Search

Ovarian cancer is the most lethal gynecologic malignancy with a stubborn mortality rate of ~65%. The persistent failure of multiline chemotherapy, and significant tumor heterogeneity, has made it challenging to improve outcomes. A target of increasing interest is the mitochondrion because of its essential role in critical cellular functions, and the significance of metabolic adaptation in chemoresistance. This review describes mitochondrial processes, including metabolic reprogramming, mitochondrial transfer and mitochondrial dynamics in ovarian cancer progression and chemoresistance. The effect of malignant ascites, or excess peritoneal fluid, on mitochondrial function is discussed. The role of photodynamic therapy (PDT) in overcoming mitochondria-mediated resistance is presented. PDT, a photochemistry-based modality, involves the light-based activation of a photosensitizer leading to the production of short-lived reactive molecular species and spatiotemporally confined photodamage to nearby organelles and biological targets. The consequential effects range from subcytotoxic priming of target cells for increased sensitivity to subsequent treatments, such as chemotherapy, to direct cell killing. This review discusses how PDT-based approaches can address key limitations of current treatments. Specifically, an overview of the mechanisms by which PDT alters mitochondrial function, and a summary of preclinical advancements and clinical PDT experience in ovarian cancer are provided.

Two cell lines, A549 (human-derived non-small-cell lung cancer) and 1c1c7 (mouse hepatoma) were photosensitized with m-THPC and irradiated under LD90 conditions. After 4 h, a pattern of cytoplasmic vacuoles had formed consistent with the initiation of paraptosis. After irradiation, there was no detectable loss of the mitochondrial membrane potential indicating no significant photodamage to mitochondria. We did, however, observe localization of m-THPC in the endoplasmic reticulum (ER), as indicated by fluorescence microscopy. Subsequent ER perturbation is known to result in initiation of paraptosis, another pathway to cell death. While an apoptotic response to m-THPC has been reported, the ability to target ER and induce paraptosis could explain the efficacy of this agent which could therefore eradicate cell types with an impaired apoptotic response.

Photodynamic therapy (PDT) is a procedure for the selective photosensitization of neoplastic tissues. Subsequent irradiation with visible light can lead to cell death along with vascular shutdown and other responses that lead to selective eradication of malignant cells. Among the cellular responses to PDT are necrosis, apoptosis, and autophagy. These pathways have generally been associated with cell death, although autophagy can also be cytoprotective. A fourth effect that has hitherto been somewhat neglected is termed "paraptosis," a lethal response that can be identified by detecting an extensive collection of cytoplasmic vacuoles. Unlike autophagy, these vacuoles are bound by single membranes. Paraptosis has been characterized as a response to misfolded endoplasmic reticulum proteins that must be "cleared" if the affected cell is to survive. At present, there is no simple biochemical test for paraptosis. This chapter describes the procedure for detection of paraptosis using phase-contrast microscopy, along with some confirmatory approaches.

"Nobody expects the Spanish Inquisition." The color red has often been associated with danger and other adverse events. In the recent literature, the protein "KillerRed" has been proposed as a means for lethally sensitizing neoplastic cells and tissues to light. A somewhat different perspective on the feasibility of this approach is offered.

Paul Strudler who facilitated the support of photodynamic therapy by an NIH study section.

Photodynamic therapy (PDT) is a light-catalyzed process that can initiate cellular death pathways from the formation of cytotoxic reactive oxygen species at sub-cellular sites. Apoptosis was the first such pathway to be identified. Autophagy can also occur and is often found to be cytoprotective. Another process termed paraptosis can also have lethal consequences, even in cells with an impaired apoptotic pathway. PDT in vivo can evoke other potentially cytotoxic processes including vascular shutdown and enhanced immunologic recognition of neoplastic cells. Using appropriate photosensitizing agents, sub-cellular PDT targeting can be directed so that the resulting interplay among assorted death and survival pathways will result in an enhanced level of photokilling.

