Cytotoxicity and adjuvant activity of cationic photosensitizers in a multidrug resistant cell line

The toxicities and phototoxicities of methylene blue (MB), toluidine blue O (TBO) and Victoria blue BO (VBBO) in a murine mammary tumour cell line (EMT6-S) and a multidrug resistant (MDR) sub-line (EMT6-R) were measured and their efficacy against the resistant sub-line was compared to that of doxorubicin and cis-platinum. The MDR cell line was considerably more susceptible to VBBO than to the conventional agent doxorubicin. VBBO was also phototoxic whereas illumination did not alter the activity of doxorubicin or of cisplatin. Both TBO and MB showed limited light activation (2-fold) in both the sensitive and resistant cell lines. Pre-treatment with VBBO prior to exposure to doxorubicin caused a two-fold increase in doxorubicin toxicity in both cell lines. MB and TBO, however, increased doxorubicin toxicity in EMT6-R cells x2 and x3 respectively, but had less effect on the sensitive cell line (increase x1.4 and x2 respectively). Thus MB and TBO may act on the MDR cell line via a different mechanism to that of VBBO.     

Extracorporeal photochemotherapy for treatment of clonal T cell proliferations

Extracorporeal photochimiotherapy (ECP) is based on the exposure of peripheral blood mononuclear cells to the photosensitizing agent (psoralen or 8MOP) and UVA radiation. Mononuclear cells are harvested by cytapheresis and reinfused to the patient after irradiation. This cell therapy has been shown to be effective in the treatment of selected diseases mediated by clonal T cells proliferation such as Sezary T cells lymphoma, rejection after solid organ transplantation and graft-versus-host disease but results obtained in autoimmune diseases are less convincing. ECP is well tolerated with very few side effects and can be combined with immunosuppressive drugs. Two methods of ECP are currently used: in the first one, the whole procedure is performed with the same equipment whereas in the second one, the cytapheresis is performed on a conventional cell separator and treated with an independent UVA irradiation: Experimental data and clinical results suggest that PCE might induced an immune response against pathological T cells clones. However, technical differences in the methods of PCE and weak knowledge on its mechanism of action impair the standardization and evaluation of this cell therapy process as well as its clinical development.     

Mitochondrial toxicity of 2,5-diaziridinyl-3,6-bis-(carboethoxyamino)-1,4-benzoquinone

2,5-Diaziridinyl-3,6-bis-(carboethoxyamino)-1,4-benzoquinone (AZQ) is an antitumor agent characterized by lipid solubility and the ability to penetrate the central nervous system. Two human glioma-derived cell lines, SNB-1 and SNB-2, proved by microcytotoxicity assay to be sensitive to AZQ, were examined with transmission electron microscopy for morphologic changes induced by this drug. AZQ was used in two ways: a) dissolved with dimethylacetamide (aqueous) at concentrations of 25 and 50 micrograms/ml and b) solid (surface plated) at 3.1 and 6.2 micrograms/mm2. A time study from 30 minutes to 48 hours was performed. Selective mitochondrial destruction was noted after 8 hours of exposure to AZQ. After 12 hours, condensation of chromatin along the periphery of the nucleus and dilation of endoplasmic reticulum were two other cellular reactions observed. Thus in addition to the known alkylating activity of AZQ, it has significant mitochondrial toxicity. This antimitochondrial effect is possibly an important factor in the antiglioma cell cytotoxicity of AZQ.     

二氢卟吩类光敏剂在光动力疗法治疗恶性肿瘤中的研究进展

肿瘤的治疗是目前医学领域最为棘手的难题之一,患者多采用手术、放化疗相结合的综合治疗方式。与传统抗癌疗法相比,光动力疗法最突出的优点是能选择性杀灭局部肿瘤而不危及正常组织。光敏剂作为光动力疗法中至关重要的因素,它的研究受到越来越多的重视。本文就近年来二氢卟吩类光敏剂在光动力疗法治疗恶性肿瘤中的研究进展作一简要综述。     

Target-selective degradation of cancer-related proteins by novel photosensitizers for molecular-targeted photodynamic therapy

