Efficient photodynamic inactivation of Leishmania parasites mediated by lipophilic water-soluble Zn(II) porphyrin ZnTnHex-2-PyP4+

BackgroundPhotodynamic inactivation (PDI) is emerging as a promising alternative for cutaneous leishmaniasis (CL). The chemotherapy currently used presents adverse effects and cases of drug resistance have been reported. ZnTnHex-2-PyP⁴⁺ is a porphyrin with a high potential as a photosensitizer (PS) for PDI, due to its photophysical properties, structural stability, and cationic/amphiphilic character that can enhance interaction with cells. This study aimed to investigate the photodynamic effects mediated by ZnTnHex-2-PyP⁴⁺ on Leishmania parasites.MethodsZnTnHex-2-PyP⁴⁺ stability was evaluated using accelerated solvolysis conditions. The photodynamic action on promastigotes was assessed by (i) viability assays, (ii) mitochondrial membrane potential evaluation, and (iii) morphological analysis. The PS-promastigote interaction was studied. PDI on amastigotes and the cytotoxicity on macrophages were also analyzed.ResultsZnTnHex-2-PyP⁴⁺, under submicromolar concentration, led to immediate inactivation of more than 95% of promastigotes. PDI promoted intense mitochondrial depolarization, loss of the fusiform shape, and plasma membrane wrinkling in promastigotes. Fluorescence microscopy revealed a punctate PS labeling in the parasite cytoplasm. PDI also led to reductions of ca. 64% in the number of amastigotes/macrophage and 70% in the infection index after a single treatment session. No noteworthy toxicity was observed on mammalian cells.ConclusionsZnTnHex-2-PyP⁴⁺ is stable against demetallation and more efficient as PS than the ethyl analogue ZnTE-2-PyP⁴⁺, indicating readiness for evaluation in in vivo studies as an alternative approach to CL.General significanceThis report highlighted promising photodynamic effects mediated by ZnTnHex-2-PyP⁴⁺ on Leishmania parasites, opening up perspectives for applications in CL pre-clinical assays and PDI of other microorganisms.     

Optimization of cellular uptake of zinc(II) 2,9,16,23-tetrakis[4-(N-methylpyridyloxy)]phthalocyanine for maximal photoinactivation of Candida albicans

Cellular uptake and photodynamic action of zinc(II) 2,9,16,23-tetrakis[4-(N-methylpyridyloxy)]phthalocyanine (ZnPPc⁴⁺) was examined in Candida albicans. In vitro investigations showed that ZnPPc⁴⁺ was rapidly bound to C. albicans cells. The binding of phthalocyanine to cells was dependent on ZnPPc⁴⁺ concentrations (1–10 μM) and cells densities (10⁶–10⁸ cells mL^?1). A high amount of ZnPPc⁴⁺ retained in the cells after two washing steps, indicating a strong interaction between the photosensitizer and C. albicans. The uptake was temperature dependent, although the difference between 37 °C and 4 °C was about 10 %. Also, the amount of ZnPPc⁴⁺ bound to C. albicans was affected when the cells were incubated for a longer time with azide and 2,4-dinitrophenol (DNP) prior to treatment with ZnPPc⁴⁺. Cell survival after irradiation was dependent on the irradiation period, ZnPPc⁴⁺ concentration and cells density. Photoinactivation of C. albicans cells was elevated even after two washing steps. The strong dependence of uptake on cell density reveals the strength and avidity of the binding of ZnPPc⁴⁺ to C. albicans cells. The accumulation behaviour of ZnPPc⁴⁺ suggests that mainly an affinity-mediated binding mechanism can be involved. Therefore, ZnPPc⁴⁺ is an interesting phthalocyanine for photodynamic inactivation (PDI) of yeasts in liquid suspensions.     

Superoxide dismutase is upregulated in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> following protoporphyrin-mediated photodynamic inactivation and does not directly influence the response to photodynamic treatment

Background  

Staphylococcus aureus, a major human pathogen causes a wide range of disease syndromes. The most dangerous are methicillin-resistant S. aureus (MRSA) strains, resistant not only to all β-lactam antibiotics but also to other antimicrobials. An alarming increase in antibiotic resistance spreading among pathogenic bacteria inclines to search for alternative therapeutic options, for which resistance can not be developed easily. Among others, photodynamic inactivation (PDI) of S. aureus is a promising option. Photodynamic inactivation is based on a concept that a non toxic chemical, called a photosensitizer upon excitation with light of an appropriate wavelength is activated. As a consequence singlet oxygen and other reactive oxygen species (e.g. superoxide anion) are produced, which are responsible for the cytotoxic effect towards bacterial cells. As strain-dependence in photodynamic inactivation of S. aureus was observed, determination of the molecular marker(s) underlying the mechanism of the bacterial response to PDI treatment would be of great clinical importance. We examined the role of superoxide dismutases (Sod) in photodynamic inactivation of S. aureus as enzymes responsible for oxidative stress resistance.     

