Reference #: OLI-1017-197331
Submit Date: 03/26/2002 20:18:50-0500

Cellular uptake, biodistribution and subcellular localization of Hypericin in human malignant cells

ABSTRACT:
The development of new-generation photosensitizers to improve photodynamic therapy (PDT) and photodynamic diagnosis (PDD) is an area of extensive research. One such compound that has been studied in our group is Hypericin (HY). To study the mechanism of action we have investigated uptake, intracellular localization, cell phototoxicity and morphological changes especially to ultrastructures following photodynamic treatment in poorly (CNE2) and moderately (TW0-1) differentiated human nasopharyngeal carcinoma (NPC) cells and also other carcinoma cells such as colon (CCL-220.1) and bladder (SD) in vitro. Following photo-irradiation phototoxicity was determined by crystal fast violet assay and apoptosis was assessed using annexin V assay. Using spectrofluorimetry and confocal laser scanning microscopy (CLSM) we have determined cellular fluorescence localization and uptake of HY. Co-labeling with HY and fluorescent dyes specific for cell organelles revealed an intracellular localization of HY in mitochondria and lysosomes predominantly. Since many photosensitizing agents in current clinical use have mitochondrial targets, HY may be a valuable addition to current protocols. In addition, our results also indicate that leakage of lysosomal cysteine protease into cytosolic compartment might be involved in the induction of apoptosis. Electron microscopy revealed damage to plasma membrane with high drug dose (>5 M); indicating a mechanism related to necrosis, whereas lower sub-lethal doses (<2.5 M) resulted in induction of apoptosis by the show of chromatin condensation, range of mitochondrial alterations and shrinkage in cell size. Our results based on mitochondrial and lysosomal localization support the idea that PDT can contribute to elimination of malignant cells by the induction of apoptosis, and can be of physiological significance.

Keywords: HY, Hypericin, Mitochondria , Lysosomes, Uptake, Localization

Reference #: MIY-1017-687419
Submit Date: 04/01/2002 12:47:20-0500

Response of leukemia cells, solid tumor cells, drug-resistant tumor cells and normal hematopoietic progenitor cells to crystal violet- and Merocyanine 540-PDT

ABSTRACT:
The purpose of this study was to determine in a preclinical purging model, how effective crystal violet (CV)-PDT is against leukemia cells, solid tumor cells, as well as drug-resistant mutant tumor cells, and if certain limitations of CV-PDT can be overcome by using CV in combination with Merocyanine 540 (MC540). When used under conditions that preserved an adequate fraction of normal human granulocyte/macrophage progenitors (CFU-GM), CV-PDT failed to achieve meaningful reductions of DU145 prostate, H69 small cell lung cancer, and MDA-MB-435S breast cancer cells. Melphalan-resistant L1210/L-PAM1 (elevated glutathione), adriamycin-resistant HL-60/ADR (MDR-mediated drug efflux), and adriamycin-resistant P388/ADR (P-glycoprotein) leukemia cells were markedly less sensitive to CV-PDT than their wild-type counterparts. Contrary to expectations, cisplatin-resistant H69/CDDP cells (elevated glutathione-S-transferase-pi, metallothionein, and glutathione) were more sensitive to CV-PDT than wild-type H69 cells. The molecular basis of the increased sensitivity of mutant H69/CDDP cells is not yet understood, but is unlikely to be directly related to the drug resistance mechanism. Sequential exposure to MC540- and CV-PDT (under conditions that preserved adequate numbers of CFU-GM) was highly effective against H69 and H69/CDDP cells, but ineffective against HL-60/ADR, P388/ADR, MDA-MB-435S, and DU145 cells. Taken together, our data suggest that with regard to their capacity to eliminate wild-type leukemia cells, CV and MC540 are roughly equivalent. With regard to its capacity to eliminate solid tumor cells from autologous stem cell grafts, CV is as ineffective if not more ineffective than MC540. While three of the four investigated drug resistance mechanisms rendered tumor cells resistant to CV-PDT, only one resistance mechanism (elevated glutathione) has so far been shown to interfere with MC540-PDT. In certain situations, clinically meaningful depletions of solid tumor cells may be obtained by using CV- and MC540-PDT in combination. Our data also show that normal hematopoietic progenitor cells are much more sensitive to CV-PDT than what one might have expected based on previous experiments with CV-1 African green monkey kidney cells. (Supported by CHF, MACC Fund, and CA77387)