Photodynamic therapy can be useful for the eradication of malignant cells at sites that are accessible to light delivery. There are few adverse effects, with many clinical reports indicating that PDT has curative potential. Patients with minimal disease, where success is more likely, are also sought by those promoting other protocols. New photosensitizing agents that initiate light-catalyzed reactions continue to be discovered. Reports describing advances in understanding fundamental aspects of photobiology are always of interest. But, implications for treatment of neoplasia and other diseases are not always justified, especially when poorly penetrating wavelengths of light are employed, often at very high light doses. Efficacy is sometimes estimated by protocols that may not accurately measure photokilling. Many reports claiming potential clinical relevance for in vitro observations are based on a limited understanding of the determinants of clinical efficacy. The future of photodynamic therapy depends on an appreciation of what can be accomplished, especially when used with other modalities, but will also depend on the goals and interests of granting agencies, pharmaceutical groups, and clinical personnel. This commentary is intended to provide some thoughts on current research efforts, especially where clinical implications are suggested, hinted at or otherwise implied.

The field of photodynamic therapy has suffered some significant losses during the past decade. Among those was James Kennedy, shown at the Innsbruck IPA conference (2011).

While photodynamic therapy (PDT) is effective for the eradication of select neoplasia and certain other pathologic conditions, it has yet to achieve wide acceptance in clinical medicine. A variety of factors contribute to this situation including relations with the pharmaceutical industry that have often been problematic. Some current studies relating to photodynamic effects are 'phenomenological', i.e., they describe phenomena that only reiterate what is already known. The net result has been a tendency of granting agencies to become disillusioned with support for PDT research. This report is intended to provide some thoughts on current research efforts that improve clinical relevance and those that do not.

When the mitochondria and/or the endoplasmic reticulum were targeted by photodynamic therapy, photodamage to the anti-apoptotic protein Bcl-2 was observed. This led to an apoptotic outcome if that death pathway was available. Lysosomal photodamage ultimately resulted in activation of the pro-apoptotic protein Bid, also leading to apoptosis. Photodamage to the plasma membrane was associated with migration of sensitizers to the cytosol and procaspase photodamage, with apoptosis impaired. Where apoptosis was unavailable because of lack of necessary components of the program, an autophagic outcome has been observed. It is also clear that autophagy can occur along with apoptosis as a PDT response, and may play a role in immunologic responses to photodamaged tumor cells.

A death mode termed paraptosis is initiated by photodamage to the endoplasmic reticulum (ER). This is characterized by an extensive pattern of cytoplasmic vacuole formation and leads to a gradual loss of cytoplasm and of viability. Many photosensitizers in clinical use target sub-cellular organelles that include the ER and can, therefore, invoke paraptosis as well as apoptosis. In this study, we explore pathways to paraptosis in OVCAR-5, an ovarian cancer cell line of human origin. At low PDT doses, the route to paraptosis follows the pattern that occurs after chemotherapy, that is, a pattern of vacuole formation that can be suppressed by MAPK antagonists or inhibition of new protein synthesis. At PDT doses clinically pertinent for tumor eradication, the pathway to paraptosis appears to be independent of these factors. In addition to morphologic changes, translocation of the nuclear protein HMGB1(high mobility group protein B1) to the cell periphery has been described as a potential marker for drug-induced paraptosis. This occurs to only a very minor extent after a lethal PDT dose that targets the ER. It appears that a lethal level of ER photodamage can initiate a different pathway to paraptosis, perhaps associated with ER protein cross-linking.

A major objective in developing new treatment approaches for lethal tumors is to reduce toxicity to normal tissues while maintaining therapeutic efficacy. Photodynamic therapy (PDT) provides a mechanistically distinct approach to treat tumors without the systemic toxicity of chemotherapy drugs. PDT involves the light-based activation of a small molecule, a photosensitizer (PS), to generate reactive molecular species (RMS) that are toxic to target tissue. Depending on the PS localization, various cellular and subcellular components can be targeted, causing selective photodamage. It has been shown that targeted lysosomal photodamage followed by, or simultaneous with, mitochondrial photodamage using two different PS results in a considerable enhancement in PDT efficacy. Here, two liposomal formulations of benzoporphyrin derivative (BPD): (1) Visudyne (clinically approved) and (2) an in-house formulation entrapping a lipid conjugate of BPD are used in combination with direct PS localization to mitochondria, endoplasmic reticulum and lysosomes, enabling simultaneous photodamage to all three organelles using a single wavelength of light. Building on findings by our group, and others, this study demonstrates, for the first time in a 3D model for ovarian cancer, that BPD-mediated photodestruction of lysosomes and mitochondria/ER significantly enhances PDT efficacy at lower light doses than treatment with either PS formulation alone.

