A Pre-implementation Study of Volumetric Modulated Arc Therapy for Same-day Planning and Treatment of Vertebral Bone Metastases Within a Rapid-access Palliative Radiotherapy Programme.

AIMS: We aimed to develop a process for same-day contouring, planning, quality assurance and delivery of volumetric modulated arc therapy (VMAT) for vertebral bone metastases within our institution's rapid-access palliative radiotherapy programme. MATERIALS AND METHODS: Two thoracic (T6-7, T3-7) and two lumbar (L2-3, L1-5) targets were contoured on computed tomography images acquired from an anthropomorphic phantom and five patient scans. Inverse planning aimed to provide coverage of a prescribed dose of 8 Gy with a combined lung V2Gy < 25% and a combined kidney mean dose <2 Gy. Serial plans were created to identify an efficient combination of six main planning variables specific to our treatment planning system: (i) voxel size (3 mm versus 5 mm), (ii) Monte Carlo statistical uncertainty (1% per calculation versus 3% per control point), (iii) fluence smoothing (medium versus high), (iv) number of iterations of segment shape changes during optimisation (1 versus 5), (v) dose calculation algorithm (Monte Carlo versus pencil beam) and (vi) number of arcs (single versus multiple). Contouring, planning, quality assurance and treatment delivery were timed. RESULTS: The combination of planning variables deemed efficient and appropriate was: a 3 mm voxel size, statistical uncertainty of 1% per calculation, medium fluence smoothing, five iterations of segment shape changes, Monte Carlo dose calculation and single full arc delivery. Patient scan contouring times ranged from 7 to 9 min (T6-7), 11-13 min (T3-7), 5-7 min (L2-3) and 8-10 min (L1-5) and planning times ranged from 9 to 15 min (T6-7), 13-25 min (T3-7), 18-25 min (L2-3) and 21-31 min (L1-5). Physics quality assurance times ranged from 15 to 21 min and beam-on times ranged from 3 to 6 min. CONCLUSIONS: The combined elements of VMAT for thoracic and lumbar vertebral bone metastases were completed in under 2 h. This new process makes same-day contouring, planning, quality assurance and treatment delivery of VMAT feasible within our rapid-access palliative radiotherapy programme.

Cleavage of DAP5 by coxsackievirus B3 2A protease facilitates viral replication and enhances apoptosis by altering translation of IRES-containing genes.

Cleavage of eukaryotic translation initiation factor 4G (eIF4G) by enterovirus proteases during infection leads to the shutoff of cellular cap-dependent translation, but does not affect the initiation of cap-independent translation of mRNAs containing an internal ribosome entry site (IRES). Death-associated protein 5 (DAP5), a structural homolog of eIF4G, is a translation initiation factor specific for IRES-containing mRNAs. Coxsackievirus B3 (CVB3) is a positive single-stranded RNA virus and a primary causal agent of human myocarditis. Its RNA genome harbors an IRES within the 5'-untranslated region and is translated by a cap-independent, IRES-driven mechanism. Previously, we have shown that DAP5 is cleaved during CVB3 infection. However, the protease responsible for cleavage, cleavage site and effects on the translation of target genes during CVB3 infection have not been investigated. In the present study, we demonstrated that viral protease 2A but not 3C is responsible for DAP5 cleavage, generating 45- and 52-kDa N- (DAP5-N) and C-terminal (DAP5-C) fragments, respectively. By site-directed mutagenesis, we found that DAP5 is cleaved at amino acid G434. Upon cleavage, DAP5-N largely translocated to the nucleus at the later time points of infection, whereas the DAP5-C largely remained in the cytoplasm. Overexpression of these DAP5 truncates demonstrated that DAP5-N retained the capability of initiating IRES-driven translation of apoptosis-associated p53, but not the prosurvival Bcl-2 (B-cell lymphoma 2) when compared with the full-length DAP5. Similarly, DAP5-N expression promoted CVB3 replication and progeny release; on the other hand, DAP5-C exerted a dominant-negative effect on cap-dependent translation. Taken together, viral protease 2A-mediated cleavage of DAP5 results in the production of two truncates that exert differential effects on protein translation of the IRES-containing genes, leading to enhanced host cell death.

