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Objective: The silicon phthalocyanine Pc 4 is a photosensitizing agent previously shown to be a promising treatment for cutaneous neoplasms using photodynamic therapy (PDT). Based on prior preclinical studies, we believe Pc 4-PDT has potential as a targeted treatment of human recurrent respiratory papillomatosis or laryngeal leukoplakia by direct injection into mucosal surfaces., Methods: This was a proof-of-concept pilot study assessing direct mucosal injection of Pc 4 into buccal and vocal fold mucosae in a rabbit model. Five New Zealand white rabbits underwent tattooing of bilateral buccal mucosae to delineate injection sites, followed by submucosal injections of control and Pc 4 solutions. Rabbits were monitored for post-injection tolerance. Punch biopsies were obtained from injected mucosa and assessed histopathologically. Once the buccal mucosa was found to be tolerant, vocal folds of three rabbits were injected. The rabbits were then sacrificed, and laryngeal tissue was assessed histopathologically., Results: All rabbits tolerated injection of Pc 4 and control solutions into buccal mucosa with no evidence of gross visual inflammatory changes and no changes in behavior or masticatory function. Histopathologic analysis of Pc 4 injected buccal and control mucosal tissue revealed mild focal histological changes and no stigmata of diffuse inflammatory reactions. The histopathologic analysis of Pc 4 injected into laryngeal tissue revealed similar findings with addition of mild eosinophilia in one sample., Conclusion: Direct mucosal injection of Pc 4 in rabbit buccal and vocal fold mucosae appears to be well tolerated with no gross inflammatory changes, and only mild histopathologic inflammatory changes observed., Level of Evidence: NA Laryngoscope, 2024., (© 2024 The American Laryngological, Rhinological and Otological Society, Inc.)

Purpose: TRC102, a small-molecule base-excision repair inhibitor, potentiates the cytotoxicity of pemetrexed and reverses resistance by binding to chemotherapy-induced abasic sites in DNA. We conducted a phase I clinical trial combining pemetrexed and TRC102 with cisplatin-radiation in stage III nonsquamous non-small cell lung cancer (NS-NSCLC)., Patients and Methods: Fifteen patients were enrolled from 2015 to 2019. The primary objective was to determine the dose-limiting toxicity and maximum tolerated dose of TRC102 in combination with pemetrexed, cisplatin, and radiotherapy. Secondary objectives were to assess toxicity, tumor response, and progression-free survival at 6 months. Based on our preclinical experiments, pemetrexed-TRC102 was given on day 1, and cisplatin/radiotherapy was initiated on day 3. This schedule was duplicated in the second cycle. After completion, two additional cycles of pemetrexed-cisplatin were given. Toxicities were assessed using NCI CTACAE versions 4/5., Results: The median age was 69 years (45-79) with the median follow-up of 25.7 months (range, 7.9-47.4). No dose-limiting toxicities and no grade 5 toxicity were seen. Hematologic and gastrointestinal toxicities were the most common side effects. No clinical radiation pneumonitis was seen. Of 15 evaluable patients, three had complete response (20%), and 12 had partial response (80%). The 6-month progression-free survival was 80%, and the 2-year overall survival was 83%., Conclusions: Pemetrexed-TRC102 combined with cisplatin/radiotherapy in NS-NSCLC is safe and well tolerated. The recommended phase II dose is 200 mg TRC102 along with cisplatin-pemetrexed. No additional safety signal was seen beyond the expected CRT risks. A phase II trial, integrating post-CRT immunotherapy with this aggressive DNA-damaging regimen, is warranted., (©2021 The Authors; Published by the American Association for Cancer Research.)

Purpose: "FLASH" radiotherapy (RT) is a potential paradigm-changing RT technology with marked tumor killing and normal tissue sparing. However, the mechanism of the FLASH effect is not well understood. We hypothesize that the ultra-high dose rate FLASH-RT significantly reduces the killing of circulating immune cells which may partially contribute to the reported FLASH effect., Methods: This computation study directly models the effect of radiation dose rate on the killing of circulating immune cells. The model considers an irradiated volume that takes up A% of cardiac output and contains B% of total blood. The irradiated blood volume and dose were calculated for various A%, B%, blood circulation time, and irradiation time (which depends on the dose rate). The linear-quadratic model was used to calculate the extent of killing of circulating immune cells at ultra-high vs. conventional dose rates., Results: A strong sparing effect on circulating blood cells by FLASH-RT was noticed; i.e., killing of circulating immune cells reduced from 90% to 100% at conventional dose rates to 5-10% at ultra-high dose rates. The threshold FLASH dose rate was determined to be ~40 Gy/s for mice in an average situation (A% = 50%), consistent with the reported FLASH dose rate in animal studies, and it was approximately one order of magnitude lower for humans than for mice. The magnitude of this sparing effect increased with the dose/fraction, reached a plateau at 30-50 Gy/fraction, and almost completely vanished at 2 Gy/fraction., Conclusion: We have calculated a strong sparing effect on circulating immune cells by FLASH-RT, which may contribute to the reported FLASH effects in animal studies., Competing Interests: Declaration of Competing Interest This work was partially supported by the USA National Cancer Institute, National Institutes of Health, R01 CA142840 (Kong). The authors declare that they have no other known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Ribonucleotide reductase (RR) catalyses the rate-limiting step of dNTP synthesis, establishing it as an important cancer target. While RR is traditionally inhibited by nucleoside-based antimetabolites, we recently discovered a naphthyl salicyl acyl hydrazone-based inhibitor (NSAH) that binds reversibly to the catalytic site (C-site). Here we report the synthesis and in vitro evaluation of 13 distinct compounds (TP1-13) with improved binding to hRR over NSAH (TP8), with lower K D 's and more predicted residue interactions. Moreover, TP6 displayed the greatest growth inhibiting effect in the Panc1 pancreatic cancer cell line with an IC 50 of 0.393 µM. This represents more than a 2-fold improvement over NSAH, making TP6 the most potent compound against pancreatic cancer emerging from the hydrazone inhibitors. NSAH was optimised by the addition of cyclic and polar groups replacing the naphthyl moiety, which occupies the phosphate-binding pocket in the C-site, establishing a new direction in inhibitor design.

Epithelial ovarian cancer is a deadly gynecologic malignancy because of its late detection, usually after local and distant metastatic spread. These cancers develop resistance to traditional chemotherapeutic agents; therefore, the development of next-generation immunotherapeutic approaches may have a significant promise in improving outcomes. A novel immunotherapeutic approach utilizing combination radiation therapy (RT) with immunostimulatory cowpea mosaic virus (CPMV) was tested in a preclinical syngeneic mouse model of ovarian carcinoma. ID8-Defb29/Vegf tumors were generated in C57BL/6 mice. Compared to placebo-treated control tumors or those treated with a single agent RT or CPMV, the combination treatment resulted in a significantly improved tumor growth delay ( p < 0.05). Additionally, immunohistochemical profiling of tumor samples after treatment with CPMV demonstrated an increase in tumor infiltrating lymphocytes (TILs). These results suggest that utilizing CPMV particles in combination with RT can turn an immunologically "cold" tumor (with low number of TILs) into an immunologically "hot" tumor. This novel combination treatment approach of RT and CPMV demonstrated the ability to control tumor growth in a preclinical ID8 ovarian cancer model, showing promise as an in situ tumor vaccine and warrants further testing., Competing Interests: The authors declare no competing financial interest.

