The effects of protoporphyrin IX-induced photodynamic therapy with and without iron chelation on human squamous carcinoma cells cultured under normoxic, hypoxic and hyperoxic conditions.

BACKGROUND: Photodynamic therapy requires the combined interaction of a photosensitiser, light and oxygen to ablate target tissue. In this study we examined the effect of iron chelation and oxygen environment manipulation on the accumulation of the clinically useful photosensitiser protoporphyrin IX (PpIX) within human squamous epithelial carcinoma cells and the subsequent ablation of these cells on irradiation. METHODS: Cells were incubated at concentrations of 5%, 20% or 40% oxygen for 24h prior to and for 3h following the administration of the PpIX precursors aminolevulinic acid (ALA), methyl aminolevulinate (MAL) or hexylaminolevulinate (HAL) with or without the iron chelator 1,2-diethyl-3-hydroxypyridin-4-one hydrochloride (CP94). PpIX accumulation was monitored using a fluorescence plate reader, cells were irradiated with 37 J/cm(2) red light and cell viability measured using the neutral red uptake assay. RESULTS: Manipulation of the oxygen environment and/or co-administration of CP94 with PpIX precursors resulted in significant changes in both PpIX accumulation and photobleaching. Incubation with 5% or 40% oxygen produced the greatest levels of PpIX and photobleaching in cells incubated with ALA/MAL. Incorporation of CP94 also resulted in significant decreases in cell viability following administration of ALA/MAL/HAL, with oxygen concentration predominantly having a significant effect in cells incubated with HAL. CONCLUSIONS: Experimentation with human squamous epithelial carcinoma cells has indicated that the iron chelator CP94 significantly increased PpIX accumulation induced by each PpIX congener investigated (ALA/MAL/HAL) at all oxygen concentrations employed (5%/20%/40%) resulting in increased levels of photobleaching and reduced cell viability on irradiation. Further detailed investigation of the complex relationship of PDT cytotoxicity at various oxygen concentrations is required. It is therefore concluded that iron chelation with CP94 is a simple protocol modification with which it may be much easier to enhance clinical PDT efficacy than the complex and less well understood process of oxygen manipulation

An in vitro comparison of the effects of the iron-chelating agents, CP94 and dexrazoxane, on protoporphyrin IX accumulation for photodynamic therapy and/or fluorescence guided resection.

Comparative StudyJournal Article"This is the peer reviewed version of the following article:Photochemistry and Photobiology, Volume 87, Issue 6, pages 1419–1426, November/December 2011, which has been published in final form at http://onlinelibrary.wiley.com/wol1/doi/10.1111/j.1751-1097.2011.00985.x/full. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving."Photodynamic therapy (PDT) utilizes the combined interaction of a photosensitizer, light and molecular oxygen to ablate tumor tissue. Maximizing the accumulation of the photosensitizer protoporphyrin IX (PpIX) within different cell types would be clinically useful. Dermatological PpIX-induced PDT regimes produce good clinical outcomes but this currently only applies when the lesion remains superficial. Also, as an adjuvant therapy for the treatment of primary brain tumors, fluorescence guided resection (FGR) and PDT can be used to highlight and destroy tumor cells unreachable by surgical resection. By employing iron chelators PpIX accumulation can be enhanced. Two iron-chelating agents, 1,2-diethyl-3-hydroxypyridin-4-one hydrochloride (CP94) and dexrazoxane, were individually combined with the porphyrin precursors aminolevulinic acid (ALA), methyl aminolevulinate (MAL) and hexyl aminolevulinate (HAL). Efficacies of the iron-chelating agents were compared by recording the PpIX fluorescence in human squamous epithelial carcinoma cells (A431) and human glioma cells (U-87 MG) every hour for up to 6 h. Coincubation of ALA/MAL/HAL with CP94 resulted in a greater accumulation of PpIX compared to that produced by coincubation of these congeners with dexrazoxane. Therefore the clinical employment of iron chelation, particularly with CP94 could potentially increase and/or accelerate the accumulation of ALA/MAL/HAL-induced PpIX for PDT or FGR

Radon and skin cancer in southwest England: an ecologic study

This is the author's post peer review version of the article. The final version is published in Epidemiology. 2012 Jan;23(1):44-52. doi: 10.1097/EDE.0b013e31823b6139.Radon, a naturally occurring radioactive gas, is a carcinogen that causes a small proportion of lung cancers among exposed populations. Theoretical models suggest that radon may also be a risk factor for skin cancer, but epidemiologic evidence for this relationship is weak. In this study, we investigated ecologic associations between environmental radon concentration and the incidence of various types of skin cancer

The cellular and molecular carcinogenic effects of radon exposure: a review.

