Binding to and photo-oxidation of cardiolipin by the phthalocyanine photosensitizer Pc 4

Abstract

Cardiolipin is a unique phospholipid of the mitochondrial inner membrane. Its peroxidation correlates with release of cytochrome c and induction of apoptosis. The phthalocyanine photosensitizer Pc 4 binds preferentially to the mitochondria and endoplasmic reticulum. Earlier Förster resonance energy transfer studies showed colocalization of Pc 4 and cardiolipin, which suggests cardiolipin as a target of photodynamic therapy (PDT) with Pc 4. Using liposomes as membrane models, we find that Pc 4 binds to cardiolipin-containing liposomes similarly to those that do not contain cardiolipin. Pc 4 binding is also studied in MCF-7c3 cells and those whose cardiolipin content was reduced by treatment with palmitate. Decreased levels of cardiolipin are quantified by thin-layer chromatography. The similar level of binding of Pc 4 to cells, irrespective of palmitate treatment, supports the lack of specificity of Pc 4 binding. Thus, factors other than cardiolipin are likely responsible for the preferential localization of Pc 4 in mitochondria. Nonetheless, cardiolipin within liposomes is readily oxidized by Pc 4 and light, yielding apparently mono- and dihydroperoxidized cardiolipin. If similar products result from exposure of cells to Pc 4-PDT, they could be part of the early events leading to apoptosis following Pc 4-PDT.

Figure 1

Increase in Pc 4 fluorescence intensity with increasing liposome concentration. In this example, the liposomes contained 45% DMPC, 45% DMPE, 10% CL and [Pc 4]=6 μM. (a) Fluorescence spectra of Pc 4 in PBS with no lipid or increasing lipid concentration [L]=0.102; 0.412; 0.630; 0.889; 1.120 and 1.5510 mM, and (b) titration curve for no lipid up to [L]=2.5 mM.

Figure 2

Fluorescence spectra of [Pc 4]=6 μM in (dashed line) liposomes at 0% CL and [L]=2.23 mM and (solid line) liposomes at 20% CL and [L]=1.33 mM.

Figure 3

Influence of mitochondrial membrane potential on the binding of Pc 4 to mitochondria. (A) Confocal images of intracellular distribution/accumulation of Pc 4. MCF-7c3 cells were plated at 2×10⁵ per 35-mm dish, allowed to attach overnight (16 to 18 h), and then treated for 1 h in one of three ways: (a) and (b) untreated cells, (c) and (d) cells heated at 65°C, and (e) and (f) cells incubated with CCCP (10 μM). After that, the medium was removed and the cultures were incubated 1 h with Pc 4 (200 nM) in a 37°C incubator. All cells were washed twice in PBS and overlaid with phenol red-free Hank’s balanced salt solution prior to imaging. (B) Mitochondrial membrane potential visualized by confocal microscopy. Cells were grown as already described, then loaded with 100 nM TMRM for 30 min at 37°C. The medium was removed, and the cells were kept in 10 nM TMRM in calcium buffer. After recording the first image, 10 μM CCCP was added to the cells in calcium buffer containing TMRM (10 nM). Control cells received 10 nM TMRM in calcium buffer but without CCCP. Imaging was performed at 5-s intervals over a period of 1 min or until depolarization was observed. Upper panel from left to right: (a) cells before adding CCCP, 10 μM; (b) and (c) 48 s and 1 min after adding CCCP, respectively; (d) 1 min after adding CCCP in a different field. Lower panel from left to right: control cells (e) 0, (f) 1, and (g) 2 min, after the start of imaging; and (h) 8 min after the start of imaging but in a different field.

Figure 4

(A) Mitochondrial membrane potential visualized by confocal microscopy. Cells were grown as described in methods for 48 h, then loaded with 100 nM TMRM for 30 min at 37°C. The medium was removed, and the cells were kept in 10 nM TMRM in calcium buffer. After recording the first image, 200 nM CCCP was added to the cells in calcium buffer containing TMRM (10 nM). Imaging was performed at 5-s intervals until depolarization was observed: (a) cells before adding CCCP; (b) and (c) 48 s and 1 min, respectively, after adding CCCP; and (d) 1 min after adding CCCP in a different field. (B) Flow cytometric evaluation of the effect of heat on cells. Graphs of side scatter (SSC) versus forward scatter (FSC) of (a) control cells kept at 37°C and (b) cells after 1 h at 65°C. Dead cells have lower FSC and higher SSC than living cells.

Figure 5

Separation of cellular lipids by TLC. MCF-7c3 cells were treated as described in Sec. 2. Phospholipids were extracted with chloroform/methanol (2:1, v/v) from cells suspended in saturated saline solution. After that, the organic phase was recovered and dried under nitrogen. Each dried extract was dissolved in 25 μL chloroform and applied to a silica TLC plate. The mobile phase consisted of methanol/chloroform/acetic acid/water (3∕0.52∕0.36∕0.12). TLC was developed by iodine vapors to detect total lipid. Commercial CL was analyzed similarly as a standard. Lanes are 1, control cells; 2 and 3, palmitate-treated cells; 4, control cells plus addition of CL standard; and 5, CL standard. CL, cardiolipin; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine.

Figure 6

ESI-MS spectra of CL extracted from liposomes. Liposomes with Pc 4 were prepared as described in Sec. 2. Control liposomes were kept in the dark in the presence of Pc 4, while experimental liposomes were irradiated with 100 mW∕cm² red light at room temperature for 40 min. The peak at m∕z 1447.8 is [M-H⁺]⁻ of tetralinoleoyl CL.