Adding Base-Excision Repair Inhibitor TRC102 to Standard Pemetrexed-Platinum-Radiation in Patients with Advanced Nonsquamous Non-Small Cell Lung Cancer: Results of a Phase I Trial.

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.

Ultra-high dose rate effect on circulating immune cells: A potential mechanism for FLASH effect?

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.

Structure-guided design of anti-cancer ribonucleotide reductase inhibitors.

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 KD's and more predicted residue interactions. Moreover, TP6 displayed the greatest growth inhibiting effect in the Panc1 pancreatic cancer cell line with an IC50 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.

Radiation Therapy Combined with Cowpea Mosaic Virus Nanoparticle in Situ Vaccination Initiates Immune-Mediated Tumor Regression.

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.

ATR/CHK1 inhibitors and cancer therapy.

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.

Cell Death Pathways Associated with Photodynamic Therapy: An Update.

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.

Potent competitive inhibition of human ribonucleotide reductase by a nonnucleoside small molecule.

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 IC50 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.

Radiosensitization of non-small-cell lung cancer cells and xenografts by the interactive effects of pemetrexed and methoxyamine.

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.

Activated T cells exhibit increased uptake of silicon phthalocyanine Pc 4 and increased susceptibility to Pc 4-photodynamic therapy-mediated cell death.

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.

Identification of Non-nucleoside Human Ribonucleotide Reductase Modulators.

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.

Cutaneous penetration of the topically applied photosensitizer Pc 4 as detected by intravital 2-photon laser scanning microscopy.

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.

Optimization of a nanomedicine-based silicon phthalocyanine 4 photodynamic therapy (Pc 4-PDT) strategy for targeted treatment of EGFR-overexpressing cancers.

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.

EGFR-mediated intracellular delivery of Pc 4 nanoformulation for targeted photodynamic therapy of cancer: in vitro studies.

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.

Photodynamic therapy with Pc 4 induces apoptosis of Candida albicans.

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.

Radiosensitization of human cervical cancer cells by inhibiting ribonucleotide reductase: enhanced radiation response at low-dose rates.

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.

Preliminary clinical and pharmacologic investigation of photodynamic therapy with the silicon phthalocyanine photosensitizer pc 4 for primary or metastatic cutaneous cancers.

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.

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

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.

Photosensitization of intact heart mitochondria by the phthalocyanine Pc 4: Correlation of structural and functional deficits with cytochrome c release.

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.

Photo-oxidation of cardiolipin and cytochrome c with bilayer-embedded Pc 4.

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.

Photodynamic therapy and cell death pathways.

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.