Keywords: Crystal violet, Merocyanine 540, Purging

Reference #: SUB-1017-330701
Submit Date: 03/28/2002 09:03:34-0500

Seeing is Believing: Non-invasive Imaging of Apoptosis induced by Photodynamic Therapy in live animals using ^99mTc-Annexin V

ABSTRACT:
Photodynamic therapy (PDT) is a strong inducer of apoptosis both in vitro and in vivo situations. This has sparked much attention to elucidate the role of apoptosis in response to PDT. Yet there exists no methodology to assess the extent of apoptosis induced by PDT non-invasively during regression of tumors in live animals or in the clinic. We developed a technology using ^99mTc-annexin V to monitor apoptosis in live animals. The radiochemical purity of the ^99mTc-annexin V was >95% as determined by ascending instant thin layer and high pressure liquid chromatograpy. To explore its usefulness for monitoring apoptosis in live animals we employed radiation induced fibrosarcoma (RIF-1) murine model that has been shown to undergo rapid onset of apoptosis following PDT treatment. C3H mice bearing RIF-1 tumors (40-60 mm³) were administered the photosensitizer Pc 4 (1.0 mg/kg b.w.) via tail vein. Forty-eight hours later the tumors were irradiated with a power density of 150 mW/cm²and a light fluence of 150 J/cm² at 672 nm using diode laser. Following 1h, 3h and 6h post-PDT treatments, ^99mTc-annexin V (150 micro Ci) was injected via tail vein into treated and untreated mice and one hour later images were obtained using Cyclone Phosphor Imager. These mice were euthanized and the radioactivity in excised organs determined and expressed as percent injected dose/organ. An avid uptake of the tracer accumulation was observed in the treated tumors, while no uptake was seen in tumors treated with light alone or Pc 4 alone. The uptake of ^99mTc-annexin V in the treated tumors at 1h, 3h and 6h was 19, 14 and 14 %, respectively, compared to 2% in the untreated tumors. Histopathology and immunohistochemistry of PDT-treated tumors confirmed the evidence of induction of apoptosis as compared to untreated tumors. This study demonstrated the utility of ^99mTc-annexin V for in vivo imaging of apoptosis in live animals induced by PDT and showed that apoptosis occurs early during the regression of tumors following PDT treatment. This novel technique could be used as a non-invasive means to detect and serially image organs/tissues undergoing apoptosis in vivo following PDT as well as other cancer treatment protocols.

Keywords: Photodynamic Therapy, Apoptosis, ^99mTc-annexin V

Reference #: ORT-1017-702322
Submit Date: 04/01/2002 16:36:25-0500

Order-dependent enhancement of ALA-PDT by chemotherapy

ABSTRACT:
BACKGROUND: Induction of differentiation enhances the efficacy of photodynamic treatment in several cell lines. We have previously demonstrated differentiation-specific increase of aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) production using a variety of differentiating agents. Because of the known association between differentiation and growth arrest we have now explored the use of chemotherapy-induced transitory growth arrest as means to enhance ALA-PDT. METHODS: We have evaluated the use of the anti-folate chemotherapeutic agent methotrexate (MTX) in combination with ALA-dependent PDT in the human prostate cancer cell line LNCaP. Cells were exposed to 1mg/L MTX for 72 hours. Then MTX was removed and cells were incubated with ALA for 4 hours. The photosensitization irradiations were done immediately after ALA exposure. Survival was assessed by MTT assay and clonogenic assay. RESULTS: MTX treatment of LNCaP cells resulted in growth arrest. Subsequent exposure to ALA for 4 hours resulted in a fourfold increased rate of PpIX formation. Consequently, lethal cytotoxicity at 24 hours after 514 nm light exposure was enhanced. Long-term survival as quantified by colony formation was also reduced with MTX pretreatment. The interaction of MTX and ALA was sequence dependent. When LNCaP cells were first exposed to ALA-PDT and then for 72 hours to methotrexate, there was no reduction of long-term survival. This indicates that the enhancement of cytotoxicity by MTX in the combined regimen is due to the increase in PpIX formation. The combination of short-term chemotherapy and subsequent ALA-PDT may help reduce dose-related toxicity of either component and thus improve existing PDT regimens for cancer treatment.

