Rachel L. Morris

The role of apoptosis in response to photodynamic therapy: what, where, why, and how

Photodynamic therapy (PDT), a treatment for cancer and for certain benign conditions, utilizes a photosensitizer and light to produce reactive oxygen in cells. PDT is primarily employed to kill tumor and other abnormal cells, so it is important to ask how this occurs. Many of the photosensitizers currently in clinical or pre-clinical studies of PDT localize in or have a major influence on mitochondria, and PDT is a strong inducer of apoptosis in many situations. The purpose of this review is to critically evaluate all of the recently published research on PDT-induced apoptosis, with a focus on studies providing mechanistic insights. Components of the mechanism whereby PDT causes cells to undergo apoptosis are becoming understood, as are the influences of several signal transduction pathways on the response. Future research should be directed to elucidating the role(s) of the multiple steps in apoptosis in directing damaged cells to an apoptotic vs. necrotic pathway and for producing tumor ablation in conjunction with tissue-level mechanisms operating in vivo.

The Peripheral Benzodiazepine Receptor in Photodynamic Therapy with the Phthalocyanine Photosensitizer Pc 4

Abstract The peripheral benzodiazepine receptor (PBR) is an 18 kDa protein of the outer mitochondrial membrane that interacts with the voltage-dependent anion channel and may participate in formation of the permeability transition pore. The physiological role of PBR is reflected in the high-affinity binding of endogenous ligands that are metabolites of both cholesterol and heme. Certain porphyrin precursors of heme can be photosensitizers for photodynamic therapy (PDT), which depends on visible light activation of porphyrin-related macrocycles. Because the apparent binding affinity of a series of porphyrin analogs for PBR paralleled their ability to photoinactivate cells, PBR has been proposed as the molecular target for porphyrin-derived photocytotoxicity. The phthalocyanine (Pc) photosensitizer Pc 4 accumulates in mitochondria and structurally resembles porphyrins. Therefore, we tested the relevance of PBR binding on Pc 4–PDT. Binding affinity was measured by competition with 3H-PK11195, a high-affinity ligand of PBR, for binding to rat kidney mitochondria (RKM) or intact Chinese hamster ovary (CHO) cells. To assess the binding of the Pc directly, we synthesized 14C-labeled Pc 4 and found that whereas Pc 4 was a competitive inhibitor of 3H-PK11195 binding to the PBR, PK11195 did not inhibit the binding of 14C–Pc 4 to RKM. Further, 14C–Pc 4 binding to RKM showed no evidence of saturation up to 10 μM. Finally, when Pc 4–loaded CHO cells were exposed to activating red light, apoptosis was induced; Pc 4–PDT was less effective in causing apoptosis in a companion cell line overexpressing the antiapoptotic protein Bcl-2. For both cell lines, PK11195 inhibited PDT-induced apoptosis; however, the inhibition was transient and did not extend to overall cell death, as determined by clonogenic assay. The results demonstrate (1) the presence of low-affinity binding sites for Pc 4 on PBR; (2) the presence of multiple binding sites for Pc 4 in RKM and CHO cells other than those that influence PK11195 binding; and (3) the ability of high supersaturating levels of PK11195 to transiently inhibit apoptosis initiated by Pc 4–PDT, with less influence on overall cell killing. We conclude that the binding of Pc 4 to PBR is less relevant to the photocytotoxicity of Pc 4–PDT than are other mitochondrial events, such as photodamage to Bcl-2 and that the observed inhibition of Pc 4–PDT–induced apoptosis by PK11195 likely occurs through a mechanism independent of PBR.