Simple Summary Despite the advances in multimodality treatment strategies, more than 30% of patients with advanced head and neck squamous cell carcinoma (HNSCC) experience recurrence of the disease that is usually derived from the residual tumor. The goal of our study is to understand the molecular basis underlying radiotherapy resistance in advanced HNSCC and to identify a mechanism-based radiosensitizer. We found that the autophagic cell survival pathway is upregulated in therapy-resistant HNSCC. Photodynamic therapy (PDT) directed at the endoplasmic reticulum (ER)/mitochondria induces programmed cell death such as paraptosis and apoptosis in an autophagic adaptor p62-dependent manner, promoting radiotoxicity. Abstract Despite recent advances in therapeutic modalities such as radiochemotherapy, the long-term prognosis for patients with advanced head and neck squamous cell carcinoma (HNSCC), especially nonviral HNSCC, remains very poor, while survival of patients with human papillomavirus (HPV)-associated HNSCC is greatly improved after radiotherapy. The goal of this study is to develop a mechanism-based treatment protocol for high-risk patients with HPV-negative HNSCC. To achieve our goal, we have investigated molecular mechanisms underlying differential radiation sensitivity between HPV-positive and -negative HNSCC cells. Here, we found that autophagy is associated with radioresistance in HPV-negative HNSCC, whereas apoptosis is associated with radiation sensitive HPV-positive HNSCC. Interestingly, we found that photodynamic therapy (PDT) directed at the endoplasmic reticulum (ER)/mitochondria initially induces paraptosis followed by apoptosis. This led to a substantial increase in radiation responsiveness in HPV-negative HNSCC, while the same PDT treatment had a minimal effect on HPV-positive cells. Here, we provide evidence that the autophagic adaptor p62 mediates signal relay for the induction of apoptosis, promoting ionizing radiation (XRT)-induced cell death in HPV-negative HNSCC. This work proposes that ER/mitochondria-targeted PDT can serve as a radiosensitizer in intrinsically radioresistant HNSCC that exhibits an increased autophagic flux.

Welcome and Introduction to SPIE Photonics West BiOS conference 11628: Optical Methods for Tumor Treatment and Detection: Mechanisms and Techniques in Photodynamic Therapy XXX

There are multiple determinants of PDT efficacy. An in vitro study showed that the head and neck tumor lines derived from patients with an HPV infection showed a substantial decrease in the degree of photokilling by PDT directed at ER/mitochondria. We previously reported that this was not correlated with altered photosensitizer uptake, sites of sub-cellular concentration, or rate of ROS formation. PDT targeted to the ER can lead to the initiation of parapotis, a death pathway associated with ER stress. This pathway appears to be operational even cell lines with an impaired apoptotic pathway and can lead to an apoptotic response. Impaired PDT-induced photokilling in HPV(+)line was associated with a decrease of both the paraptotic response to ER photodamage and the loss of mitochondrial membrane potential (ΔΨm) after mitochondrial photodamage. This may explain, in part, the impaired response to PDT when BPD (benzoporphyrin derivative, Vidsudyne) was the photosensitizing agent.

A concurrent human papilloma virus (HPV) infection potentiates the efficacy of ionizing radiation for treatment of head and neck cancer by promoting apoptosis. Studies in cell culture indicated an opposite effect for photodynamic therapy (PDT) when this leads to mitochondrial and ER photodamage. The explanation for this difference in PDT efficacy remains to be established. While apoptosis was impaired in HPV(−) cells, such cells can be killed via photodamage directed at the ER: this leads to a non-apoptotic death pathway termed paraptosis. No differences in photosensitizer uptake or reactive oxygen species (ROS) production were observed in HPV(+) vs. HPV(−) tumors. We now provide evidence that death pathways initiated by ER/mitochondrial photodamage leading to either paraptosis or apoptosis are impaired in an HPV(+) head and neck cell line. These results illustrate the complex determinants of PDT efficacy, a topic that has yet to be fully explored.

Efficacy of photodynamic therapy (PDT) with hypericin was compared using two cell lines in monolayer culture. Photokilling was substantially impaired in the A549 (human-derived lung tumor) when compared with the 1c1c7 murine hepatoma. The efficacy of PDT with regard to photokilling was correlated with the steady-state intracellular hypericin concentration. There was otherwise no difference in subcellular targets or the pathway to cell death: paraptosis. In a prior study involving cell lines derived from patients with head and neck cancer, differences in response to PDT had shown no correlation with photosensitizer uptake.