Proteins are key players in many biological events including cancers. The development of novel photosensitizers for the selective degradation of cancer-related proteins has attracted much attention in the fields of photodynamic therapy for various cancers. In this review article, several novel photosensitizers, which selectively degrade cancer-related proteins under photoirradiation and mild conditions without any further additives, are introduced. This novel class of photosensitizers promises bright prospects for finding molecular-targeted drugs for cancer photodynamic therapy in the near future. ( Cancer Sci 2009; 100: 1581–1584)     

Evaluation of sulfonated aluminum phthalocyanines for use in photochemotherapy. Cellular uptake studies

Cellular uptake of aluminum phthalocyanine sulfonated to different degree was studied by means of fluorescence measurements and HPLC chromatography. These results were correlated to the lipophilic property of each drug measured as the distribution of the drug between a lipophilic phase (Triton X-114) and an aqueous phase. All the sulfonated aluminum phthalocyanines were taken up into cells to a higher extent than porphyrins of a similar lipophilicity. The cellular uptake of monosulfonated aluminum phthalocyanine was 10-fold higher than the cellular uptake of tetrasulfonated aluminum phthalocyanine and at least 50% higher than tetra(3-hydroxy-phenyl)porphin which is so far the porphyrin shown to be taken up into cells to the highest extent.     

Improved targeting of photosensitizers by intratumoral administration of immunoconjugates

Biodistribution of technetium (99mTc) labeled hematoporphyrin derivative (HpD, Photosan-3) conjugated to a monoclonal antibody to carcinoembryonic antigen (anti-CEA) was compared following intravenous (i.v.) and intratumoral (i.t.) administration in solid Ehrlich ascites tumor bearing mice. Images of mice at different time intervals were acquired after injection of radiolabeled PS-3 in either conjugated or unconjugated forms. Quantitative estimation of the radiolabel in different tissues was performed by selecting the different region of interests (ROIs). Maximum accumulation of both free and antibody conjugated PS-3 following i.v. administration was observed in liver followed by tumor. Tumor/muscle (T/N) ratio was more with free PS-3 compared to conjugated PS-3. Pharmacokinetics of free and conjugated PS-3 was also different with faster accumulation of conjugated PS-3 in the tumor. With intratumoral administration of anti-CEA-PS-3-99mTc, specific accumulation and retention of the sensitizer was observed in the tumor tissue. Since, direct injection of antibody conjugated photosensitizer into the tumor resulted in longer retention of the dye in the tumor with no accumulation in the normal tissues, the present results imply that the toxicity to normal tissues could be reduced significantly with selective destruction of the tumor following photodynamic treatment with the use of i.t. administration of specific antibodies conjugated to photosensitizers.     

Rhodamine dyes as potential agents for photochemotherapy of cancer in human bladder carcinoma cells

The phototoxicity in vitro of rhodamine 123 and tetrabromo rhodamine 123 (TBR) was compared, in order to assess their photochemotherapeutic potential. Exposure to 514.5-nm radiation from an argon ion laser caused phototoxicity in MGH-U1 bladder carcinoma cells previously treated with either dye at 10 microM for 30 min. As assessed by colony formation and cellular morphology, TBR was markedly more phototoxic than rhodamine 123, reflecting increased intersystem crossing of TBR to the triplet manifold via spin-orbital coupling induced by the heavy bromine atoms. Photoreactions of TBR very efficiently generated singlet oxygen (1O2) in solution; furthermore, irradiation of TBR-treated cells was significantly more toxic when performed in the presence of deuterium oxide, an enhancer of damage caused by 1O2. Retention of fluorescence in TBR-treated cells was enhanced by irradiation, indicating that a stable photoproduct may be formed in reaction with cellular components.     

Pharmacokinetics, toxicity, and efficacy of ends-modified raf antisense oligodeoxyribonucleotide encapsulated in a novel cationic liposome.