Registration of micro-PET and high-resolution MR images of mice for monitoring photodynamic therapy

We are investigating imaging techniques to study the rapid biochemical and physiological response of tumors to photodynamic therapy (PDT). Positron emission tomography (PET) can provide physiological and functional images of cancers. While MRI can provide high resolution anatomical images and generate serial, noninvasive, in vivo observations of morphological changes. In this study, we investigate image registration methods to combine MRI and micro-PET (μPET) images for improved tumor monitoring. We acquired high resolution MR and PET 18F-fluorodeoxyglucose (FDG) images from mice with RIF-1 tumors. We used rigid body registration with three translations and three angular variables. We used normalized mutual information as the similarity measure. To assess the quality of registration, we performed slice by slice review of both image volumes, manually segmented feature organs such as the left and right kidneys and the bladder in each slice, and computed the distance between corresponding centroids of the organs. We also used visual inspection techniques such as color overlay displays. Over 40 volume registration experiments were performed with MR and μPET images acquired from three C3H mice. The color overlays showed that the MR images and the PET images matched well. The distance between corresponding centroids of organs was 1.5 ± 0.4 mm which is about 2 pixels of μPET. In conclusion, registration of high resolution MR and μPET images of mice may be useful to combine anatomical and functional information that could be used for the potential application in photodynamic therapy.

Photodynamic Therapy-Induced Apoptosis

Photodynamic therapy (PDT) is a novel treatment for cancer and certain non-malignant conditions, which employs a photosensitive drug followed by light in the visible range to produce an oxidative stress and cell death in the targeted tissue. The photosensitizers (PSs) are most commonly porphyrins or related hydrophobic macrocycles that localize in one or more intracellular membranes and that are activated by long-wavelength (red) visible light. Singlet molecular oxygen and other reactive oxygen species are the primary damaging species produced by PDT, and these oxidize cellular substrates, including lipids and proteins of the membranes in which the PS resides. PDT is an efficient inducer of apoptosis; this has been demonstrated in many cell types and with many different PSs. Once induced, apoptosis follows recognized paths but most commonly the intrinsic (mitochondrial) pathway, mediated by activated caspases and resulting in DNA fragmentation and morphological apoptosis. The aspects of apoptosis that are unique to PDT are the molecular targets, the types of initial cellular damage, and the immediate consequences of that damage. Many PSs target mitochondria, resulting in changes in the permeability transition pore complex, the apoptotic proteins Bcl-2 and Bcl-xL, and/or phospholipids, especially cardiolipin. Some PSs also target the endoplasmic reticulum (ER), damaging calcium pumps and resulting in the efflux of stored calcium into the cytosol and subsequently mitochondria. With PSs that localize in lysosomes, photoactivation damages the lysosomal membrane, causing release of cathepsins and other factors that can activate apoptosis mediators such as Bid that in turn promote mitochondrial apoptosis. Although the plasma membrane is not commonly a direct target of most PSs, it can be secondarily affected by the release of ligands of membrane receptors, such as Fas ligand. Evidence supporting the roles of each of the organelles in PDT-induced apoptosis are reviewed, and a model is proposed in which mitochondrial damage by PDT directly activates apoptosis and damage to the ER and/or the lysosome promotes the central mitochondrial pathway of apoptosis.

From molecular PDT damage to cellular PDT responses: attempts at bridging the gap on the role of Bcl-2