Effects of photodynamic therapy (PDT) using the anthraquinone hypericin were explored with OVCAR-5 cells in vitro. Irradiation resulted in ER > lysosomal photodamage. Paraptosis was identified as a primary death pathway resulting from ER perturbation. This is characterized by an extensive pattern of cytoplasmic vacuole formation. As the PDT dose increased, apoptotic death was also detected. The cytoprotective effect of autophagy, observed when certain other subcellular sites are PDT targets, appears to be absent. These results, together with prior evidence that paraptosis can be lethal to cells with an impaired apoptotic pathway, suggest a role for agents with this targeting profile in photodynamic therapy. A limitation to be overcome for hypericin is a suboptimal absorbance profile.

With the rapidly emerging designs and applications of light-activated, photodynamic therapy (PDT)-based nanoconstructs, photonanomedicines (PNMs), an unmet need exists to establish whether conventional methods of photochemical and photophysical characterization of photosensitizers are relevant for evaluating new PNMs in order to intelligently guide their design. As a model system, we build on the clinical formulation of benzoporphyrin derivative (BPD), Visudyne(®), by developing a panel of nanolipid formulations entrapping new lipidated chemical variants of BPD with differing chemical, photochemical and photophysical properties. These are 16:0 and 20:0 lysophosphocholine-BPD (16:0/20:0 BPD-PC), DSPE-PEG-BPD and BPD-cholesterol. We show that Visudyne(®) was the most phototoxic formulation to OVCAR-5 cells, and the least effective was liposomal DSPE-PEG-BPD. However, these differences did not match their optical, photophysical and photochemical properties, as the static BPD quenching was highest in Visudyne, which also exhibited the lowest generation of singlet oxygen production. Furthermore, we establish that OVCAR-5 cell phototoxicity also does not correlate with rates of photosensitizer photobleaching and fluorescence quantum yields in any nanolipid formulations. These findings warrant critical future studies into subcellular targets and molecular mechanisms of phototoxicity of photodynamic nanoconstructs, as more reliable prognostic surrogates for predicting efficacy in order to appropriately and intelligently guide their design.

Photodamage to the endoplasmic reticulum (ER) can initiate a death pathway termed paraptosis. The "canonical" model of paraptosis, initiated by certain drugs and other stimuli, requires a brief interval of protein synthesis, involves the action of MAP kinases and can be followed by biochemical markers. The latter include changes in expression of AIP-1/Alix and IGF-1R proteins and translocation of HMGB-1 from nucleus to plasma membrane. There is also a report indicating that an enhanced level of autophagy can impair death by paraptosis. The pathway to paraptosis follows the canonical pathway when ER photodamage is minor (

The ability of photosensitizing agents to create photodamage at specific sub-cellular sites has proved useful for characterizing pathway(s) to cell death and for selecting optimal targets for anti-tumor efficacy. Both apoptosis and autophagy can occur after photodamage directed at mitochondria, lysosomes or the ER, with the balance often a determinant of overall efficacy. A combination of lysosomal + mitochondrial targets is associated with enhanced efficacy. More recently, ER photodamage was found to evoke a mainly unexplored mode of photokilling that involves extensive cytoplasmic vacuole formation but does not represent autophagy. This has been termed ‘paraptosis’ and appears to be a reaction to the appearance of misfolded ER proteins. This report is designed to summarize current knowledge relating to death pathways and update information relating to paraptosis as a PDT response.

In prior studies, we have identified the ability of low-level lysosomal photodamage to potentiate the phototoxic effect of subsequent photodamage to mitochondria. The mechanism involves calpain-mediated cleavage of the autophagy-associated protein ATG5 to form a pro-apoptotic fragment (tATG5). In this report, we explore the permissible time lag between the two targeting procedures along with the effect of simultaneously targeting both lysosomes and mitochondria. This was found to be as effective as the sequential protocol with no gap between the irradiation steps. Inhibition of calpain reversed the enhanced efficacy of the ‘simultaneous’ protocol. It appears that even a minor level of lysosomal photodamage can have a significant effect on the efficacy of subsequent mitochondrial photodamage. We propose that these results may explain the efficacy of Photofrin, a photosensitizing product that also targets both lysosomes and mitochondria for photodamage.

