging of a glioma using peripheral benzodiazepine receptor ligands. Proc. Natl. Acad. Sci. U.S.A. 84:891–895.

    Article  Google Scholar 

  38. Katz Y, Ben-Baruch G, Kloog Y, Menczer J, and Gavish M. (1990) Increased density of peripheral benzodiazepine-binding sites in ovarian carcinomas as compared with benign ovarian tumours and normal ovaries. Clin. Sci. (Colch.) 78:155–158.

    Article  CAS  Google Scholar 

  39. Katz Y, Eitan A, and Gavish M. (1990) Increase in peripheral benzodiazepine binding sites in colonic adenocarcinoma. Oncology 47:139–142.

    Article  CAS  PubMed  Google Scholar 

  40. Batra S, and Alenfall J. (1994) Characterization of peripheral benzodiazepine receptors in rat prostatic adenocarcinoma. Prostate 24:269–278.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by USPHS grant DA-00266, Research Scientist Award DA-00074 to S.H.S., ES-07076 to J.R.W., and a grant of the W.M. Keck Foundation (S.H.S.). A.V. was supported by a grant from The Defense and Veterans Head Injury Program.

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The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Army or the Department of Defense.

Communicated by S. H. Snyder.

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Verma, A., Facchina, S.L., Hirsch, D.J. et al. Photodynamic Tumor Therapy: Mitochondrial Benzodiazepine Receptors as a Therapeutic Target. Mol Med 4, 40–45 (1998). https://doi.org/10.1007/BF03401728

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Keywords

  • Mitochondrial Benzodiazepine Receptor (MBR)
  • Photodynamic Therapy
  • PK11195 Binding
  • Porphyrin-like Compounds
  • Porphyrin PDT