Expression of the anti-apoptotic proteins Bcl-2 and/or Bcl-xL is greatly elevated in many advanced cancers, especially those resistant to standard therapies, such as radiation or chemotherapy. It has been suggested that those two proteins would be attractive targets for the development of new cancer treatments. Photodynamic therapy (PDT) with photosensitizers that localize in or target mitochondria, such as the phthalocyanine Pc 4, specifically attack the anti-apoptotic protein Bcl-2, generating a variety of oxidized, complexed, and cleaved photoproducts. The closely related protein Bcl-xL is also a target of Pc 4-PDT. In a recent study employing transient transfection of an expression vector encoding deletion mutants of Bcl-2, we identified the membrane anchorage regions of the protein that are required to form the photosensitive target. In spite of the demonstrated photodamage to Bcl-2 (and Bcl-xL), how the photodamage translates into changes in the sensitivity of cells to PDT-induced apoptosis or other modes of cell death is not clear, and it also remains unclear how elevated amounts of anti-apoptotic proteins in tumors might make them more or less responsive to PDT. In the present study, we have studied the PDT response of MCF7 human breast cancer cells overexpressing wild-type Bcl-2 or certain deletion mutants either in a transient or stable mode. We show that cells expressing modestly elevated amounts (<10-fold increase) of Bcl-2 and in which the pro-apoptotic protein Bax is not upregulated do not differ from the parental cells with respect to PDT-induced cell killing. In contrast, cells expressing higher amounts (>50-fold increase) of Bcl-2 or certain mutants are made significantly more resistant to the induction of apoptosis and the loss of clonogenicity upon exposure to Pc 4-PDT. In the presence of high levels of Bcl-2, extensive photodamage requires higher PDT doses. We conclude that Pc 4-PDT targets Bcl-2 and Bcl-xL, eliminating one mechanism that protects the tumor cells from other types of therapy. However, it is possible that cells expressing very high levels of the anti-apoptotic proteins might still be resistant to PDT. The data suggest that PDT with a non-vascular-targeting photosensitizer might be effective in a combination treatment in which Bcl-2 and Bcl-xL are first photodamaged before delivery of a second agent.

Pc 4 photodynamic therapy of U87 (human glioma) orthotopic tumor in nude rat brain

Introduction: Photodynamic therapy (PDT) for Barrett’s esophagus, advanced esophageal cancer, and both early and late inoperable lung carcinoma is now FDA-approved using the first generation photosensitizer PhotofrinTM (Axcan Pharma, Birmingham, AL). Photofrin-mediated PDT of glioma is now in Phase III clinical trials. A variety of second generation photosensitizers have been developed to provide improved: (1) specificity for the target tissue, (2) tumoricidal capability, and (3) rapid clearance the vascular compartment, skin, and eyes. The phthalocyanine Pc 4 is a second generation photosensitizer that is in early phase I clinical trials for skin cancer. We have undertaken a preclinical study that seeks to determine if Pc 4-mediated PDT can be of benefit for the intra-operative localization and treatment of glioma. Methods: Using a stereotactic frame, 250,000 U87 cells were injected via Hamilton syringe through a craniotomy, and the dura, 1-2 mm below the cortical surface of nude (athymic) rat brains (N=91). The craniotomy was filled with a piece of surgical PVC and the scalp closed. After two weeks of tumor growth, the animals received 0.5 mg/kg Pc 4 via tail vein injection. One day later the scalp was re-incised, and the PVC removed. The tumor was then illuminated with either 5 or 30 Joule/cm2 of 672-nm light from a diode laser at 50 mW/cm2. The animals were sacrificed one day later and the brain was cold-perfused with formaldehyde. Two thirds of the explanted brains are now being histologically surveyed for necrosis after staining with hematoxylin and eosin and for apoptosis via immunohistochemistry (i.e., TUNEL assay). The other third were analyzed by HPLC-mass spectrometry for the presence of drug in tumor, normal brain, and plasma at sacrifice. Initial histological results show PDT-induced apoptosis and necrosis confined to the growing (live) portion of the tumor. Preliminary analysis shows an average selectivity of Pc 4 uptake in the bulk tumor to be 3.8 times greater than in normal brain tissue. Discussion: The observed specificity and tumoricidal activity of Pc 4 warrants further preclinical studies to determine the preferred Pc 4 drug and light dose for future glioma patient clinical trials.