Serpina3n accelerates tissue repair in a diabetic mouse model of delayed wound healing.

Chronic, non-healing wounds are a major complication of diabetes and are characterized by chronic inflammation and excessive protease activity. Although once thought to function primarily as a pro-apoptotic serine protease, granzyme B (GzmB) can also accumulate in the extracellular matrix (ECM) during chronic inflammation and cleave ECM proteins that are essential for proper wound healing, including fibronectin. We hypothesized that GzmB contributes to the pathogenesis of impaired diabetic wound healing through excessive ECM degradation. In the present study, the murine serine protease inhibitor, serpina3n (SA3N), was administered to excisional wounds created on the dorsum of genetically induced type-II diabetic mice. Wound closure was monitored and skin wound samples were collected for analyses. Wound closure, including both re-epithelialization and contraction, were significantly increased in SA3N-treated wounds. Histological and immunohistochemical analyses of SA3N-treated wounds revealed a more mature, proliferative granulation tissue phenotype as indicated by increased cell proliferation, vascularization, fibroblast maturation and differentiation, and collagen deposition. Skin homogenates from SA3N-treated wounds also exhibited greater levels of full-length intact fibronectin compared with that of vehicle wounds. In addition, GzmB-induced detachment of mouse embryonic fibroblasts correlated with a rounded and clustered phenotype that was prevented by SA3N. In summary, topical administration of SA3N accelerated wound healing. Our findings suggest that GzmB contributes to the pathogenesis of diabetic wound healing through the proteolytic cleavage of fibronectin that is essential for normal wound closure, and that SA3N promotes granulation tissue maturation and collagen deposition.

Granzyme B degrades extracellular matrix and contributes to delayed wound closure in apolipoprotein E knockout mice.

Chronic inflammation and excessive protease activity have a major role in the persistence of non-healing wounds. Granzyme B (GzmB) is a serine protease expressed during chronic inflammation that, in conjunction with perforin, has a well-established role in initiating apoptotic cell death. GzmB is also capable of acting extracellularly, independent of perforin and can degrade several extracellular matrix (ECM) proteins that are critical during wound healing. We used apolipoprotein E (ApoE) knockout (AKO) mice as a novel model of chronic inflammation and impaired wound healing to investigate the role of GzmB in chronic wounds. Wild-type and AKO mice were grown to 7 weeks (young) or 37 weeks (old) of age on a regular chow or high-fat diet (HFD), given a 1-cm diameter full thickness wound on their mid dorsum and allowed to heal for 16 days. Old AKO mice fed a HFD exhibited reduced wound closure, delayed contraction, chronic inflammation and altered ECM remodeling. Conversely, GzmB/ApoE double knockout mice displayed improved wound closure and contraction rates. In addition, murine GzmB was found to degrade both fibronectin and vitronectin derived from healthy mouse granulation tissue. In addition, GzmB-mediated degradation of fibronectin generated a fragment similar in size to that observed in non-healing mouse wounds. These results provide the first direct evidence that GzmB contributes to chronic wound healing in part through degradation of ECM.

SU-E-T-22: Is the Residual Range a Universal Quantity to Specify the Quality of Modulated Proton Beams?