Keywords: photodynamic therapy, aminolevulinic acid, methotrexate, prostate cancer

Reference #: HOO-1017-689577
Submit Date: 04/01/2002 13:09:55-0500

Determination of Therapeutic Ratio for Photodynamic Therapy

ABSTRACT:
Introduction: Photodynamic Therapy (PDT) tissue damage is induced by both vascular occlusion and direct cellular toxicity. The tolerance of the normal tissues adjacent to a tumor limit the ultimate dose of PDT. Therefore for PDT to be a successful cancer treatment modality, a difference in the normal tissue : tumor tolerance ratio (therapeutic ratio) should be optimized. We have used quantitative histology and photosensitizer dosimetry to determine therapeutic ratio in murine tumor/normal muscle model. Methods: Using a murine adenocarcinoma (MTGB) and normal muscle we have investigated the level of tumor and muscle damage at similar dose levels. Sixty-two mice were divided into six groups using four different laser exposure times (18, 36, 54, 72 J/cm2) and the same photosensitizer dose (Aluminum Pthalocyanine @ 1.0 mg/kg). The PDT treatment was initiated when the tumor volume reached 0.2 cc3. A real-time surface probe fluorescence detector (Aurora Optics Inc.) was used to assess tissue fluorescence (photosensitizer level) in realtime. After a four hours incubation both the tumor area and the muscle area were treated. All mice were sacrificed forty-eight hours post treatment and the tumor and muscle tissue taken for quantitative and qualitative histological assessment. Results: Preliminary results suggest that the all PDT doses resulted in skin necrosis and that the highest dose resulted in significant damage to the tumor and normal muscle. However, the two intermediate doses appear to demonstrate significant muscle tissues damage without accompanying tumor damage. This is interesting since the light dose in the tumor is theoretically higher than that in the underlying muscle. We hypothesize that the difference in the tissue damage level for the tumor and muscle are related to the level of photosensitizer and/or oxygen at the time of treatment. Through the use of realtime photosensitizer level determination and quantitative histopathology, our current studies will address the importance of identifying and understanding a therapeutic ratio for PDT.

Keywords: photodynamic theraph, PDT, therapeutic ratio

Reference #: TEI-1017-410153
Submit Date: 03/29/2002 01:40:22-0500

Endoplasmic reticulum and Golgi apparatus are the preferential sites of Foscan® localization in cultured tumor cells

ABSTRACT:
Intracellular photosensitizer localization significantly influences the mechanism of response to photodynamic therapy, since the primary sites of damage are closely related to the specific sensitizer distribution. We investigated the subcellular distribution of Foscan®, a second generation sensitizer, in the MCF-7 human breast adenocarcinoma cell line in vitro by means of microspectrofluorimetry and confocal laser scanning microscopy. MCF-7 cells were co-labeled with Foscan® (1.5 M, 3h) and various organelle probes. Results of measurements performed after co-incubation of cells with Lucifer Yellow (LY), a vital probe of lysosomes, showed that the fluorescence topographic profile of Foscan® did not match that obtained with LY. Similarly, distinctly different fluorescence topographic profiles were recorded after co-staining with Foscan® and the mitochondrial probe rhodamine 123. The comparison of the Foscan® and Bodipy FL C₅-ceramide topographic profiles indicated a strong localization of Foscan® in the endoplasmic reticulum and in the Golgi apparatus. Foscan®'s localization in the endoplasmic reticulum was unambiguously confirmed by Foscan® /DiOC₆ double staining, which showed that the Foscan® fluorescence topographic profile co-localized perfectly with that obtained for DiOC₆, a specific endoplasmic reticulum marker. The patterns of fluorescence derived from confocal microscopy studies were consistent with predominant localization of Foscan® in these organelles. To the best of our knowledge, this is the first study to unambiguously demonstrate that the endoplasmic reticulum and the Golgi apparatus are preferential sites of Foscan® accumulation. Further, the activity of the NADPH cytochrome c reductase, an endoplasmic reticulum marker enzyme measured immediately after PDT, demonstrated a fluence-dependent degradation, suggesting that these organelles could be primary sites of photodamage.

Keywords: Foscan®, intracellular localization, microspectrofluorometry, confocal microscopy

Reference #: SEL-1017-693726
Submit Date: 04/01/2002 13:51:48-0500

Photochemical disruption of endocytic vesicles before delivery of drugs: a new strategy for cancer therapy