Considerations on the role of cardiolipin in cellular responses to PDT

Cardiolipin is a unique phospholipid containing two phosphatidyl glycerol moieties and four fatty acids per molecule. It is found exclusively in the mitochondrial inner membrane and at the contact sites between the inner and outer membranes. The acridine derivative, nonyl-acridine orange (NAO), is a highly specific probe of cardiolipin, with a binding affinity approximately two orders of magnitude greater than that for binding to other anionic phospholipids. We recently reported that when NAO is bound in the mitochondria of human prostate cancer PC-3 cells and activated at 488 nm, NAO could transfer fluorescence resonance energy to the phthalocyanine photosensitizer Pc 4. This observation indicates that one site of Pc 4 binding is very near to NAO and therefore very near to cardiolipin. The average distance between the two fluorophores was calculated to be 7 nm. In the present study, we have extended the observation to the endogenously synthesized photosensitizer, protoporphyrin IX, an intermediate in heme biosynthesis that is used for photodynamic therapy of several types of malignant and non-malignant conditions. Protoporphyrin IX is generated in the mitochondria but is known to bind to other cellular sites as well, especially the endoplasmic reticulum. The ability of this molecule to accept resonance energy from NAO in cells is consistent with a localization of at least some of the molecules in the mitochondria either on the inner membrane, the site of cardiolipin, or within about 10 nm of it. Since protoporphyrin IX binds with high affinity to the peripheral benzodiazepine receptor, a component of the permeability transition pore complex that forms at contact sites between the inner and outer membranes, our observations provide evidence for the close association of several critical molecules for mitochondrial functions and suggest that cardiolipin may be an early oxidative target during PDT with at least two photosensitizers.

Mitochondrial targets of photodynamic therapy and their contribution to cell death

In response to photodynamic therapy (PDT), many cells in culture or within experimental tumors are eliminated by apoptosis. PDT with photosensitizers that localize in or target mitochondria, such as the phthalocyanine Pc 4, causes prompt release of cytochrome c into the cytoplasm and activation of caspases-9 and -3, among other caspases, that are responsible for initiating cell degradation. Some cells appear resistant to apoptosis after PDT; however, if they have sustained sufficient damage, they will die by a necrotic process or through a different apoptotic pathway. In the case of PDT, the distinction between apoptosis and necrosis may be less important than the mechanism that triggers both processes, since critical lethal damage appears to occur during treatment and does not require the major steps in apoptosis to be expressed. We earlier showed, for example, that human breast cancer MCF-7 cells that lack caspase-3 are resistant to the induction of apoptosis by PDT, but are just as sensitive to the loss of clonogenicity as MCF-7 cells stably expressing transfected procaspase-3. Many photosensitizers that target mitochondria specifically attack the anti-apoptotic protein Bcl-2, generating a variety of crosslinked and cleaved photoproducts. Recent evidence suggests that the closely related protein Bcl-xL is also a target of Pc 4-PDT. Transient transfection of an expression vector encoding deletion mutants of Bcl-2 have identified the critical sensitive site in the protein that is required for photodamage. This region contains two alpha helices that form a secondary membrane anchorage site and are thought to be responsible for pore formation by Bcl-2. As specific protein targets are identified, we are becoming better able to model the critical events in PDT-induced cell death.

PDT-induced apoptosis: what are the critical molecular targets

Early molecular damages have been studied in a series of human tumor and rodent cell lines following photodynamic therapy (PDT) sensitized by the silicon phthalocyanine Pc 4. Pc 4 preferentially localizes in mitochondria, and upon photoirradiation, immediate photodamage to the anti-apoptotic oncoprotein Bcl-2 is observed. The loss of the native 26-kDa protein, as evidenced by western blot analysis, is accompanied by the generation of a 23-kDa fragment from a small portion of the molecules as well as a variety of higher molecular weight protein bands indicative of photochemical crosslinking of Bcl-2 to itself, to (pro-apoptotic) homologs, or to other nearby proteins. The changes in Bcl-2 are apparent immediately upon exposure of Pc 4-loaded cells to activating red light, occur in the cold, and are not dependent upon caspase-3 or other proteases. Crosslinking is also observed for the intermediate filament protein vimentin. The results implicate Bcl-2 (and perhaps vimentin) as important molecular targets that lead to apoptosis in Pc 4-PDT-treated cells.