The cell cycle checkpoint proteins ataxia-telangiectasia-mutated-and-Rad3-related kinase (ATR) and its major downstream effector checkpoint kinase 1 (CHK1) prevent the entry of cells with damaged or incompletely replicated DNA into mitosis when the cells are challenged by DNA damaging agents, such as radiation therapy (RT) or chemotherapeutic drugs, that are the major modalities to treat cancer. This regulation is particularly evident in cells with a defective G1 checkpoint, a common feature of cancer cells, due to p53 mutations. In addition, ATR and/or CHK1 suppress replication stress (RS) by inhibiting excess origin firing, particularly in cells with activated oncogenes. Those functions of ATR/CHK1 make them ideal therapeutic targets. ATR/CHK1 inhibitors have been developed and are currently used either as single agents or paired with radiotherapy or a variety of genotoxic chemotherapies in preclinical and clinical studies. Here, we review the status of the development of ATR and CHK1 inhibitors. We also discuss the potential mechanisms by which ATR and CHK1 inhibition induces cell killing in the presence or absence of exogenous DNA damaging agents, such as RT and chemotherapeutic agents. Lastly, we discuss synthetic lethality interactions between the inhibition of ATR/CHK1 and defects in other DNA damage response (DDR) pathways/genes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Photodynamic therapy (PDT) has the potential to make a significant impact on cancer treatment. PDT can sensitize malignant tissues to light, leading to a highly selective effect if an appropriate light dose can be delivered. Variations in light distribution and drug delivery, along with impaired efficacy in hypoxic regions, can reduce the overall tumor response. There is also evidence that malignant cells surviving PDT may become more aggressive than the initial tumor population. Promotion of more effective direct tumor eradication is therefore an important goal. While a list of properties for the "ideal" photosensitizing agent often includes formulation, pharmacologic and photophysical elements, we propose that subcellular targeting is also an important consideration. Perspectives relating to optimizing PDT efficacy are offered here. These relate to death pathways initiated by photodamage to particular subcellular organelles., (© 2017 The American Society of Photobiology.)

Human ribonucleotide reductase (hRR) is crucial for DNA replication and maintenance of a balanced dNTP pool, and is an established cancer target. Nucleoside analogs such as gemcitabine diphosphate and clofarabine nucleotides target the large subunit (hRRM1) of hRR. These drugs have a poor therapeutic index due to toxicity caused by additional effects, including DNA chain termination. The discovery of nonnucleoside, reversible, small-molecule inhibitors with greater specificity against hRRM1 is a key step in the development of more effective treatments for cancer. Here, we report the identification and characterization of a unique nonnucleoside small-molecule hRR inhibitor, naphthyl salicylic acyl hydrazone (NSAH), using virtual screening, binding affinity, inhibition, and cell toxicity assays. NSAH binds to hRRM1 with an apparent dissociation constant of 37 µM, and steady-state kinetics reveal a competitive mode of inhibition. A 2.66-Å resolution crystal structure of NSAH in complex with hRRM1 demonstrates that NSAH functions by binding at the catalytic site (C-site) where it makes both common and unique contacts with the enzyme compared with NDP substrates. Importantly, the IC 50 for NSAH is within twofold of gemcitabine for growth inhibition of multiple cancer cell lines, while demonstrating little cytotoxicity against normal mobilized peripheral blood progenitor cells. NSAH depresses dGTP and dATP levels in the dNTP pool causing S-phase arrest, providing evidence for RR inhibition in cells. This report of a nonnucleoside reversible inhibitor binding at the catalytic site of hRRM1 provides a starting point for the design of a unique class of hRR inhibitors., Competing Interests: The authors declare no conflict of interest.

Background and Purpose: The anti-folate pemetrexed is a radiosensitizer. In pre-clinical models, pemetrexed is more effective along with the base-excision-repair inhibitor methoxyamine. We tested whether methoxyamine enhances pemetrexed-mediated radiosensitization of lung adenocarcinoma cells and xenografts., Materials and Methods: A549 and H1299 cells were evaluated for cell cycle distribution by flow cytometry, radiosensitization by clonogenic assay, and DNA repair by neutral comet assay and repair protein activation. H460 cells were included in some studies. Xenografts in nude mice received drug(s) and/or radiation, and tumor growth was monitored by caliper and in vivo toxicity by animal weight., Results: Exposure to pemetrexed/methoxyamine for 24 (H1299, H460) or 48 (A549)hours before irradiation resulted in accumulation of cells near the radiosensitive G1/S border; dose-enhancement factors of 1.62±0.19, 1.97±0.25, and 1.67±0.30, respectively; reduction of mean inactivation dose by 32%, 30%, and 46%, respectively; and significant reductions of SF2 and SF4 (p<0.05). Radiosensitization was associated with rapid DNA double-strand-break rejoining and increased levels of DNA-PKcs. Both tumor-growth rate and tumor-growth delay were significantly improved by adding methoxyamine to pemetrexed pre-irradiation (p<0.0001); no mice lost weight during treatment., Conclusions: Addition of methoxyamine to pemetrexed and fractionated radiotherapy may improve outcome for patients with locally advanced non-squamous non-small-cell lung cancer., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Photodynamic therapy (PDT) is an emerging treatment for malignant and inflammatory dermal disorders. Photoirradiation of the silicon phthalocyanine (Pc) 4 photosensitizer with red light generates singlet oxygen and other reactive oxygen species to induce cell death. We previously reported that Pc 4-PDT elicited cell death in lymphoid-derived (Jurkat) and epithelial-derived (A431) cell lines in vitro, and furthermore that Jurkat cells were more sensitive than A431 cells to treatment. In this study, we examined the effectiveness of Pc 4-PDT on primary human CD3(+) T cells in vitro. Fluorometric analyses of lysed T cells confirmed the dose-dependent uptake of Pc 4 in non-stimulated and stimulated T cells. Flow cytometric analyses measuring annexin V and propidium iodide (PI) demonstrated a dose-dependent increase of T cell apoptosis (6.6-59.9%) at Pc 4 doses ranging from 0-300 nM. Following T cell stimulation through the T cell receptor using a combination of anti-CD3 and anti-CD28 antibodies, activated T cells exhibited increased susceptibility to Pc 4-PDT-induced apoptosis (10.6-81.2%) as determined by Pc 4 fluorescence in each cell, in both non-stimulated and stimulated T cells, Pc 4 uptake increased with Pc 4 dose up to 300 nM as assessed by flow cytometry. The mean fluorescence intensity (MFI) of Pc 4 uptake measured in stimulated T cells was significantly increased over the uptake of resting T cells at each dose of Pc 4 tested (50, 100, 150 and 300 nM, p < 0.001 between 50 and 150 nM, n = 8). Treg uptake was diminished relative to other T cells. Cutaneous T cell lymphoma (CTCL) T cells appeared to take up somewhat more Pc 4 than normal resting T cells at 100 and 150 nm Pc 4. Confocal imaging revealed that Pc 4 localized in cytoplasmic organelles, with approximately half of the Pc 4 co-localized with mitochondria in T cells. Thus, Pc 4-PDT exerts an enhanced apoptotic effect on activated CD3(+) T cells that may be exploited in targeting T cell-mediated skin diseases, such as cutaneous T cell lymphoma (CTCL) or psoriasis.

Ribonucleotide reductase (RR) catalyzes the rate-limiting step of dNTP synthesis and is an established cancer target. Drugs targeting RR are mainly nucleoside in nature. In this study, we sought to identify non-nucleoside small-molecule inhibitors of RR. Using virtual screening, binding affinity, inhibition, and cell toxicity, we have discovered a class of small molecules that alter the equilibrium of inactive hexamers of RR, leading to its inhibition. Several unique chemical categories, including a phthalimide derivative, show micromolar IC50s and KDs while demonstrating cytotoxicity. A crystal structure of an active phthalimide binding at the targeted interface supports the noncompetitive mode of inhibition determined by kinetic studies. Furthermore, the phthalimide shifts the equilibrium from dimer to hexamer. Together, these data identify several novel non-nucleoside inhibitors of human RR which act by stabilizing the inactive form of the enzyme.