PURPOSE: To investigate the validity of using the residual range as a universal quantity to specify the quality of modulated proton beams. METHODS: We used TOPAS (Tool for Particle Simulation), an application of the Geant4 toolkit, to simulate absorbed dose and stopping-power distributions from a commercial passive scattering nozzle. We used the standard physics lists from Geant4 in the simulations. All particles were included, as well as physics models for nuclear interactions. No variance reduction techniques were used. Dose and averaged stopping-power as functions of depth were scored in a water box with 320 scoring volumes of 15 × 15 × 0.1 cm3 . Stopping-power spectra were scored in a15 × 15 × 0.1 cm3 volume located in the middle of SOBPs. All particles were considered in the dose scoring. Only protons (primary and secondary) were considered in the scoring of stopping-power. RESULTS: For the same residual range, differences in averaged stopping-power values of up to 13% were observed for a 200 MeV beam with modulations of 4 cm and 8 cm, respectively. Simulations of four modulated proton energies with the same SOBP of 8 cm showed differences of up to 13% in the averaged stopping-power values even in the SOBP region. We also simulated stopping power spectra in the middle of 8 cm SOBPs for four modulated proton energies. The averaged stopping-power values calculated from the spectra were within 3%, however, their distributions were very different with full width at half-maximum 150% larger for the 250 MeV beam compared to that of the 140 MeV beam. CONCLUSION: Large differences in the averaged stopping-power values and stopping-power spectra were observed for the same residual range. Determining whether these differences have a significant effect on the response of radiation detectors exposed to proton beams requires further investigation. Natural Sciences and Engineering Research Council of Canada and Ontario Graduate Scholarship Program, Ontario Ministry of Training, Colleges and Universities.

Poster - Thur Eve - 43: Is faster always better? An evaluation of frame rate effects on continuous acquisition mode EPID imaging for dose verification.

INTRODUCTION: As radiotherapy moves towards intensity modulated arc therapy (arc-IMRT), there is a need for electronic portal imaging (EPID) to move towards continuous acquisition (cine) mode for dosimetric verification purposes. However, as the EPID resolution and frame rate (fps) increase, so does the computational burden of image processing. We investigated the reliability of cine mode EPID imaging in IMRT as a function of frame rate. METHODS: We acquired EPID images continuously while running an IMRT plan (6MV photons, 150MU, dose rate = 300MU/min) with frame rates ranging from 1-12 fps, as well as a single integrated mode image. Each cine dataset was then averaged to form a single image, which was compared with the integrated mode image by means of a pixel-by-pixel absolute value subtraction. RESULTS: Although a greater frame rate gave better agreement with the integrated mode image in all cases, the relative benefit diminished with increasing frame rate. In particular, for the IMRT plan delivered, there was little benefit of imaging faster than 6 fps, and virtually no benefit in increasing from 9 to 12 fps. In contrast, 12 fps produces twice the number of images as 6 fps which significantly increases the image processing and data storage burdens. CONCLUSION: Increasing frame rate in cine mode EPID imaging may be beneficial in some cases, but there is likely a threshold level above which no relevant additional information is obtained. Further research to determine the ideal frame rate for any particular IMRT or arc-IMRT plan is warranted.

Serpina3n attenuates granzyme B-mediated decorin cleavage and rupture in a murine model of aortic aneurysm.

Granzyme B (GZMB) is a proapoptotic serine protease that is released by cytotoxic lymphocytes. However, GZMB can also be produced by other cell types and is capable of cleaving extracellular matrix (ECM) proteins. GZMB contributes to abdominal aortic aneurysm (AAA) through an extracellular, perforin-independent mechanism involving ECM cleavage. The murine serine protease inhibitor, Serpina3n (SA3N), is an extracellular inhibitor of GZMB. In the present study, administration of SA3N was assessed using a mouse Angiotensin II-induced AAA model. Mice were injected with SA3N (0-120 µg/kg) before pump implantation. A significant dose-dependent reduction in the frequency of aortic rupture and death was observed in mice that received SA3N treatment compared with controls. Reduced degradation of the proteoglycan decorin was observed while collagen density was increased in the aortas of mice receiving SA3N treatment compared with controls. In vitro studies confirmed that decorin, which regulates collagen spacing and fibrillogenesis, is cleaved by GZMB and that its cleavage can be prevented by SA3N. In conclusion, SA3N inhibits GZMB-mediated decorin degradation leading to enhanced collagen remodelling and reinforcement of the adventitia, thereby reducing the overall rate of rupture and death in a mouse model of AAA.