ABSTRACT:
The development of methods for specific delivery of drugs is an important issue for many disease therapy approaches. Most of macromolecular drugs are taken into the cell through endocytosis and, being unable to escape from endocytic vesicles, eventually are degraded there, which hinders their therapeutic usefulness. We have recently developed a drug delivery method, called photochemical internalization, which is based up on the photodynamic induced rupture of endocytic vesicles specifically within illuminated sites e.g. tumors. Here we present a new molecular medicine delivery concept based on the photochemical internalization-principle: photochemical disruption of endocytic vesicles before delivery of macromolecules, leading to an instant endosomal release instead of detrimental stay of the molecules in endocytic vesicles. Previously we have shown that illumination applied after the treatment with macromolecules substantially improved their biological effect both in vitro and in vivo. Here we demonstrate that exposure to light before delivery of protein toxin gelonin improves gelonin effect in vitro much more than light after. However, in vitro transfection with reporter genes delivered by non-viral and adenoviral vectors is increased more than 10-and six-fold, respectively, by both photochemical internalization strategies. The possible cellular mechanisms involved, and the potential of this new method for practical application of photochemical internalization concept in cancer therapy will be discussed.

Keywords: Photochemical Internalization, Drug delivery, Gene therapy, Endocytosis

Reference #: RHA-1019-498651
Submit Date: 04/22/2002 10:52:31-0500

Vitamin D and sunscreen use

ABSTRACT:
With skin cancer incidence rate rising throughout the world, health authorities have recommended the regular use of sunscreen. In Canada, 95% of parents use sunscreen to protect their children in summer. There is increasing concern regarding the risk of vitamin D deficiency and sunscreen use. Sunscreens with high sun protection factor (SPF 25) may block up to 98% of UVB rays required for cutaneous vitamin D synthesis. Vitamin D is an essential hormone for bone integrity and calcium homeostasis. A study was conducted between May 1999 and April 2000 in Quebec City area (47°N) in children aged 3 to 6 years. The objectives were to measure blood levels of vitamin D (25-OHD) during summer(S) and winter(W), and to assess factors associated with serum 25-OHD. Children were enrolled on a voluntary basis with the collaboration of paediatricians. A standardized questionnaire was completed to get information on UV exposure and sunscreens use. A food frequency questionnaire was administered by interviewer to estimate vitamin D intake. Blood samples were taken and analysed for 25-OHD, PTH, and calcium. Final sample included 76 children. The mean vitamin D intake was 9.5 mg/d in summer and 11.1 mg/d in winter. 88.5% of the children in summer and 91.8% in winter had vitamin D intake higher than the recommended daily amount (5 mg/d) for their age. The mean level of serum 25-OHD was higher in summer (97.3 nmol/l) than in winter (77.3 nmol/l). Although sunscreens (SPF 30) were worn by 92% at home and 97 % at the day care centre, the total amount used during summer was less than 240 ml in 63%. The serum 25-OHD(S) was inversely associated with the frequency of sunscreen use. A inverse relationship was also found between serum 25-OHD(S) and the proportion of the total body surface protected by a sunscreen. Serum 25-OHD(S) was positively correlated with the time spent outside. The vitamin D intake in summer was not associated with serum 25-OHD whereas a positive correlation was observed in winter (r=0.23; p<0.05). The results suggest that vitamin D status in a northern population of children with adequate vitamin D intake could be decreased by regular use of sunscreens. However, it remains unclear if the amount of sunscreen applied as recommended (2ml/cm2) could increase the risk of vitamin D deficiency. (Grant by Fonds de la recherche en santé du Québec).

Keywords: Sunscreen, Vitamin D, UV exposure

Reference #: DAL-1017-313470
Submit Date: 03/28/2002 04:51:04-0500

DNA-binding and DNA-photodamaging properties of naphtoquinolizinium derivatives

ABSTRACT:
The association of cationic aromatic dyes to DNA has attracted considerable interest. Such a complex formation results a significant modification of the DNA structure which may have a profound influence on the gene expression. In most cases, the binding interactions are easily detected by a change of the absorption and emission spectra of the dye upon DNA addition. This modification of the absorption and emission may also be used to mark, to probe or to characterize the nucleic acid structure. While the association of cationic dyes to DNA, and thus the DNA modification, is a reversible process, the DNA-damage, which frequently occurs on ligth irradiation of dye-DNA complexes, is irreversible. The latter DNA damage often leads to cell death or mutations, and must be avoided in healthy systems. However, this photoinduced DNA-damage may be applied in photochemotherapy to remove unwanted cells. We have observed recently that benzoquinolizinium derivatives (acridizinium) exhibit DNA-binding and DNA-photodamaging properties. During our studies of the influence of the substitution pattern on the DNA interaction of quinolizinium derivatives, we became interested in two naphthoquinolizinium derivatives. Moreover, we anticipated, that the presence of a fourth aromatic ring in the napthoquinolizinium derivatives in comparison to the tryciclic acridizinium salts, may enhance the interaction between DNA base pairs and the tetracyclic system. We have now conducted a detailed study on the binding interactions of the naphtoquinolizinium derivatives with DNA along with their propensity for photoinduced DNA-damage. Herein, these photobiological features will be presented and discussed.