The fundamental mechanism of photodynamic therapy (PDT)-induced cell death has been characterized, but early critical PDT events in vivo remain incompletely defined. With the recent development in advanced fluorescence imaging modalities, such as intravital 2-photon laser scanning microscopy (2P-LSM), researchers are now able to investigate and visualize biological processes with high resolution in real time. This powerful imaging technology allows deep tissue visualization with single-cell resolution, thus providing dynamic information on the 3-dimensional architectural makeup of the tissue. The main goal of this study was to determine the cutaneous penetration of a topically applied photosensitizer, the silicon phthalocyanine Pc 4, into the skin of live animals and to assess the effective absorption of Pc 4 through the skin barrier. Our 2P-LSM images indicate that Pc 4 penetrates to the epidermal/dermal junction of mouse skin. The data also indicate that the degree of Pc 4 absorption is dose dependent. These findings represent initial steps that may help in improving the clinical utilization of topical Pc 4-PDT., (Published by Elsevier B.V.)

The current clinical mainstays for cancer treatment, namely, surgical resection, chemotherapy, and radiotherapy, can cause significant trauma, systemic toxicity, and functional/cosmetic debilitation of tissue, especially if repetitive treatment becomes necessary due to tumor recurrence. Hence there is significant clinical interest in alternate treatment strategies like photodynamic therapy (PDT) which can effectively and selectively eradicate tumors and can be safely repeated if needed. We have previously demonstrated that the second-generation photosensitizer Pc 4 (silicon phthalocyanine 4) can be formulated within polymeric micelles, and these micelles can be specifically targeted to EGFR-overexpressing cancer cells using GE11 peptide ligands, to enhance cell-specific Pc 4 delivery and internalization. In the current study, we report on the in vitro optimization of the EGFR-targeting, Pc 4 loading of the micellar nanoformulation, along with optimization of the corresponding photoirradiation conditions to maximize Pc 4 delivery, internalization, and subsequent PDT-induced cytotoxicity in EGFR-overexpressing cells in vitro. In our studies, absorption and fluorescence spectroscopy were used to monitor the cell-specific uptake of the GE11-decorated Pc 4-loaded micelles and the cytotoxic singlet oxygen production from the micelle-encapsulated Pc 4, to determine the optimum ligand density and Pc 4 loading. It was found that the micelle formulations bearing 10 mol % of GE11-modified polymer component resulted in the highest cellular uptake in EGFR-overexpressing A431 cells within the shortest incubation periods. Also, the loading of ∼ 50 μg of Pc 4 per mg of polymer in these micellar formulations resulted in the highest levels of singlet oxygen production. When formulations bearing these optimized parameters were tested in vitro on A431 cells for PDT effect, a formulation dose containing 400 nM Pc 4 and photoirradiation duration of 400 s at a fluence of 200 mJ/cm(2) yielded close to 100% cell death.

In photodynamic therapy (PDT), the light activation of a photosensitizer leads to the generation of reactive oxygen species that can trigger various mechanisms of cell death. Harnessing this process within cancer cells enables minimally invasive yet targeted cancer treatment. With this rationale, here we demonstrate tumor-targeted delivery of a highly hydrophobic photosensitizer Pc 4 loaded within biocompatible poly(ethylene glycol)-poly(ɛ-caprolactone) block co-polymer micelles. The micelles were surface-modified with epidermal growth factor receptor (EGFR)-targeting GE11 peptides for active targeting of EGFR-overexpressing cancer cells, in vitro. Pc 4-loaded EGFR-targeted micelles were incubated with EGFR-overexpressing A431 epidermoid carcinoma cells for various time periods, to determine Pc 4 uptake by epifluorescence microscopy. The cells were subsequently photoirradiated, and PDT-induced cell death for various incubation periods was determined by MTT assay and fluorescence Live/Dead assay. Our results indicate that active EGFR targeting of the Pc 4-loaded micelles accelerates intracellular uptake of the drug. Consequently, this enhances the PDT-induced cytotoxicity within shorter time periods., From the Clinical Editor: Photodynamic cancer therapy using Pc 4, a light activated and highly hydrophobic photosensitizer is demonstrated in this paper in vitro. Pc 4 was delivered in block-copolymer micelles surface-modified with GE11 peptides targeting EGFR-overexpressing cancer cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

Background:  Photodynamic therapy (PDT) has been shown to be effective in the treatment of malignancies of a variety of organ systems, including the lungs, bladder, gastrointestinal tract and skin. Cutaneous lesions serve as ideal targets of PDT because of the accessibility of the skin to light. To achieve optimum results, the photosensitizer must be delivered effectively into the target layers of the skin within a practical timeframe, via noninvasive methods., Aim: To determine whether topical application of a second-generation photosensitizer, silicon phthalocyanine (Pc) 4 [SiPc(OSi(CH3)2 (CH2)3 N(CH3)2)(OH)], results in effective penetration of the skin barrier., Methods: Penetration of Pc 4 was evaluated using standard Franz-type vertical diffusion cell experiments on surrogate materials (silicone membranes) and laser-scanning confocal microscopy of normal skin biopsy samples from human volunteers., Results: The Franz diffusion data indicate that Pc 4 formulated in an ethanol/propylene glycol solution (70/30%, v/v) can penetrate the membrane at a flux that is appreciable and relatively invariant. Using the same formulation, Pc 4 uptake could be detected in human skin via laser-scanning confocal microscopy., Conclusion: After topical application, Pc 4 is absorbed into the epidermis in as little as 1 h, and the absorption increased with increasing time and dose. Pc 4 can be effectively delivered into human skin via topical application. The data also suggest that the degree of penetration is time- and dose-dependent., (© The Author(s). CED © 2011 British Association of Dermatologists.)

Purpose: To test whether pharmacologic inhibition of ribonucleotide reductase (RNR) by 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, NSC #663249) enhances radiation sensitivity during low-dose-rate ionizing radiation provided by a novel purpose-built iridium-192 cell irradiator., Methods and Materials: The cells were exposed to low-dose-rate radiation (11, 23, 37, 67 cGy/h) using a custom-fabricated cell irradiator or to high-dose-rate radiation (330 cGy/min) using a conventional cell irradiator. The radiation sensitivity of human cervical (CaSki, C33-a) cancer cells with or without RNR inhibition by 3-AP was evaluated using a clonogenic survival and an RNR activity assay. Alteration in the cell cycle distribution was monitored using flow cytometry., Results: Increasing radiation sensitivity of both CaSki and C33-a cells was observed with the incremental increase in radiation dose rates. 3-AP treatment led to enhanced radiation sensitivity in both cell lines, eliminating differences in cell cytotoxicity from the radiation dose rate. RNR blockade by 3-AP during low-dose-rate irradiation was associated with low RNR activity and extended G(1)-phase cell cycle arrest., Conclusions: We conclude that RNR inhibition by 3-AP impedes DNA damage repair mechanisms that rely on deoxyribonucleotide production and thereby increases radiation sensitivity of human cervical cancers to low-dose-rate radiation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The high prevalence of drug resistance necessitates the development of novel antifungal agents against infections caused by opportunistic fungal pathogens, such as Candida albicans. Elucidation of apoptosis in yeast-like fungi may provide a basis for future therapies. In mammalian cells, photodynamic therapy (PDT) has been demonstrated to generate reactive oxygen species, leading to immediate oxidative modifications of biological molecules and resulting in apoptotic cell death. In this report, we assess the in vitro cytotoxicity and mechanism of PDT, using the photosensitizer Pc 4, in planktonic C. albicans. Confocal image analysis confirmed that Pc 4 localizes to cytosolic organelles, including mitochondria. A colony formation assay showed that 1.0 μM Pc 4 followed by light at 2.0 J cm(-2) reduced cell survival by 4 logs. XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide) assay revealed that Pc 4-PDT impaired fungal metabolic activity, which was confirmed using the FUN-1 (2-chloro-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenylquinolinium iodide) fluorescence probe. Furthermore, we observed changes in nuclear morphology characteristic of apoptosis, which were substantiated by increased externalization of phosphatidylserine and DNA fragmentation following Pc 4-PDT. These data indicate that Pc 4-PDT can induce apoptosis in C. albicans. Therefore, a better understanding of the process will be helpful, as PDT may become a useful treatment option for candidiasis., (© 2011 The Authors. Photochemistry and Photobiology © 2011 The American Society of Photobiology.)