Granzymes in age-related cardiovascular and pulmonary diseases.

Chronic inflammation is a hallmark of age-related cardiovascular and pulmonary diseases. Granzymes are a family of serine proteases that have been traditionally viewed as initiators of immune-mediated cell death. However, recent findings suggest that the pathophysiological role of granzymes is complex. Emerging functions for granzymes in extracellular matrix degradation, autoimmunity, and inflammation suggests a multifactorial mechanism by which these enzymes are capable of mediating tissue damage. Recent discoveries showing that granzymes can be produced and secreted by nonimmune cells during disease provide an additional layer of intricacy. This review examines the emerging biochemical and clinical evidence pertaining to intracellular and/or extracellular granzymes in the pathogenesis of aging and cardiopulmonary diseases.

Moderate exercise attenuates caspase-3 activity, oxidative stress, and inhibits progression of diabetic renal disease in db/db mice.

Diabetic nephropathy, the leading cause of end-stage renal disease, is characterized by a proapoptotic and prooxidative environment. The mechanisms by which lifestyle interventions, such as exercise, benefit diabetic nephropathy are unknown. We hypothesized that exercise inhibits early diabetic nephropathy via attenuation of the mitochondrial apoptotic pathway and oxidative damage. Type 2 diabetic db/db and normoglycemic wild-type mice were exercised for an hour everyday at a moderate intensity for 7 wk, following which renal function, morphology, apoptotic signaling, and oxidative stress were evaluated. Exercise reduced body weight, albuminuria, and pathological glomerular expansion in db/db mice independent of hyperglycemic status. Changes in renal morphology were also related to reduced caspase-3 (main effector caspase in renal apoptosis), caspase-8 (main initiator caspase of the "extrinsic" pathway) activities, and TNF-alpha expression. A role for the mitochondrial apoptotic pathway was unlikely as both caspase-9 activity (initiator caspase of this pathway) and expression of regulatory proteins such as Bax and Bcl-2 were unchanged. Kidneys from db/db mice also produced higher levels of superoxides and had greater oxidative damage concurrent with downregulation of superoxide dismutase (SOD) 1 and 3. Interestingly, although exercise also increased superoxides, there was also upregulation of multiple SODs that likely inhibited lipid (hydroperoxides) and protein (carbonyls and nitrotyrosine) oxidation in db/db kidneys. In conclusion, exercise can inhibit progression of early diabetic nephropathy independent of hyperglycemia. Reductions in caspase-3 and caspase-8 activities, with parallel improvements in SOD expression and reduced oxidative damage, could underlie the beneficial effects of exercise in diabetic kidney disease.

Cytochrome p450 2C enzymes contribute to peritransplant ischemic injury and cardiac allograft vasculopathy.

Peritransplant ischemia and reperfusion (I/R) injury contributes to posttransplant vascular dysfunction and cardiac allograft vasculopathy (CAV). We have previously shown that cytochrome p450 (CYP) 2C inhibition significantly reduces I/R-induced myocardial infarction and postischemic vascular dysfunction. In the latter study, pretreatment with sulfaphenazole (SP), a specific inhibitor of CYP 2C, restored postischemic NO-mediated, endothelium-dependent vasodilation and reduced vascular superoxide production. Given the association between I/R injury, early vascular dysfunction and CAV, we hypothesized that CYP 2C may also contribute to the onset of CAV. Lewis-to-Fisher rat heterotopic heart transplants were performed. Donors and recipients were treated with 5 mg/kg SP or vehicle control 1 h prior to surgery. SP did not affect posttransplant morbidity, mortality or weight gain. Coronary blood vessels from rats treated with SP exhibited significantly reduced luminal narrowing and demonstrated a corresponding decrease in smooth muscle cell (SMC) proliferation compared to controls. SP did not reduce diffuse, focal, epicardial, endocardial or perivascular immune infiltration nor did it significantly alter TUNEL positivity in myocardial, endothelial or SMC populations. In conclusion, CYP 2C contributes to SMC proliferation CAV without affecting general immune infiltration.