Journal ArticleResearch Support, Non-U.S. Gov'tReviewOpen access articleRadon-222 is a naturally occurring radioactive gas that is responsible for approximately half of the human annual background radiation exposure globally. Chronic exposure to radon and its decay products is estimated to be the second leading cause of lung cancer behind smoking, and links to other forms of neoplasms have been postulated. Ionizing radiation emitted during the radioactive decay of radon and its progeny can induce a variety of cytogenetic effects that can be biologically damaging and result in an increased risk of carcinogenesis. Suggested effects produced as a result of alpha particle exposure from radon include mutations, chromosome aberrations, generation of reactive oxygen species, modification of the cell cycle, up or down regulation of cytokines and the increased production of proteins associated with cell-cycle regulation and carcinogenesis. A number of potential biomarkers of exposure, including translocations at codon 249 of TP53 in addition to HPRT mutations, have been suggested although, in conclusion, the evidence for such hotspots is insufficient. There is also substantial evidence of bystander effects, which may provide complications when calculating risk estimates as a result of exposure, particularly at low doses where cellular responses often appear to deviate from the linear, no-threshold hypothesis. At low doses, effects may also be dependent on cellular conditions as opposed to dose. The cellular and molecular carcinogenic effects of radon exposure have been observed to be both numerous and complex and the elevated chronic exposure of man may therefore pose a significant public health risk that may extend beyond the association with lung carcinogenesis

Effect of an oxygen pressure injection (OPI) device on the oxygen saturation of patients during dermatological methyl aminolevulinate photodynamic therapy

Journal Article“The final publication is available at Springer via http://dx.doi.org/10.1007/s10103-012-1188-y"Methyl aminolevulinate photodynamic therapy (MAL-PDT) (a topical treatment used for a number of precancerous skin conditions) utilizes the combined interaction of a photosensitizer (protoporphyrin IX (PpIX)), light of the appropriate wavelength, and molecular oxygen to produce singlet oxygen and other reactive oxygen species which induce cell death. During treatment, localized oxygen depletion occurs and is thought to contribute to decreased efficacy. The aim of this study was to investigate whether an oxygen pressure injection (OPI) device had an effect on localized oxygen saturation levels and/or PpIX fluorescence of skin lesions during MAL-PDT. This study employed an OPI device to apply oxygen under pressure to the skin lesions of patients undergoing standard MAL-PDT. Optical reflectance spectrometry and fluorescence imaging were used to noninvasively monitor the localized oxygen saturation and PpIX fluorescence of the treatment area, respectively. No significant changes in oxygen saturation were observed when these data were combined for the group with OPI and compared to the group that received standard MAL-PDT without OPI. Additionally, no significant difference in PpIX photobleaching or clinical outcome at 3 months between the groups of patients was observed, although the group that received standard MAL-PDT demonstrated a significant increase (p < 0.05) in PpIX fluorescence initially and both groups produced a significant decrease (p < 0.05) after light irradiation. In conclusion, with this sample size, this OPI device was not found to be an effective method with which to improve tissue oxygenation during MAL-PDT. Further investigation is therefore required to find a more effective method of MAL-PDT enhancement. © 2012 Springer-Verlag London Ltd

The time-dependent accumulation of protoporphyrin IX fluorescence in nodular basal cell carcinoma following application of methyl aminolevulinate with an oxygen pressure injection device

Clinical TrialJournal ArticleAuthor's post-print is subject to a Creative Commons Attribution Non-Commercial No Derivatives LicenseTopical protoporphyrin (PpIX)-induced photodynamic therapy (PDT) relies on the penetration of the prodrug into the skin lesion and subsequent accumulation of the photosensitizer. Methyl aminolevulinate (MAL)-PDT is an established treatment for thinner and superficial non-melanoma skin cancers (NMSCs) but for the treatment of the thicker nodular basal cell carcinoma (nBCC) enhanced penetration of the prodrug is required. This study employed a new higher pressure, oxygen pressure injection (OPI) device, at the time of Metvix® application with a view to enhancing the penetration of MAL into the tumors. Each patient had Metvix® applied to a single nBCC followed by application of a higher pressure OPI device. Following different time intervals (0, 30, 60, 120 or 180 min) the tumors were excised. The maximum depth and area of MAL penetration achieved in each lesion was measured using PpIX fluorescence microscopy. As expected, an increase in the depth of MAL-induced PpIX accumulation and area of tumor sensitized was observed over time; when the Metvix® cream was applied for 0, 30, 60, 120 and 180 min the median depth of PpIX fluorescence was 0%, 21%, 26.5%, 75.5% and 90%, respectively and the median area of tumor sensitized was 0%, 4%, 6%, 19% and 60%, respectively. As the investigation presented here did not include a control arm, the relative depths of fluorescence observed in this study were statistically compared (using the non-parametric Mann Whitney U test) with the results of our previous study where patients had Metvix® cream applied either with or without the standard pressure OPI device. When the higher pressure OPI device was employed compared to without OPI this increase was observed to be greater following 30, 120, and 180 min although overall not significantly (p=0.835). In addition, no significant difference between the higher pressure OPI device employed here and the previously investigated standard pressure OPI device was observed (p=0.403). However, when the results for both OPI devices were combined and compared to the standard treatment (no OPI employed) group, although the difference did not reach significance (p=0.531) a consistent and substantial increase in the depth of PpIX fluorescence was observed, therefore employment of an OPI device during topical MAL-PDT protocols warrants further investigation as a technique for enhancing MAL penetration