Photodynamic therapy (PDT) for cutaneous malignancies has been found to be an effective treatment with a range of photosensitizers. The phthalocyanine Pc 4 was developed initially for PDT of primary or metastatic cancers in the skin. A Phase I trial was initiated to evaluate the safety and pharmacokinetic profiles of systemically administered Pc 4 followed by red light (Pc 4-PDT) in cutaneous malignancies. A dose-escalation study of Pc 4 (starting dose 0.135 mg/m(2)) at a fixed light fluence (135 J/cm(2) of 675-nm light) was initiated in patients with primary or metastatic cutaneous malignancies with the aim of establishing the maximum tolerated dose (MTD). Blood samples were taken at intervals over the first 60 h post-PDT for pharmacokinetic analysis, and patients were evaluated for toxicity and tumor response. A total of three patients (two females with breast cancer and one male with cutaneous T-cell lymphoma) were enrolled and treated over the dose range of 0.135 mg/m(2) (first dose level) to 0.54 mg/m(2) (third dose level). Grade 3 erythema within the photoirradiated area was induced in patient 2, and transient tumor regression in patient 3, in spite of the low photosensitizer doses. Pharmacokinetic observations fit a three-compartment exponential elimination model with an initial rapid distribution phase (∼0.2 h) and relatively long terminal elimination phase (∼28 h), Because of restrictive exclusion criteria and resultant poor accrual, the trial was closed before MTD could be reached. While the limited accrual to this initial Phase I study did not establish the MTD nor establish a complete pharmacokinetic and safety profile of intravenous Pc 4-PDT, these preliminary data support further Phase I testing of this new photosensitizer.

Background: Photodynamic therapy (PDT) is a non-invasive treatment for non-melanoma skin cancer. However, PDT systems currently used clinically have limitations such as pain and superficial tissue penetration. The silicon phthalocyanine Pc 4 is a second-generation photosensitizer with peak absorption in the far red at 675 nm., Objective: To assess the safety and tolerability of topically applied Pc 4 followed by red light (Pc 4-PDT) in treating cutaneous neoplasms., Study Design/materials and Methods: Forty three adults with a diagnosis of neoplasms including actinic keratoses, Bowen's disease, squamous cell carcinoma, basal cell carcinoma, or mycosis fungoides were treated with a single administration of Pc 4-PDT and followed for 14 days. The study utilized a light and Pc 4 dose escalation design in sequential groups of three subjects each., Results: Pc 4-PDT was well tolerated with no significant local toxicity or increased photosensitivity. It has promising biologic effects, particularly in mycosis fungoides where 14 of 35 subjects demonstrated a clinical response, which correlates with Pc 4-PDT-induced apoptosis, as measured by increased active caspase-3 in the treated skin lesions., Conclusions: Pc 4-PDT is a safe and tolerable treatment modality that effectively triggers apoptosis in cutaneous neoplasms such as mycosis fungoides., (Copyright © 2010 Wiley-Liss, Inc.)

Singlet oxygen, (1)O(2), is produced by absorption of red light by the phthalocyanine dye Pc 4, followed by energy transfer to dissolved triplet molecular oxygen, (3)O(2). In tissues, Pc 4 concentrates in lipid bilayers, and particularly in mitochondrial membranes, because of its positive charge. Illumination of cells and tissues with red light after uptake of Pc 4 results in cell death. The potential initial chemical steps that result in cellular dysfunction have been characterized in this study. Both unsaturated acyl chains of phospholipids and proteins are identified as targets of oxidation. Tetra-linoleoyl cardiolipin was oxidized in both liposomes and mitochondria after Pc 4-mediated (1)O(2) generation. Evidence for the formation of both mono- and bis-hydroperoxide adducts of single linoleoyl side chains is provided by ESI-MS and ESI-MS/MS. Similarly, illumination of Pc 4 in liposomes and mitochondria resulted in cytochrome c oxidation as detected by oxidation of His 26 in the peptide H(26)*KTGPNLHGLFGK, further supporting the potential use of this peptide as a biomarker for the presence of mitochondrial oxidative stress characteristic of (1)O(2) in vivo (J. Kim et al., Free Radic. Biol. Med. 44:1700-1711; 2008). These observations provide evidence that formation of lipid hydroperoxides and/or protein oxidation can be the initial chemical steps in Pc 4-mediated induction of apoptosis in photodynamic therapy., (Copyright 2010 Elsevier Inc. All rights reserved.)

Photodynamic therapy (PDT) with lysosome-targeted photosensitizers induces the intrinsic pathway of apoptosis via the cleavage and activation of the BH3-only protein Bid by proteolytic enzymes released from photodisrupted lysosomes. To investigate the role of Bid in apoptosis induction and the role of damaged lysosomes on cell killing by lysosome-targeted PDT, we compared the responses of wild type and Bid-knock-out murine embryonic fibroblasts toward a mitochondrion/endoplasmic reticulum-binding photosensitizer, Pc 4, and a lysosome-targeted sensitizer, Pc 181. Whereas apoptosis and overall cell killing were induced equally well by Pc 4-PDT in both cell lines, Bid(-/-) cells were relatively resistant to induction of apoptosis and to overall killing following PDT with Pc 181, particularly at low PDT doses. Thus, Bid is critical for the induction of apoptosis caused by PDT with the lysosome-specific sensitizers, but dispensable for PDT targeted to other membranes., (© 2010 The Authors. Journal Compilation. The American Society of Photobiology.)

Singlet oxygen is produced by the absorption of red light by the phthalocyanine dye Pc 4, followed by energy transfer to dissolved triplet oxygen. Mitochondria preincubated with Pc 4 were illuminated by red light and the damage to mitochondrial structure and function by the generated singlet oxygen was studied. At early illumination times (3-5 min of red light exposure), State 3 respiration was inhibited (50%), whereas State 4 activity increased, resulting in effectively complete uncoupling. Individual complex activities were measured and only complex IV activity was significantly reduced and exhibited a dose response, whereas the activities of electron transport complexes I, II, and III were not significantly affected. Cytochrome c release was an increasing function of irradiation time, with 30% being released after 5 min of illumination. Mitochondrial expansion along with changes in the structure of the cristae were observed by transmission electron microscopy after 5 min of irradiation, with an increase in large vacuoles and membrane rupture occurring after more extensive exposures., (Copyright 2010 Elsevier Inc. All rights reserved.)

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.

The silicon phthalocyanine Pc 4 is a second-generation photosensitizer that has several properties superior to other photosensitizers currently approved by the FDA, and it has shown significant promise for photodynamic therapy (PDT) in several cancer cells in vitro and model tumor systems in vivo. However, because of the high hydrophobicity of Pc 4, its formulation for in vivo delivery and favorable biodistribution become challenging. To this end, we are studying encapsulation and delivery of Pc 4 in block copolymer micelles. Here, we report the development of biocompatible PEG-PCL micelle nanoparticles, encapsulation of Pc 4 within the micelle core by hydrophobic association with the PCL block, and in vitro PDT studies of the micelle-formulated Pc 4 in MCF-7c3 human breast cancer cells. Our studies demonstrate efficient encapsulation of Pc 4 in the micelles, intracellular uptake of the micelle-formulated Pc 4 in cells, and significant cytotoxic effect of the formulation upon photoirradiation. Quantitative estimation of the extent of Pc 4 loading in the micelles and the photocytotoxicity of the micelle-incorporated Pc 4 demonstrate the promise of our approach to develop a biocompatible nanomedicine platform for tumor-targeted delivery of Pc 4 for site-selective PDT.