Rapid induction of apoptosis in human keratinocytes with the photosensitizer QLT0074 via a direct mitochondrial action.

QLT0074 is a newly introduced, porphyrin-derivative for use in photodynamic therapy (PDT). In the current study, the intracellular distribution of QLT0074 and the mode of cell death induced by photosensitization with this compound in vitro were assessed for transformed human HaCaT keratinocytes. Fluorescence microscopy studies indicated a distribution of the drug to the cytoplasm, nuclear membrane and mitochondria of these cells. In the absence of light, QLT0074 produced no evidence of apoptosis-related biochemical changes or affected cell viability. When combined with blue light exposure, cytotoxicity was exerted in a QLT0074- and light-dose-related manner. Appearance of the mitochondrial protein cytochrome c in the cytosolic fraction and expression of the apoptosis-associated mitochondrial 7A6 antigen were demonstrable following photosensitization at nano-molar levels of QLT0074. Evidence of processing of the apoptosis-effector molecules caspase-3, -6, -7, -8 and -9 as well as cleavage of the caspase-3 substrate poly (ADP-ribose) polymerase (PARP) were demonstrable subsequent to cytochrome c release after PDT. Treatment with the anti-oxidant pyrrolidine dithiocarbamate (PDTC) inhibited cytochrome c release, caspase-3 activation and PARP cleavage associated with PDT thereby supporting the contention that QLT0074 induces apoptosis through the generation of reactive oxygen species upon light activation. QLT0074 is a potent photosensitizer with the capacity to directly initiate apoptosis by acting upon mitochondria.

Bcl-2 increases emptying of endoplasmic reticulum Ca2+ stores during photodynamic therapy-induced apoptosis.

Photodynamic therapy (PDT) is clinically approved for the treatment of several types of cancer as well as age-related macular degeneration, the leading cause of blindness in the elderly. PDT using the photosensitizer verteporfin has been previously shown to induce rapid apoptosis via a mitochondrial-caspase activation pathway. The impact of PDT on other cellular organelles such as the endoplasmic reticulum (ER) is undefined. The effect of PDT on intracellular Ca2+ ([Ca2+]i) in control and Bcl-2-overexpressing HeLa cells was assessed. A greater [Ca2+]i transient was observed for Bcl-2 overexpressing cells in response to PDT. The PDT-induced Ca2+ release was due to the emptying of Ca2+ from ER and possibly mitochondrial stores and was not due to an influx of Ca2+ from the medium. For Bcl-2-transfected cells, the release of Ca2+ was incomplete as determined by a further [Ca2+]i transient produced by the addition of the Ca2+ ionophore ionomycin after PDT. Furthermore, extrusion of Ca2+ was not hindered while ER-mediated sequestration of Ca2+ was impaired after PDT. Impairment of ER-mediated sequestration of Ca2+ may be due to the immediate caspase-independent depletion of sarco/endoplasmic reticulum Ca2+ ATPase-2 (SERCA2) that occurred in response to PDT in birth HeLa/Neo and Bcl-2 overexpressed HeLa cells. In summary, PDT induced the rapid degradation of SERCA2 and release of ER and mitochondrial Ca2+ stores. Although overexpression of Bcl-2 did not protect against SERCA2 degradation, it may influence the release of Ca2+ from ER and mitochondrial stores in PDT-treated cells.