Raf-1 protein serine threonine kinase plays an important role in cell survival and proliferation. Antisense inhibition of Raf-1 expression has been shown to enhance the cytotoxic effects of radiation and anticancer drugs. Here we have evaluated the toxicity, pharmacokinetics, and antitumor efficacy of a novel formulation of liposome-entrapped raf antisense oligodeoxyribonucleotide (LErafAON). The LErafAON preparation showed high liposome entrapment efficiency of rafAON (>85%) and stability at room temperature. In CD2F1 mice, administration of LErafAON produced no morbidity/mortality (5-35 mg/kg/dose, i.v., x12). Dose-related elevations in liver enzymes (alanine aminotransferase and aspartate aminotransferase) and histopathological changes in liver were noted in LErafAON and blank liposome groups. No morbidity/mortality and changes in clinical chemistry or histopathology were observed in New Zealand white rabbits (3.75 mg/kg/dose, i.v., x8; 6.5 mg/kg/dose, i.v., x6) or in cynomolgous monkeys (3.75 or 6.25 mg/kg/dose, i.v., x9). Transient decrease in total hemolytic complement activity (approximately 62-74%) and increases in C3a (approximately 3-fold) and Bb levels (approximately 5-12-fold) were observed in LErafAON and blank liposome groups of monkeys. A 30 mg/kg i.v. dose of LErafAON in human prostate tumor (PC-3)-bearing BALB/c athymic mice gave a terminal plasma half-life of 27 h, and intact rafAON could be detected in plasma and in normal and tumor tissues for up to at least 48 h. In monkeys, the terminal plasma half-life of 30.36 +/- 23.87 h was observed at an i.v. dose of 6.25 mg/kg. LErafAON (25 mg/kg/dose, i.v., x10) or ionizing radiation (3.8 Gy/day, x5) treatment of PC-3 tumor-bearing athymic mice led to tumor growth arrest, whereas a combination of LErafAON and ionizing radiation treatments resulted in tumor regression. LErafAON treatment caused inhibition of Raf-1 protein expression in normal and tumor tissues in these mice (>50%, versus controls). These data have formed a basis of the clinical Phase I studies of LErafAON for cancer treatment.     

Extracorporeal photochemotherapy in the treatment of severe graft-versus-host disease

Advances in posttransplant immunosuppression have to the present not been able to prevent the development of graft-versus-host disease (GVHD) in patients given related or unrelated stem cell grafts for cure of hematologic diseases. Patients with GVHD not responding to first line therapy with corticosteroids remain at high risk of death due to severe infections or organ failure. Extracorporeal exposure of peripheral blood mononuclear cells to the photosensitizing agent 8-methoxypsoralen and ultraviolet A radiation has been shown to be effective in treatment of selected T-cell mediated diseases, including cutaneous T-cell lymphoma and rejection after organ transplantation. Extracorporeal photochemotherapy (ECP) is also a safe and efficacious adjunct therapy for both acute and chronic extensive GVHD with skin and visceral involvement and resistance to conventional immunosuppressive therapy. A multicenter randomized study should help define the impact of ECP in the treatment of GVHD and overall survival of these patients.     

Hypocrellins as photosensitizers for photodynamic therapy: a screening evaluation and pharmacokinetic study

Hypocrellin compounds were selected as potential photosensitizers for photodynamic therapy (PDT) owing to their high quantum yields of singlet oxygen (¹O₂), and facility for site-directed chemical modification to enhance phototoxicity, pharmacokinetics, solubility, and light absorption in the red spectral region, among other properties. Parent hypocrellins A and B share an absorption peak at 658 nm. These molecules may therefore be considered useful progenitors of derivatives which absorb more strongly in the red, considering that the ideal sensitizer should absorb in the 650–800 nm range, beyond the absorption range of hemoglobin and melanin, and where light penetration in tissues is maximized through reduced scattering. A series of pure, monomeric hypocrellin derivatives was tested for properties of dark cytotoxicity and photosensitizing potential by clonogenic assay in monolayer cultures of EMT6/Ed murine tumor cells. Their respective toxicities are reported on a molar basis. The in vitro screening assay has, to date, resulted in the selection of four hypocrellin derivatives for further development as photosensitizers for PDT. Cellular uptake for photosensitizing doses of selected compounds was determined by fluorimetry. Dose escalation studies in rodents indicate that potentially photosensitizing doses promote no demonstrable systemic toxicity. Received: 19 May 1994/Accepted: 21 March 1995     

Mechanism of action and spectrum of cell types susceptible to doxorubicin photochemotherapy