Efficacy of ionizing radiation (I/R) was compared with phototoxic effects of photodynamic therapy (PDT) in vitro using two cell lines derived from patients with head and neck squamous cell carcinoma (HNSCC). A cell line derived from a donor with a human papilloma virus (HPV) infection was more responsive to I/R but significantly less responsive to PDT than a cell line derived from an HPV-free patient. Cell death after I/R in the HPV(+) cell line was associated with increased DEVDase activity, a hallmark of apoptosis. The HPV(−) line was considerably less responsive to I/R, with DEVDase activity greatly reduced, suggesting an impaired apoptotic program. In contrast, the HPV(−) cells were readily killed by PDT when the ER was among the targets for photodamage. While DEVDase activity was enhanced, the death pathway appears to involve paraptosis until the degree of photodamage reached the LD(99) range. These data suggest that PDT-induced paraptosis can be a death pathway for cells with an impaired apoptotic program. This image demonstrates the ability of photodynamic therapy (PDT) directed at ER/mitochondria to eradicate a head & neck cancer cell line (WSU12) from a patient with a human papilloma virus infection. These cells were relatively unresponsive to ionizing radiation, typical of HPV (+) tumors. In contrast, cells from an HPV (−) patient (UP154) were responsive to ionizing radiation but not to PDT.

Photodynamic therapy (PDT) involves the selective sensitization of tissues to light. A major advance in the field occurred when Thomas Dougherty at the Roswell Park Cancer Institute initiated a series of clinical studies that eventually led to FDA approval of the procedure. This report contains a summary of Dougherty’s contributions and an assessment of where this has led, along with a summary of implications for future drug development.

Exploiting differences in photosensitizer (PS) localization and mechanisms of action with sequential or simultaneous activation protocols has been shown to improve photodynamic therapy (PDT) efficacy. Various sub-cellular, cellular and stromal components can be targeted, causing selective photodamage. Previous reports have shown that rationally targeting non-overlapping tumor compartments or sub-cellular sites considerably enhances outcomes from PDT. The current presentation describes the benefits of simultaneously targeting lysosomes and mitochondria/endoplasmic reticulum using lipid-anchored and entrapped liposomal preparations of benzoporphyrin derivative, respectively, with an emphasis on results in 3D models of ovarian cancer.

While many have contributed to the field of ‘photodynamic therapy’, Thomas J. Dougherty played the central role. Without his efforts, it seems unlikely that PDT would have advanced much beyond being a medical curiosity. This summary discusses both his many contributions to the field and the wide range of discoveries made possible by his pioneering studies that brought PDT to the attention of the funding agencies.

Without the efforts of Tom Dougherty, it is unlikely that photodynamic therapy would have been more than a minor biomedical curiosity. His contributions to the field not only involved demonstrating the clinical relevance of the procedure but organizing that eventually led to FDA approval. This report summarizes his contributions to the field and illustrates the scope of discoveries that became feasible after his work brought PDT to the attention of others and to assorted funding agencies.

Two death pathways have been associated with photodynamic therapy (PDT): apoptosis and paraptosis. The former is characterized by caspase activation and nuclear fragmentation while the latter is associated with an unfolded protein response leading to an extensive pattern of cytoplasmic vacuole formation. In this study, we explore some of the determinants of a response involving paraptosis after photodynamic therapy in cell culture using a human-derived ovarian cancer cell line: OVCAR-5 cells. Experimental results are consistent with the hypothesis that paraptosis is evoked by ER photodamage. This leads to an extensive pattern of cytoplasmic vacuolization. While activation of JNK and related MAP kinases has been associated with paraptosis, the JNK antagonist SP600125 did not prevent a paraptotic response to ER photodamage in OVCAR-5. Translocation of the nuclear HMGB1 to the cell periphery had been proposed as a potential marker for paraptosis. We found this effect was not a reliable marker for paraptosis. Targeting the ER for photodamage may, however, be a useful approach to eradicating malignant cells with an impaired route to apoptosis.

Photodynamic therapy (PDT) offers a new approach to selective tumor eradication. Modifications designed to increase and optimize efficacy continue to emerge. Selective photodamage to malignant cells and their environment can bring about tumor cell destruction, shutdown of the tumor vasculature, stimulation of immunologic anti-tumor effects and potentiation of other therapeutic effects. Current development of combination protocols may provide a better rationale for integration of PDT into clinical practice. An example described here is the ability of a sequential (two-sensitizer) PDT protocol to enhance the efficacy of photokilling. The first step involves low-level lysosomal photodamage that has been shown to promote the apoptotic response to subsequent photodynamic effects directed at mitochondria. In this report, we demonstrate the ability of Photofrin, an FDA-approved photosensitizing agent, to serve as either the first or second element of the sequential protocol.