Fas ligand and TRAIL augment the effect of photodynamic therapy on the induction of apoptosis in JURKAT cells.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) and Fas ligand (FasL) trigger apoptosis by stimulating the formation of a death inducing signaling complex at the cytoplasmic terminus of their respective receptors. Photodynamic therapy (PDT) is an approved treatment for several types of cancer as well as for age-related macular degeneration and is under investigation for different cancer, ocular, autoimmune and cardiovascular indications. The effect of low dose PDT in combination with TRAIL and FasL on Jurkat lymphoma cell apoptosis was examined. Individually, TRAIL, FasL, and PDT could induce apoptosis in these cells. However, at suboptimal levels of PDT, the number of cells undergoing apoptosis was increased when recombinant FasL and/or TRAIL were added. Additive effects of these treatments were evident for different apoptosis parameters including DNA fragmentation, caspase processing and activity and caspase substrate degradation. Overall, these results provide evidence that PDT-treated cells may be more likely to undergo apoptosis when also exposed to receptor-mediated signals delivered by factors such as TRAIL or FasL. For PDT, immune cell-mediated death receptor ligation may represent a way whereby tumor cells that have withstood the direct effects of photosensitization may be eliminated.

Endothelial cell apoptosis: biochemical characteristics and potential implications for atherosclerosis.

The high turnover of endothelial cells (EC) in atherosclerosis suggests that an increase in the frequency of both cell proliferation and cell death is important in the pathogenesis of this common disorder. Further, increased apoptosis of EC, smooth muscle cells (SMC) and immune cells has been observed in atheromatous plaques. Many pro-atherogenic factors, including oxidized low-density lipoproteins, angiotensin II and oxidative stress, can induce EC apoptosis. Such damage to the endothelium may be an initiating event in atherogenesis since EC apoptosis may compromise vasoregulation, increase SMC proliferation, SMC migration and blood coagulation. In addition, EC overlying vascular lesions have been shown to increase their expression of pro-apoptotic proteins, such as Fas and Bax, while decreasing levels of anti-apoptotic factors. Therefore, understanding EC apoptotic pathways that are altered in atherosclerosis may enable a greater understanding of disease pathogenesis and foster the development of new therapies. The present discussion outlines the biochemical characteristics of EC apoptosis and the role that altered regulation of apoptosis plays in vasculopathy.

Mechanism of colon cancer cell apoptosis mediated by pyropheophorbide-a methylester photosensitization.

Pyropheophorbide-a methylester (PPME) is a second generation of photosensitizers used in photodynamic therapy (PDT). We demonstrated that PPME photosensitization triggered apoptosis of colon cancer cells as measured by using several classical parameters such as DNA laddering, PARP cleavage, caspase activation and mitochondrial release of cytochrome c. Preincubation of cells with N-acetyl cysteine (NAC) or pyrolidine dithiocarbamate (PDTC) protected against apoptosis mediated by PPME photosensitization showing that reactive oxygen species (ROS) are involved as second messengers. On the other hand, photosensitization carried out in the presence of deuterium oxide (D2O) which enhances singlet oxygen (1O2) lifetime only increases necrosis without affecting apoptosis. Since PPME was localized in the endoplasmic reticulum (ER)/Golgi system and lysosomes, other messengers than ROS were tested such as calcium, Bid, Bap31, phosphorylated Bcl-2 and caspase-12 but none was clearly identified as being involved in triggering cytochrome c release from mitochondria. On the other hand, we demonstrated that the transduction pathways leading to NF-kappaB activation and apoptosis were clearly independent although NF-kappaB was shown to counteract apoptosis mediated by PPME photosensitization.

Mitochondrial release of apoptosis-inducing factor and cytochrome c during smooth muscle cell apoptosis.

Photodynamic therapy (PDT) is under investigation for the treatment of intimal hyperplastia in conditions such as atherosclerosis and restenosis. Although smooth muscle cells (SMCs) may be a key target for treatment, the effects of PDT on these cells are poorly characterized. In the present study, apoptosis was induced in primary human aortic SMCs by the combination of the photosensitizer verteporfin and visible light. After PDT, an increase in mitochondrial cytochrome c (cyt c) and apoptosis-inducing factor (AIF) levels were detected in the cytosol immediately and their levels increased steadily up to 2 hours. Cytosolic levels of the pro-apoptotic Bcl-2 family member Bax decreased reciprocally throughout this period, but this change did not occur before cyt c release. Confocal microscopy revealed a diffuse staining pattern of cyt c within apoptotic cells as compared to a distinct mitochondrial staining in normal cells. AIF translocated from mitochondria to the nucleus during the progression of apoptosis. After cyt c release, caspase-9 and caspase-3 processing was visible by 1 hour and caspase-6, -7, and -8 processing was apparent by 2 hours after PDT. In summary, these results demonstrate for the first time the cellular redistribution of mitochondrial AIF during SMC apoptosis, as well as the early release of cyt c and the subsequent activation of multiple caspases during PDT-induced SMC apoptosis.