Objectives/hypothesis: To evaluate the efficacy of photodynamic therapy (PDT) with the phthalocyanine photosensitizer Pc 4 for treating an animal model of recurrent respiratory papillomatosis (RRP)., Methods: Rabbit skin was grafted onto the dorsum of severe combined immunodeficient mice, two xenografts per animal. After the graft healed, it was inoculated with cottontail rabbit papillomavirus (CRPV). When papillomas developed, Pc 4 (0.6 or 1.0 mg/kg) was administered systemically, and 48 hours later, one papilloma of the two on each animal was exposed to 675-nm photoactivating light at either 100 or 150 J/cm(2). In addition to the contralateral tumors, which received Pc 4 but no light, other controls included animals receiving light only or neither agent. Response was assessed by measuring papilloma size with a caliper. Some papillomas and residual skin were harvested for histological assessment., Results: For the lower-dose PDT regimens, papilloma growth rates were not significantly different from the controls. In contrast, 13 of 15 papillomas receiving the higher Pc 4 dose (1.0 mg/kg) and the higher light fluence (150 J/cm(2)) regressed completely and did not regrow within the observation period of up to 79 days. The response of these papillomas was significantly different from the controls (P < .001). Histological analysis confirmed the absence of residual tumor following complete response and replacement with near-normal epithelium., Conclusions: Pc 4-PDT is highly effective in treating virally induced (CRPV) papillomas in a murine model of RRP, and thus warrants further study as a treatment for HPV-induced papillomas.

Photodynamic therapy (PDT) uses a photosensitizer, light and oxygen to produce extensive oxidative damage to organelles housing the photosensitizer. Although PDT is an efficient trigger of apoptosis, it also induces autophagy in many kinds of cells. Autophagy can serve as both a cell survival and a cell death mechanism. Our previous study indicates that autophagy contributes to cell death after PDT, especially in apoptosis-deficient cells. Here, we provide further evidence to support the role of autophagy in cell killing after PDT. Autophagy was blocked by knockdown of one essential factor, LC3 or Atg7, in MCF-7 cells. The cells were exposed to a range of doses of PDT sensitized by the phthalocyanine Pc 4; steps in autophagy were monitored by western blotting for LC3-II and by fluorescence microscopy for the uptake of monodansylcadaverine or for the distribution of transfected GFP-LC3; and overall cell death was monitored by MTT assay and by clonogenic assay. We find that blocking autophagy increased the survival of MCF-7 cells after PDT and increased the shoulder on the dose-response curve. In response to Pc 4-PDT, Atg7-deficient MCF-7 cells remained capable of robust accumulation of LC3-II, but were defective in comparison to Atg7(+) cells in the formation of autophagosomes. We conclude that apoptosis-deficient cells rely on autophagy for cell death after Pc 4-PDT and that the strong activation of LC3 maturation in response to PDT could occur even in cells with limited or no Atg7 expression.

Photodynamic therapy (PDT) is a procedure that has applications in the selective eradication of neoplasia where sites of malignant lesions are clearly delineated. It is a two-step process whereby cells are first sensitized to light and then photoirradiated. This results in the formation of singlet molecular oxygen and other reactive oxygen species that can cause photodamage at sites where the photosensitizing agent has localized. Photosensitizers found to be clinically useful show affinity for the endoplasmic reticulum (ER), mitochondria, lysosomes, or combinations of these sites. The induction of apoptosis and/or autophagy in photosensitized cells is a common outcome of PDT. This report explores the following issues: (1) Does the induction of autophagy in PDT protocols occur independent of, or in association with, apoptosis? (2) Does the resulting autophagy play a prosurvival or prodeath role? (3) Do photosensitizers damage/inactivate specific proteins that are components of, or that modulate the autophagic process? (4) Can an autophagic response be mounted in cells in which lysosomes are specifically photodamaged? In brief, autophagy can occur independently of apoptosis in PDT protocols, and appears to play a prosurvival role in apoptosis competent cells, and a prodeath role in apoptosis incompetent cells. Mitochondrial and ER-localized sensitizers cause selective photodamage to some (i.e., Bcl-2, Bcl-x(L), mTOR) proteins involved in the apoptotic/autophagic process. Finally, an aborted autophagic response occurs in cells with photodamaged lysosomes. Whereas autophagosomes form, digestion of their cargo is compromised because of the absence of functional lysosomes.

Our current focus on the effects of Photodynamic Therapy (PDT) using silicon phthalocyanine Pc 4 photosensitizer on malignant T lymphocytes arose due to preclinical observations that Jurkat cells, common surrogate for human T cell lymphoma, were more sensitive to Pc 4-PDT-induced killing than epidermoid carcinoma A431 cells. Mycosis fungoides (MF) as well as Sezary syndrome (SS) are variants of cutaneous T-cell lymphoma (CTCL) in which malignant T-cells invade the epidermis. In this study, we investigated the cytotoxicity of Pc 4-PDT in peripheral blood cells obtained from patients with SS and in skin biopsies of patients with MF. Our data suggest that Pc 4-PDT preferentially induces apoptosis of CD4(+)CD7(-) malignant T-lymphocytes in the blood relative to CD11b(+) monocytes and nonmalignant T-cells. In vivo Pc 4-PDT of MF skin also photodamages the antiapoptotic protein Bcl-2.

Photodynamic therapy (PDT) is the term used to describe the irradiation of photosensitized cells or tissue with phototoxic consequences. This process can result in the rapid initiation of not only apoptosis, an irreversible death pathway, but also autophagy. The procedures described here are designed to characterize the correlation between the PDT dose vs. survival of cells in vitro, the apoptotic effects of photodamage, and the extent of an autophagic response. These are assessed by clonogenic assays, observation of condensed chromatin characteristic of apoptosis, activation of "executioner" caspases, and the autophagic flux as indicated by comparing accumulation of the LC3-II protein under conditions where processing of autophagosomes is retarded vs. is not retarded.

The phthalocyanine photosensitizer Pc 4 has been shown to bind preferentially to mitochondrial and endoplasmic reticulum membranes. Upon photoirradiation of Pc 4-loaded cells, membrane components, especially Bcl-2, are photodamaged and apoptosis, as indicated by activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase, is triggered. A series of analogs of Pc 4 were synthesized, and the results demonstrate that Pcs with the aminopropylsiloxy ligand of Pc 4 or a similar one on one side of the Pc ring and a second large axial ligand on the other side of the ring have unexpected properties, including enhanced cell uptake, greater monomerization resulting in greater intracellular fluorescence and three-fold higher affinity constants for liposomes. The hydroxyl-bearing axial ligands tend to reduce aggregation of the Pc and direct it to lysosomes, resulting in four to six times more killing of cells, as defined by loss of clonogenicity, than with Pc 4. Whereas Pc 4-PDT photodamages Bcl-2 and Bcl-xL, Pc 181-PDT causes much less photodamage to Bcl-2 over the same dose-response range relative to cell killing, with earlier cleavage of Bid and slower caspase-3-dependent apoptosis. Therefore, within this series of photosensitizers, these hydroxyl-bearing axial ligands are less aggregated than is Pc 4, tend to localize to lysosomes and are more effective in overall cell killing than is Pc 4, but induce apoptosis more slowly and by a modified pathway.