Relative domain folding and stability of a membrane transport protein

There is a limited understanding of the folding of multidomain membrane proteins. Lactose permease (LacY) of Escherichia coli is an archetypal member of the major facilitator superfamily of membrane transport proteins, which contain two domains of six transmembrane helices each. We exploit chemical denaturation to determine the unfolding free energy of LacY and employ Trp residues as site-specific thermodynamic probes. Single Trp LacY mutants are created with the individual Trps situated at mirror image positions on the two LacY domains. The changes in Trp fluorescence induced by urea denaturation are used to construct denaturation curves from which unfolding free energies can be determined. The majority of the single Trp tracers report the same stability and an unfolding free energy of approximately + 2 kcal mol- 1. There is one exception; the fluorescence of W33 at the cytoplasmic end of helix I on the N domain is unaffected by urea. In contrast, the equivalent position on the first helix, VII, of the C-terminal domain exhibits wild-type stability, with the single Trp tracer at position 243 on helix VII reporting an unfolding free energy of + 2 kcal mol- 1. This indicates that the region of the N domain of LacY at position 33 on helix I has enhanced stability to urea, when compared the corresponding location at the start of the C domain. We also find evidence for a potential network of stabilising interactions across the domain interface, which reduces accessibility to the hydrophilic substrate binding pocket between the two domains

SF3B1 hotspot mutations confer sensitivity to PARP inhibition by eliciting a defective replication stress response.

SF3B1 hotspot mutations are associated with a poor prognosis in several tumor types and lead to global disruption of canonical splicing. Through synthetic lethal drug screens, we identify that SF3B1 mutant (SF3B1MUT) cells are selectively sensitive to poly (ADP-ribose) polymerase inhibitors (PARPi), independent of hotspot mutation and tumor site. SF3B1MUT cells display a defective response to PARPi-induced replication stress that occurs via downregulation of the cyclin-dependent kinase 2 interacting protein (CINP), leading to increased replication fork origin firing and loss of phosphorylated CHK1 (pCHK1; S317) induction. This results in subsequent failure to resolve DNA replication intermediates and G2/M cell cycle arrest. These defects are rescued through CINP overexpression, or further targeted by a combination of ataxia-telangiectasia mutated and PARP inhibition. In vivo, PARPi produce profound antitumor effects in multiple SF3B1MUT cancer models and eliminate distant metastases. These data provide the rationale for testing the clinical efficacy of PARPi in a biomarker-driven, homologous recombination proficient, patient population

Use of model systems to understand the etiology of fragile X-associated primary ovarian insufficiency (FXPOI)

Fragile X-associated primary ovarian insufficiency (FXPOI) is among the family of disorders caused by the expansion of a CGG repeat sequence in the 5' untranslated region of the X-linked gene FMR1. About 20% of women who carry the premutation allele (55 to 200 unmethylated CGG repeats) develop hypergonadotropic hypogonadism and cease menstruating before age 40. Some proportion of those who are still cycling show hormonal profiles indicative of ovarian dysfunction. FXPOI leads to subfertility and an increased risk of medical conditions associated with early estrogen deficiency. Little progress has been made in understanding the etiology of this clinically significant disorder. Understanding the molecular mechanisms of FXPOI requires a detailed knowledge of ovarian FMR1 mRNA and FMRP’s function. In humans, non-invasive methods to discriminate the mechanisms of the premutation on ovarian function are not available, thus necessitating the development of model systems. Vertebrate (mouse and rat) and invertebrate (Drosophila melanogaster) animal studies for the FMR1 premutation and ovarian function exist and have been instrumental in advancing our understanding of the disease phenotype. For example, rodent models have shown that FMRP is highly expressed in oocytes where it is important for folliculogenesis. The two premutation mouse models studied to date show evidence of ovarian dysfunction and, together, suggest that the long repeat in the transcript itself may have some pathological effect quite apart from any effect of the toxic protein. Further, ovarian morphology in young animals appears normal and the primordial follicle pool size does not differ from that of wild-type animals. However, there is a progressive premature decline in the levels of most follicle classes. Observations also include granulosa cell abnormalities and altered gene expression patterns. Further comparisons of these models are now needed to gain insight into the etiology of the ovarian dysfunction. Premutation model systems in non-human primates and those based on induced pluripotent stem cells show particular promise and will complement current models. Here, we review the characterization of the current models and describe the development and potential of the new models. Finally, we will discuss some of the molecular mechanisms that might be responsible for FXPOI