Apoptosis induction by the photosensitizer verteporfin: identification of mitochondrial adenine nucleotide translocator as a critical target.

We report that the photosensitizer verteporfin kills lymphoma cells by an apoptotic process involving a dissipation of the mitochondrial inner transmembrane potential (deltapsim). Light-activated verteporfin-induced apoptosis was abolished by transfection with Bcl-2, a procedure reported to inhibit the mitochondrial permeability transition pore complex (PTPC). Verteporfin triggered the deltapsim loss in isolated mitochondria in vitro, and this effect was suppressed by bongrekic acid and cyclosporin A. Verteporfin plus light also permeabilized proteoliposomes containing the semipurified PTPC or the purified PTPC component adenine nucleotide translocator (ANT), yet had no effect on protein-free control liposomes. Verteporfin phototoxicity on ANT proteoliposomes was mediated by reactive oxygen species and was prevented by recombinant Bcl-2 or the adenine nucleotides ATP and ADP. In conclusion, verteporfin belongs to a class of clinically used chemotherapeutic agents acting on PTPC and ANT.

Photodynamic therapy: shedding light on the biochemical pathways regulating porphyrin-mediated cell death.

Photodynamic therapy (PDT) is a clinically approved treatment for the ocular condition age-related macular degeneration, and certain types of cancer. PDT is also under investigation for other ocular, as well as, immune-mediated and cardiovascular indications. PDT is a two step procedure. In the first step, the photosensitizer, usually a porphyrin derivative, is administered and taken up by cells. The second step involves activation of the photosensitizer with a specific wavelength of visible light. Exposure to light of an activating wavelength generates reactive oxygen species within cells containing photosensitizer. PDT with porphyrin photosensitizers induces rapid apoptotic cell death, an event which may be attributed to the close association of these compounds with mitochondria. Thus, PDT is an attractive method to treat ailments such as cancer, viral infections, autoimmune disorders and certain cardiovascular diseases in which the apoptotic program may be compromised. The present review examines the cellular events triggered at lethal and sublethal PDT doses and their relationship to the subsequent effects exerted upon cells.

Apoptosis associated with esophageal adenocarcinoma: influence of photodynamic therapy.

Tumor cell death in vitro by photodynamic therapy (PDT) has been related to the induction of apoptosis. We measured and compared changes in apoptosis and caspase 3 activity, an effector of apoptosis, in normal and neoplastic esophageal tissues during PDT. Apoptosis index, caspase 3 cleavage activity, pro-caspase 3, p53, and bcl-2 levels were measured in normal and neoplastic tissues of patients with esophageal adenocarcinoma before, during, and after PDT with Photofrin. The apoptotic index was greater in carcinoma tissue compared to adjacent normal tissues. In concert, pro-caspase 3 immunoreactivity was absent and caspase 3-like cleavage activity was over 30-fold greater in carcinoma tissue compared to normal esophageal tissues. These parameters were unaffected by PDT. Variable changes in bcl-2 and p53 immunoreactivity were noted in normal and carcinoma tissues during PDT. Greater levels of apoptosis and caspase 3 activity are hallmarks of esophageal adenocarcinoma compared to normal esophageal tissue. These differences were unaffected by PDT. This may be due to the fact that tissues were obtained 72 h post-PDT therapy. Changes in these parameters may have occurred early after PDT therapy. An assessment of apoptosis and caspase 3 activity prior to 72 h post-PDT may provide further insight into the mechanism involved, although no sustained effects on these parameters by PDT were noted.