In photodynamic therapy (PDT), visible light activates a photosensitizing drug added to a tissue, resulting in singlet oxygen formation and cell death. Assessed by confocal microscopy, the photosensitizer phthalocyanine 4 (Pc 4) localizes primarily to mitochondrial membranes in cancer cells, resulting in mitochondria-mediated cell death. A Pc 4 derivative, Pc 181, accumulates into lysosomes. In comparison to Pc 4, Pc 181 was a more effective photosensitizer promoting killing cancer cells after PDT. The mode of cell death after Pc 181-PDT is predominantly apoptosis, and pancaspase and caspase-3 inhibitors prevent onset of the cell death. To assess further how lysosomes contribute to PDT, we monitored cell killing of A431cells after PDT in the presence and absence of bafilomycin, an inhibitor of the acidic vacuolar proton pump that collapses the pH gradient of the lysosomal/endosomal compartment. Bafilomycin by itself did not induce toxicity but greatly enhanced Pc 4-PDT-induced cell killing. In comparison to Pc 4, less enhancement of cell killing by bafilomycin occurred after Pc 181-PDT at photosensitizer doses producing equivalent cell killing in the absence of bafilomycin. These results indicate that lysosomal disruption can augment PDT with Pc 4, which targets predominantly mitochondria, but less so after PDT with Pc 181, since Pc 181 already targets lysosomes.

Photodynamic therapy (PDT) involves the irradiation of photosensitized cells with light. Depending on localization of the photosensitizing agent, the process can induce photodamage to the endoplasmic reticulum (ER), mitochondria, plasma membrane, and/or lysosomes. When ER or mitochondria are targeted, antiapoptotic proteins of the Bcl-2 family are especially sensitive to photodamage. Both apoptosis and autophagy can occur after PDT, autophagy being associated with enhanced survival at low levels of photodamage to some cells. Autophagy can become a cell-death pathway if apoptosis is inhibited or when cells attempt to recycle damaged constituents beyond their capacity for recovery. While techniques associated with characterization of autophagy are generally applicable, PDT introduces additional factors related to unknown sites of photodamage that may alter autophagic pathways. This chapter discusses issues that may arise in assessing autophagy after cellular photodamage.

10-N-Nonyl acridine orange (NAO) is used as a mitochondrial probe because of its high affinity for cardiolipin (CL). Targeting of NAO may also depend on mitochondrial membrane potential. As the nonyl group has been considered essential for targeting, a systematic study of alkyl chain length was undertaken; three analogues (10-methyl-, 10-hexyl-, and 10-hexadecyl-acridine orange) were synthesized and their properties studied in phospholipid monolayers and breast cancer cells. The shortest and longest alkyl chains reduced targeting, whereas the hexyl group was superior to the nonyl group, allowing very clear and specific targeting to mitochondria at concentrations of 20-100 nM, where no evidence of toxicity was apparent. Additional studies in wild-type and cardiolipin-deficient yeast cells suggested that cellular binding was not absolutely dependent upon cardiolipin.

Singlet oxygen ((1)O(2)) is a reactive oxygen species that may be generated in biological systems. Photodynamic therapy generates (1)O(2) by photoexcitation of sensitizers resulting in intracellular oxidative stress and induction of apoptosis. (1)O(2) oxidizes amino acid side chains of proteins and inactivates enzymes when generated in vitro. Among proteogenic amino acids, His, Tyr, Met, Cys, and Trp are known to be oxidized by (1)O(2) at physiological pH. However, there is a lack of direct evidence of oxidation of proteins by (1)O(2). Because (1)O(2) is difficult to detect in cells, identifying oxidized cellular products uniquely derived from (1)O(2) could serve as a marker of its presence. In the present study, (1)O(2) reactions with model peptides analyzed by tandem mass spectrometry provide insight into the mass of prominent adducts formed with the reactive amino acids. Analysis by MALDI-TOF and tandem mass spectrometry of peptides of cytochrome c exposed to (1)O(2) generated by photoexcitation of the phthalocyanine Pc 4 showed unique oxidation products, which might be used as markers of the presence of (1)O(2) in the mitochondrial intermembrane space. Differences in the elemental composition of the oxidized amino acid residues observed with cytochrome c and the model peptides suggest that the protein environment can affect the oxidation pathway.

To examine the clinical applicability of Pc 4, a promising second-generation photosensitizer, for the photodynamic treatment of lymphocyte-mediated skin diseases, we studied the A431 and Jurkat cell lines, commonly used as surrogates for human keratinocyte-derived carcinomas and lymphocytes, respectively. As revealed by ethyl acetate extraction and absorption spectrophotometry, uptake of Pc 4 into the two cell lines was linear with Pc 4 concentration and similar on a per cell basis but greater in Jurkat cells on a per mass basis. Flow cytometry showed that uptake was linear at low doses; variations in the dose-response for uptake measured by fluorescence supported differential aggregation of Pc 4 in the two cell types. As detected by confocal microscopy, Pc 4 localized to mitochondria and endoplasmic reticulum in both cell lines. Jurkat cells were much more sensitive to the lethal effects of phthalocyanine photodynamic therapy (Pc 4-PDT) than were A431 cells, as measured by a tetrazolium dye reduction assay, and more readily underwent morphological apoptosis. In a search for molecular factors to explain the greater photosensitivity of Jurkat cells, the fate of important Bcl-2 family members was monitored. Jurkat cells were more sensitive to the induction of immediate photodamage to Bcl-2, but the difference was insufficient to account fully for their greater sensitivity. The antiapoptotic protein Mcl-1 was extensively cleaved in a dose- and caspase-dependent manner in Jurkat, but not in A431, cells exposed to Pc 4-PDT. Thus, the greater killing by Pc 4-PDT in Jurkat compared with A431 cells correlated with greater Bcl-2 photodamage and more strongly to the more extensive Mcl-1 degradation. Pc 4-PDT may offer therapeutic advantages in targeting inflammatory cells over normal keratinocytes in the treatment of T-cell-mediated skin diseases, such as cutaneous lymphomas, dermatitis, lichenoid tissue reactions and psoriasis, and it will be instructive to evaluate the role of Bcl-2 family proteins, especially Mcl-1, in the therapeutic response.

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.

Photodynamic therapy (PDT) induces apoptosis in many cell types. Recent reports identified autophagy as an alternative cell-death process following PDT. Here we investigated the occurrence of autophagy after PDT with the photosensitizer Pc 4 in human cancer cells that are deficient in the pro-apoptotic factor Bax (human prostate cancer DU145) or the apoptosis mediator caspase-3 (human breast cancer MCF-7v) and in apoptosis-competent cells (MCF-7c3 stably overexpressing human pro-caspase-3 and Chinese hamster ovary CHO 5A100). Further, each cell line was also studied with and without stably overexpressed Bcl-2. By electron microscopy and immunoblot analysis, autophagy was observed in all cells studied, whether or not they were capable of typical apoptosis or overexpressed Bcl-2. Bcl-2 overexpression protected against PDT-induced apoptosis and loss of clonogenicity in apoptosis-competent cells (MCF-7c3 and CHO); however, it did not protect against the development of autophagy or against loss of clonogenicity in apoptosis-deficient cells (MCF-7v and DU145). The results show that autophagy may be the dominant cell death pathway following PDT in cells that are incapable of undergoing normal apoptosis. In such cells, Bcl-2 does not protect against autophagic death.