Keywords: DNA-photodamage, intercalator, Photonucleases, naphtoquinolizinium

Reference #: BER-1019-320547
Submit Date: 04/20/2002 11:22:26-0500

Gene environment interaction in melanoma

ABSTRACT:
Our inability to correctly classify cutaneous melanoma risk among populations has led to the idea that the identification of interactions of specific genetic features with environmental exposures will give more precise information about risk. Ultraviolet radiation is the major environmental exposure associated with the development of melanoma. Many biochemical pathways are involved and because genetic polymorphisms are abundant and are known to affect gene function in some few cases, we are searching known pathways for single nucleotide polymorphisms (SNPs) that may functionally interact with ultraviolet radiation to increase the risk of developing melanoma. Although multiple pathways are important in the development of melanoma, our group is focusing on three pathways and their interaction with each other and with ultraviolet radiation exposure: cell cycle control, pigmentation, and DNA repair. In the cell cycle control pathway, we are screening for and sequencing INK4A mutations and polymorphisms. INK4A is also known as CDKN2A, MTS1, and p16. In the pigmentation pathway, we are sequencing the melanocortin receptor gene, MC1R, frequently shown to be associated with risk factors for melanoma and with melanoma itself. Furthermore, the recent literature shows that MC1R strongly modifies the penetrance and age at diagnosis for INK4A carriers. In the DNA repair pathway we are genotyping newly-identified polymorphisms in the nucleotide excision repair pathway, known to have major responsibility for repairing UVB-induced damage, and in the base excision repair pathway, know to have major responsibility for repairing reactive oxygen species. We are identifying mutations and polymorphisms in genes that appear to be associated with melanoma in an international population-based melanoma study. This study is taking place in the United States, Canada, Italy, and Australia. Four thousand individuals with melanoma will donate DNA samples and complete questionnaires covering ultraviolet radiation exposure over their lifetime. This study consists of a novel case-control design where the cases are individuals with multiple primary melanoma and the controls are individuals with single primary melanoma. Data from ongoing work will be presented.

Keywords: melanoma, genetics, ultraviolet radiation, interaction

Reference #: IND-1017-694068
Submit Date: 04/01/2002 14:23:20-0500

Effect of molecular structure on the phototoxicity of triarylmethane dyes towards tumor and normal cells

ABSTRACT:
The conceptual basis for the development of mitochondrial targeting as a novel therapeutic strategy for (photo)chemotherapy of neoplastic diseases rests on the observation that enhanced mitochondrial membrane potential is a prevalent tumor cell phenotype. Because transmembrane potentials are negative on the inner side of both the plasma and mitochondrial membranes, extensively conjugated cationic molecules displaying appropriate structural features are electrophoretically driven through these membranes and tend to accumulate inside energized mitochondria. The higher mitochondrial membrane potential typical of tumor cells thus opens a window for the selective destruction of these cells via mitochondrial targeting. While several cationic dyes naturally accrue in the mitochondria of living cells, only a small subset of these mitochondrial dyes induce the photochemical destruction of tumor cells with desirable selectivity. In this report we describe how the molecular structure of a series of closely related cationic triarylmethane (TAM+) photosensitizers modulates their selective phototoxicity towards tumor cells. Our results indicate that the selective phototoxicity of TAM+ dyes towards tumor cells is primarily controlled by the lipophilic/hydrophilic character of the photosensitizer. While the more hydrophilic TAM+ dyes (e.g., Bromo Crystal Violet, Crystal violet, Methyl Violet 2B, Pararosaniline) show a high degree of tumor cell selectivity, those showing higher lipophilic character (e.g., Ethyl Violet, Victoria Pure Blue BO, Victoria Blue R) do not. Comparison of 1-octanol/water partition coefficients indicates that only those dyes with a lipophilic/hydrophilic character close to that of the mitochondria-specific fluorescent marker Rhodamine-123 are tumor selective. This observation suggests that only the dyes that stain the mitochondria with close to absolute specificity can promote the photoinduced destruction of tumor cells with a high degree of selectivity. The staining of a variety of subcellular compartments (other than mitochondria) by the more lipophilic dyes apparently precludes tumor selectivity from taking place.

Keywords: triarylmethanes, photochemotherapy, mitochondria, targeting