We have shown that, whereas argon ion laser irradiation alone is not cytocidal for L929 cells, it greatly increases the cytotoxicity of intracellular doxorubicin. The present study showed that light enhancement of doxorubicin cytotoxicity was not restricted to stock L929 cells, but could also be demonstrated using L929 cells selected for doxorubicin resistance and several standard cell lines that are relatively resistant to doxorubicin prior to selection. Light-enhanced cytotoxicity resulted in extensive nuclear DNA loss and was strongly inhibited by anoxia. These findings suggest that the mechanism by which light exposure enhances doxorubicin cytotoxicity involves DNA damage by intranuclear generation of reactive oxygen species. Received: 10 June 1996 / Accepted: 16 November 1996     

Photodynamic eradication of amelanotic melanoma of the hamster with fast acting photosensitizers

Three porphycenes with fast pharmacokinetics were tested for their ability to photosensitize amelanotic hamster melanoma A-Mel-3 at short time intervals after injection. Laser light irradiation was performed at the time of maximal photosensitizer level in tumor tissue. Photodynamic therapy as short as 5 min after injection led to complete local tumor remission at a dosage of 1.4 μmol/kg for the porphycene CBPn. In comparison, Photofrin required 8.4 μmol/kg for local tumor remission in 5 of 6 animals with 24 hr accumulation time after injection. We propose a swift photodynamic protocol which can compete favorably with conventional techniques of tumor treatment. © 1996 Wiley-Liss, Inc.     

New photosensitizers for photodynamic therapy: combined effect of metallopurpurin derivatives and light on transplantable bladder tumors

A series of metallopurpurins was tested for their photodynamic activity against transplantable N-[4-(5-nitro-2-furyl)-2-thiazoyl]formamide-induced urothelial tumors growing in male Fischer CDF (F344/CrlBR) rats. Histological examination of tumors in animals treated with the metallopurpurins and red light (greater than 590 nm, 360 J/cm2) revealed tumor necrosis 24 h after completion of therapy. Control tumors showed no histological change. In 30-day tumor regrowth studies, 70% of animals treated with the metallopurpurin derivative SnET2 were free of tumors while 50% of the animals treated with the free-base purpurin ET2 were free of tumor. Metallopurpurins have intense absorptions in the red region of the visible spectrum, a region with good tissue penetration. The metallopurpurins are easily prepared from the corresponding purpurins with a high degree of purity. This study demonstrates the potential of these photosensitizers for photodynamic cancer therapy.     

Survival of patients with cutaneous T-cell lymphoma after treatment with extracorporeal photochemotherapy

Few studies have assessed the long-term outcome of patients with cutaneous T-cell lymphoma (CTCL) treated with extracorporeal photochemotherapy (ECP). Our objective was to assess the efficacy, safety, and survival of a cohort of patients with refractory CTCL in stages Ib to III who were treated with ECP. A retrospective study was performed. Twenty patients (19 male, 1 female) aged 38 to 87 years with CTCL of the mycosis fungoides type (n=17) and primary cutaneous Ki-1 lymphoma (n=2) were treated twice a month. Sixteen had an adjunctive treatment with interferon alpha (IFN alpha) s.c. 3 times a week in the maximal tolerable dosage (i.e. up to 21x106). A complete response was achieved in 10 patients, a partial response in three and a stable disease in seven patients (response rate 65.0%). The overall survival was 29.4+/-16.0 months, the event-free survival was 26.2+/-12.4 months, and the progression-free survival was 23. 4+/-12.2 months. Four patients died of causes unrelated to CTCL and two patient died of CTCL. Median survival time was 26 months. No severe side effects were noted. ECP is a safe alternative therapy for CTCL. In particular when combined with IFN alpha it can induce long-term remissions.     

ABCG2-mediated transport of photosensitizers: potential impact on photodynamic therapy