Photodynamic therapy (PDT) is emerging as a promising non-invasive treatment for cancers. PDT involves either local or systemic administration of a photosensitizing drug, which preferentially localizes within the tumor, followed by illumination of the involved organ with light, usually from a laser source. Here, we provide a selective overview of our experience with PDT at Case Western Reserve University, specifically with the silicon phthalocyanine photosensitizer Pc 4. We first review our in vitro studies evaluating the mechanism of cell killing by Pc 4-PDT. Then we briefly describe our clinical experience in a Phase I trial of Pc 4-PDT and our preliminary translational studies evaluating the mechanisms behind tumor responses. Preclinical work identified (a) cardiolipin and the anti-apoptotic proteins Bcl-2 and Bcl-xL as targets of Pc 4-PDT, (b) the intrinsic pathway of apoptosis, with the key participation of caspase-3, as a central response of many human cancer cells to Pc 4-PDT, (c) signaling pathways that could modify apoptosis, and (d) a formulation by which Pc 4 could be applied topically to human skin and penetrate at least through the basal layer of the epidermis. Clinical-translational studies enabled us to develop an immunohistochemical assay for caspase-3 activation, using biopsies from patients treated with topical Pc 4 in a Phase I PDT trial for cutaneous T-cell lymphoma. Results suggest that this assay may be used as an early biomarker of clinical response.

Introduction: High-field magnetic resonance imaging (MRI) is an emerging technique that provides a powerful, non-invasive tool for in vivo studies of cancer therapy in animal models. Photodynamic therapy (PDT) is a relatively new treatment modality for prostate cancer, the second leading cause of cancer mortality in American males. The goal of this study was to evaluate the response of human prostate tumor cells growing as xenografts in athymic nude mice to Pc 4-sensitized PDT., Materials and Methods: PC-3, a cell line derived from a human prostate malignant tumor, was injected intradermally on the back flanks of athymic nude mice. Two tumors were initiated on each mouse. One was treated and the other served as the control. A second-generation photosensitizing drug Pc 4 (0.6 mg/kg body weight) was delivered to each animal by tail vein injection 48 hours before laser illumination (672 nm, 100 mW/cm(2), 150 J/cm(2)). A dedicated high-field (9.4 T) small-animal MR scanner was used for image acquisitions. A multi-slice multi-echo (MSME) technique, permitting noninvasive in vivo assessment of potential therapeutic effects, was used to measure the T2 values and tumor volumes. Animals were scanned immediately before and after PDT and 24 hours after PDT. T2 values were computed and analyzed for the tumor regions., Results: For the treated tumors, the T2 values significantly increased (P<0.002) 24 hours after PDT (68.2+/- 8.5 milliseconds), compared to the pre-PDT values (55.8+/-6.6 milliseconds). For the control tumors, there was no significant difference (P = 0.53) between the pre-PDT (52.5+/-6.1 milliseconds) and 24-hour post-PDT (54.3+/-6.4 milliseconds) values. Histologic analysis showed that PDT-treated tumors demonstrated necrosis and inflammation that was not seen in the control., Discussion: Changes in tumor T2 values measured by multi-slice multi-echo MR imaging provide an assay that could be useful for clinical monitoring of photodynamic therapy of prostate tumors., (2007 Wiley-Liss, Inc)

Photodynamic therapy (PDT) is an efficient inducer of apoptosis in many types of cells, except in cells deficient in one or more of the factors that mediate apoptosis. Recent reports have identified autophagy as a potential alternative cell death process following PDT. Here we investigated the occurrence of autophagy after PDT with the photosensitizer Pc 4 in human cancer cells that are deficient in the pro-apoptotic factor Bax (human prostate cancer DU145 cells) or the apoptosis mediator caspase-3 (human breast cancer MCF-7v cells) and in apoptosis-competent cells (MCF-7c3 cells that stably overexpress human pro-caspase-3 and Chinese hamster ovary CHO 5A100 cells). Further, each of the cell lines was also studied with and without stably overexpressed Bcl-2. Autophagy was identified by electron microscopic observation of the presence of double-membrane-delineated autophagosomal vesicles in the cytosol and by immunoblot observation of the Pc 4-PDT dose- and time-dependent increase in the level of LC3-II, a component of the autophagosomal membrane. Autophagy was observed in all of the cell lines studied, whether or not they were capable of typical apoptosis and whether or not they overexpressed Bcl-2. The presence of stably overexpressed Bcl-2 in the cells protected against PDT-induced apoptosis and loss of clonogenicity in apoptosis-competent cells (MCF-7c3 and CHO 5A100 cells). In contrast, Bcl-2 overexpression did not protect against the development of autophagy in any of the cell lines or against loss of clonogenicity in apoptosis-deficient cells (MCF-7v and DU145 cells). Furthermore, 3-methyladenine and wortmannin, inhibitors of autophagy, provided greater protection against loss of viability to apoptosis-deficient than to apoptosis-competent cells. The results show that autophagy occurs during cell death following PDT in human cancer cells competent or not for normal apoptosis. Only the apoptosis-competent cells are protected by Bcl-2 against cell death.

We are developing in vivo small animal imaging techniques that can measure early effects of photodynamic therapy (PDT) for prostate cancer. PDT is an emerging therapeutic modality that continues to show promise in the treatment of cancer. At our institution, a new second-generation photosensitizing drug, the silicon phthalocyanine Pc 4, has been developed and evaluated at the Case Comprehensive Cancer Center. In this study, we are developing magnetic resonance imaging (MRI) techniques that provide therapy monitoring and early assessment of tumor response to PDT. We generated human prostate cancer xenografts in athymic nude mice. For the imaging experiments, we used a high-field 9.4-T small animal MR scanner (Bruker Biospec). High-resolution MR images were acquired from the treated and control tumors pre- and post-PDT and 24 hr after PDT. We utilized multi-slice multi-echo (MSME) MR sequences. During imaging acquisitions, the animals were anesthetized with a continuous supply of 2% isoflurane in oxygen and were continuously monitored for respiration and temperature. After imaging experiments, we manually segmented the tumors on each image slice for quantitative image analyses. We computed three-dimensional T2 maps for the tumor regions from the MSME images. We plotted the histograms of the T2 maps for each tumor pre- and post-PDT and 24 hr after PDT. After the imaging and PDT experiments, we dissected the tumor tissues and used the histologic slides to validate the MR images. In this study, six mice with human prostate cancer tumors were imaged and treated at the Case Center for Imaging Research. The T2 values of treated tumors increased by 24 ± 14% 24 hr after the therapy. The control tumors did not demonstrate significant changes of the T2 values. Inflammation and necrosis were observed within the treated tumors 24 hour after the treatment. Preliminary results show that Pc 4-PDT is effective for the treatment of human prostate cancer in mice. The small animal MR imaging provides a useful tool to evaluate early tumor response to photodynamic therapy in mice.

ATX-s10 is a novel and second-generation photosensitizer for photodynamic therapy (PDT). In order to conduct clinical trials of ATX-s10-PDT and/or extend its clinical applications, it is very important to elucidate the mechanisms of the action of ATX-s10-PDT. We examined the apoptic response against ATX-s10-PDT using a Bcl-2 or Bcl-2 mutant overexpressing cells. Using fluorescent microscopy, ATX-s10 localized not only to mitochondria but also to lysosomes and possibly other intracellular organelles, but not to the plasma membrane or the nucleus. These results suggest that ATX-s10-PDT can damage mitochondria and lysosomes. By Western blot analysis, ATX-s10-PDT damaged Bcl-2, which localized preferentially at mitochondrial membranes, and caused Bcl-2 to cross-link immediately after laser irradiation. However, ATX-s10-PDT was not able to rapidly induce morphologically typical apoptosis (i.e. chromatin condensation and fragmentation) as PDT using mitochondria targeted photosensitizers, such as phthalocyanine 4 (Pc 4). Pharmacological inhibitions of lysosomal cytokine protease cathepsins, such as cathepsin B and D, protected MCF-7c3 cells (human breast cancer cells expressing stably transfected procaspase-3) from apoptosis caused by ATX-s10-PDT. Overexpression of wild-type Bcl-2 or Bcl-2Delta33-54 resulted in relative resistance of cells to ATX-s10-PDT, as assessed by the degree of morphological apoptosis or loss of clonogenicity. We conclude that lysosomal damage by ATX-s10-PDT can initiate apoptotic response and this apoptotic pathway can be regulated by photodamage to Bcl-2 via mitochondrial damage.

We are investigating imaging techniques to study the tumor response to photodynamic therapy (PDT). Positron emission tomography (PET) can provide physiological and functional information. High-resolution magnetic resonance imaging (MRI) can provide anatomical and morphological changes. Image registration can combine MRI and PET images for improved tumor monitoring. In this study, we acquired high-resolution MRI and microPET 18F-fluorodeoxyglucose (FDG) images from C3H mice with RIF-1 tumors that were treated with Pc 4-based PDT. We developed two registration methods for this application. For registration of the whole mouse body, we used an automatic three-dimensional, normalized mutual information algorithm. For tumor registration, we developed a finite element model (FEM)-based deformable registration scheme. To assess the quality of whole body 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. Over 40 volume registration experiments were performed with MRI and microPET images. The distance between corresponding centroids of organs was 1.5 +/- 0.4 mm which is about 2 pixels of microPET images. The mean volume overlap ratios for tumors were 94.7% and 86.3% for the deformable and rigid registration methods, respectively. Registration of high-resolution MRI and microPET images combines anatomical and functional information of the tumors and provides a useful tool for evaluating photodynamic therapy.

Cell death following photodynamic therapy (PDT) with the photosensitizer Pc 4 involves the intrinsic pathway of apoptosis. To evaluate the importance of Bax in apoptosis after PDT, we compared the PDT responses of Bax-proficient (Bax(+/-)) and Bax knock-out (BaxKO) HCT116 human colon cancer cells. PDT induced a slow apoptotic process in HCT Bax(+/-) cells following a long delay in the activation of Bax and release of cytochrome c from mitochondria. Although cytochrome c was not released from mitochondria following PDT in BaxKO cells, an alternative mechanism of caspase-dependent apoptosis with extensive chromatin and DNA degradation was found in these cells. This alternative process was less efficient and slower than the normal apoptotic process observed in Bax(+/-) cells. Early events upon PDT, such as the loss of mitochondrial membrane potential, photodamage to Bcl-2, and activation of p38 MAP kinase, were observed in both HCT116 cell lines. In spite of differences in the efficiency and mode of apoptosis induced by PDT in the Bax(+/-) and BaxKO cells, they were found to be equally sensitive to killing by PDT, as determined by loss of clonogenicity. Thus, for Pc 4-PDT, the commitment to cell death occurs prior to and independent of Bax activation, but the process of cellular disassembly differs in Bax-expressing vs. non-expressing cells.

The antiapoptotic Bcl-2-family proteins, Bcl-2 and Bcl-xL, are recognized phototargets of photodynamic therapy (PDT) with the mitochondrion-targeting phthalocyanine photosensitizer Pc 4. In the present study, we found that myeloid cell leukemia 1 (Mcl-1), another antiapoptotic member of the Bcl-2 family, was not photodamaged in Pc 4-PDT-treated human carcinoma cells MCF-7c3, MDA-MB468, DU145, and A431, although Mcl-1 turnover was observed after exposure of HeLa or MCF-7c3 cells to a supralethal dose of UVC. In contrast, when human lymphoma U937 and Jurkat cells were treated with Pc 4-PDT, staurosporine (STS) or UVC, Mcl-1 was cleaved to generate a 28-kDa fragment over a 2-4 h period. The cleavage of Mcl-1 was accompanied by the activation of caspases-3, -9, and -8. The broad-specificity caspase inhibitor z-VAD-fmk completely blocked Mcl-1 cleavage induced by PDT, STS or UVC, providing evidence for Mcl-1 as a substrate for caspases. Western blot analysis localized Mcl-1 to mitochondria, ER, and cytosol of both MCF-7c3 and U937 cells, suggesting that Mcl-1 protein, unlike Bcl-2 and Bcl-xL, is not a target for Pc 4-PDT, probably due to its localization to sites removed from those of Pc 4 binding. The 28-kDa cleaved fragment of Mcl-1, which has proapoptotic activity, was produced in PDT-treated lymphoid-derived cells, but not in cells of epithelial origin, suggesting that PDT-induced rapid and extensive apoptosis in lymphoma cells may result in part from the sensitivity of their Mcl-1 to caspase cleavage, removing an important negative control on apoptosis.

Background and Objectives: As a potential therapy for malignant glioma, we tested the phthalocyanine photosensitizer Pc 4 for: (1) rapid clearance from the vasculature, (2) specificity for glioma, and (3) tumoricidal photosensitizing capability., Study Design/materials and Methods: Parenchymal injection of U87 cells into athymic rat brains (N = 100) was followed after 12 days by tail vein injection of 0.5 mg/kg Pc 4. After 1 day, the tumor was illuminated with either 5 (N = 11) or 30 (N = 16) J/cm(2) red light at 672 nm. Sacrifice was 1 day later. The brains from these 27 animals underwent H&E (necrosis) and TUNEL assay (apoptosis) histology. Pc 4 concentration of explanted brains and tumors (N = 16), and all blood samples (N = 52) were determined by HPLC-MS 1 day post Pc 4 administration., Results: Tumor-specific apoptosis was almost uniformly seen; however, necrosis was found mostly in the high-light-dose group. Pc 4 concentration in bulk tumor averaged 3.8 times greater than in normal brain., Conclusions: These results warrant expanding this pre-clinical study to seek effective baseline Pc 4 drug- and light-doses and infusion-to-photoirradiation timing that would be necessary for a Pc 4-mediated PDT clinical trial for glioma patients., (Copyright 2005 Wiley-Liss, Inc.)

The antiapoptotic oncoprotein Bcl-2 is now a recognized phototarget of photodynamic therapy (PDT) with the phthalocyanine Pc 4 and with other mitochondrion-targeting photosensitizers. Photodamage, observed on Western blots as the loss of the native 26-kDa Bcl-2 protein, is PDT dose dependent and occurs in multiple cell lines, in the cold, and immediately upon photoirradiation. In our initial study, no photochemical damage was observed to Bcl-xL, in spite of its similarity in size, sequence, location and function to Bcl-2. The original study used a commercial anti-Bcl-xS/L antibody. We have revisited this issue by examining Western blots developed using one of three epitope-specific anti-Bcl-xL antibodies from commercial sources, a polyclonal antibody generated to the entire protein, as well as the antibody used previously. All five Bcl-xL antibodies recognized bacterially expressed Bcl-xL, but not Bcl-2, whereas an anti-Bcl-2 antibody recognized Bcl-2 and not Bcl-xL. All five Bcl-xL antibodies recognized at least one protein migrating at approximately 30 kDa; two of the antibodies recognized an additional band, migrating at approximately 33 or approximately 24 kDa. We now observe Pc 4-PDT-induced photodamage to all Bcl-xL-related proteins, except the 33-kDa species, in several human cancer cell lines. The results indicate that, in addition to the expected quantitative differences that may reflect exposure of individual epitopes, the antibodies also detect proteins of different apparent molecular weights that may be distinct isoforms or post-translationally modified forms of Bcl-xL. No evidence for PDT-induced phosphorylation or degradation was observed. Bcl-xL localized to mitochondria was considerably more sensitive to photodamage than was Bcl-xL in the cytosol, indicating that as previously found for Bcl-2, Bcl-xL must be membrane localized to be photosensitive.