In photodynamic therapy (PDT), a tumor-selective photosensitizer is administered followed by activation of the photosensitizer by exposure to a light source of a given wavelength. This, in turn, generates reactive oxygen species that induce cellular apoptosis and necrosis in tumor tissue. Based on our earlier finding that the photosensitizer pheophorbide a is an ABCG2 substrate, we explored the ability of ABCG2 to transport photosensitizers with a structure similar to that of pheophorbide a. ABCG2-overexpressing NCI-H1650 MX50 bronchoalveolar carcinoma cells were found to have reduced intracellular accumulation of pyropheophorbide a methyl ester and chlorin e6 compared to parental cells as measured by flow cytometry. The ABCG2 inhibitor fumitremorgin C was found to abrogate ABCG2-mediated transport. Intracellular fluorescence of hematoporphyrin IX, meso-tetra(3-hydroxyphenyl)porphyrin, and meso-tetra(3-hydroxyphenyl)chlorin was not substantially affected by ABCG2. ABCG2-overexpressing cells also displayed decreased intracellular fluorescence of protoporphyrin IX generated by exogenous application of 5-aminolevulinic acid. Mutations at amino acid 482 in the ABCG2 protein known to affect substrate specificity were not found to impact transport of the photosensitizers. In cytotoxicity assays, ABCG2-transfected HEK-293 cells were 11-fold, 30-fold, 4-fold, and >7-fold resistant to PDT with pheophorbide a, pyropheophorbide a methyl ester, chlorin e6, and 5-aminolevulinic acid, respectively. ABCG2-transfected cells were not resistant to PDT with meso-tetra(3-hydroxyphenyl) chlorin. Neither multidrug resistance-associated protein 1 expression nor P-glycoprotein expression appreciably decreased the intracellular fluorescence of any of the photosensitizers examined as determined by flow cytometry. The results presented here implicate ABCG2 as a possible cause for cellular resistance to photodynamic therapy.     

Lysosomal localization and mechanism of uptake of Nile blue photosensitizers in tumor cells

Nile blue derivatives have been shown to be potentially effective photosensitizers for photodynamic therapy of malignant tumors. Results of a previous study suggested that the high accumulation of these dyes in cells may be the result of dye aggregation, partition in membrane lipids, and/or sequestration in subcellular organelles. In this report, results of studies are presented from an investigation of the subcellular localization and mechanism of accumulation of these dyes in cells in vitro. A video-enhanced fluorescence microscopy was used, and a punctate pattern of fluorescence was seen, most of which was localized in the perinuclear region with extracellular dye concentrations between 1 to 100 nM. These particles resembled characteristic particles identified by standard lysosomal dyes. At higher dye concentrations (1 microM or above), fluorescence in the perinuclear region was too intense to resolve into discrete cellular structures, while fluorescence in other cellular structures including mitochondria and cytomembranes was visible. At even higher dye concentrations (10-100 microM), Nile blue derivatives were seen with a light microscope as blue particles, the size and location of which resembled the punctate fluorescence described above. Results which further suggest that the lysosome is the main site of dye localization include (a) histochemical staining of dye-loaded cells with the lysosomal marker enzyme acid phosphatase, which showed similar localization of the enzyme-staining and dye-containing particles, (b) phototreatment of dye-loaded cells which obliterated the majority of the acid phosphatase-stained particles, and (c) treatments with agents affecting the membrane pH gradient reduced the uptake and enhanced the efflux of dyes, while agents that alter cellular membrane potentials had no effect on dye accumulation. The uptake of the dyes was partially inhibited by inhibitors of oxidative phosphorylation indicating that at least part of the process is energy dependent. These findings, together with previous results showing that the cellular uptake of these dyes is highly concentrative and proportional to the extracellular dye concentration over a wide range, are consistent with the hypothesis that the dyes are mainly localized in the lysosomes via an ion-trapping mechanism. Results of the present study also suggest that the lysosomes may be an intracellular target for photodynamic killing of tumor cells mediated by Nile blue photosensitizers and that lysosomotropic photosensitization may be a strategy for effective and selective destruction of tumor cells.     

原发性肺癌线粒体DNA控制区微卫星不稳定性的研究

[目的]探讨线粒体DNA控制区微卫星不稳定性（mitochondrial microsatellite instability,mtMSI）与肺癌的相关性。[方法]采用PCR-SSCP（PCR single-stranded conformation polymorphism,PCR-SSCP）和序列测定技术对37例肺癌组织及癌旁组织线粒体DNA控制区的3个微卫星位点进行了分析。[结果]37例肺癌样本中共检出mtMSI12例（32.4%）,肺癌mtMSI发生率与患者性别、年龄及肿瘤的病理类型无相关性（P〉0.05）,吸烟组（包括曾经吸烟者）与非吸烟组mtMSI发生率两者有显著差异（P〈0.05）。[结论]线粒体DNA控制区的遗传不稳定性在一些肺癌的发生中可能起一定作用。