In Vitro Photodynamic Inactivation of Plant-Pathogenic Fungi Colletotrichum acutatum and Colletotrichum gloeosporioides with Novel Phenothiazinium Photosensitizers

The increasing tolerance to currently used fungicides in both clinical and agricultural areas is of great concern. The nonconventional light-based approach of antimicrobial photodynamic treatment (APDT) is a promising alternative to conventional fungicides. We evaluated the effects of APDT with four phenothiazinium derivatives (methylene blue [MB], new methylene blue N [NMBN], toluidine blue O [TBO], and the novel pentacyclic phenothiazinium photosensitizer [PS] S137) on conidia of three fungal species (Colletotrichum acutatum, Colletotrichum gloeosporioides, and Aspergillus nidulans). The efficacy of APDT with each PS was determined, initially, based on photosensitizer MICs. Additionally, the effects of APDT with two selected PSs (NMBN and S137) on survival of conidia were evaluated. The subcellular localization of the PS in C. acutatum conidia was determined. The effects of photodynamic treatments on leaves of the plant host Citrus sinensis were also investigated. APDT with S137 showed the lowest MIC. MICs for S137 were 5 μM for the three fungal species when a fluence of 25 J cm⁻² was used. APDT with NMBN (50 μM) and S137 (10 μM) resulted in a reduction in the survival of the conidia of all species of approximately 5 logs with fluences of ≥15 J cm⁻². Washing of the conidia before light exposure did not prevent photodynamic inactivation. Both NMBN and S137 accumulated in cytoplasmic structures, such as lipid bodies, of C. acutatum conidia. No damage to orange tree leaves was observed after APDT.     

Synthesis of 2-morpholinetetraphenylporphyrins and their photodynamic activities

A series of 2-morpholinetetraphenylporphyrins functionalized with various substituents (Cl, Me, MeO group) at 4-phenyl position were prepared via nucleophilic substitution of 2-nitroporphyrin copper derivatives with morpholine by refluxing under a nitrogen atmosphere and then demetalization. Their basic photophysical properties, intracellular localization, cytotoxicities in vitro and in vivo were also investigated. All synthesized photosensitizers exhibited longer maxima absorption wavelengths than Hematoporphyrin monomethyl ether (HMME). They showed low dark cytotoxicity compared with that of HMME and were more phototoxic than HMME against Eca-109 cells in vitro. M3 also exhibited better photodynamic antitumor efficacy on BALB/c nude mice at a lower concentration. Therefore, M3 is a promising antitumor photosensitizer in photodynamic therapy application.     

Synthesis and biological evaluation of 173-dicarboxylethyl-pyropheophorbide-a amide derivatives for photodynamic therapy

Three novel 17³-dicarboxylethyl-pyropheophorbide-a amide derivatives as photosensitizers for photodynamic therapy (PDT) were synthesized from pyropheophorbide-a (Ppa). Their photophysical and photochemical properties, intracellular localization, photocytotoxicity in vitro and in vivo were investigated. All target compounds exhibited low cytotoxicity in the dark and remarkable photocytotoxicity against human esophageal cancer cells. Among them, 1a showed highest singlet oxygen quantum yield. Upon light activation, 1a exhibited significant photocytotoxicity. After PDT treatment, the growth of Eca-109 tumor in nude mice was significantly inhibited. Therefore, 1a is a powerful and promising antitumor photosensitizer for PDT.     

Efficient Photodynamic Therapy against Gram-Positive and Gram-Negative Bacteria Using THPTS,a Cationic Photosensitizer Excited by Infrared Wavelength

The worldwide rise in the rates of antibiotic resistance of bacteria underlines the need for alternative antibacterial agents. A promising approach to kill antibiotic-resistant bacteria uses light in combination with a photosensitizer to induce a phototoxic reaction. Concentrations of 1, 10 and 100µM of tetrahydroporphyrin-tetratosylat (THPTS) and different incubation times (30, 90 and 180min) were used to measure photodynamic efficiency against two Gram-positive strains of S.aureus (MSSA and MRSA), and two Gram-negative strains of E.coli and P.aeruginosa. We found that phototoxicity of the drug is independent of the antibiotic resistance pattern when incubated in PBS for the investigated strains. Also, an incubation with 100µM THPTS followed by illumination, yielded a 6lg (≥99.999%) decrease in the viable numbers of all bacteria strains tested, indicating that the THPTS drug has a high degree of photodynamic inactivation. We then modulated incubation time, photosensitizer concentration and monitored the effect of serum on the THPTS activity. In doing so, we established the conditions to obtain the strongest bactericidal effect. Our results suggest that this new and highly pure synthetic compound should improve the efficiency of photodynamic therapy against multiresistant bacteria and has a significant potential for clinical applications in the treatment of nosocomial infections.     

Evaluation of the antifungal photodynamic activity of Thymophylla pentachaeta extracts against Candida albicans and its virulence factors

BackgroundCandida albicans is one of the most common causative of opportunistic infections. Treatment of candidiasis is challenging considering the few antifungal drugs available and the increase in resistance. Antimicrobial photodynamic therapy (aPDT) is a recently developed therapeutic option that combines a non-toxic photosensitizer (PS) and light to kill the microbial pathogens. Targeting virulence, defined as the ability of a pathogen to cause overt disease, represents another attractive target for the development of novel antifungal agents. Thymophylla pentachaeta (DC.) Small var. belenidium (DC.) is an endemic plant from Argentina in which the presence of thiophenes, biologically active compounds whose antifungal activity is enhanced by irradiation with Ultraviolet A (UVA), have been already described.PurposeThe purpose of this study was to evaluate the photodynamic antifungal activity of hexane (Hex), dichloromethane (DCM), ethyl acetate (EtOAc) and methanol (MeOH) extracts from T. pentachaeta var. belenidium and their inhibitory effects on C. albicans virulence factors as well as biofilm formation and eradication.Study Design/MethodsAntifungal photodynamic activity of Hex, DCM, EtOAc and MeOH extracts from different parts of the plant were assessed with the microbroth dilution, bioautography and the time-kill assays, under light and darkness conditions. The capacities of the most active extracts of inhibiting Candida virulence factors (adherence to epithelial cells, germ tube and pseudomycelium formation and hydrolytic enzyme secretion) were assessed. In addition, the activity against biofilm formation and eradication has been investigated by reaction with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) that quantifies living cells in these structures.ResultsHex and DCM extracts from T. pentachaeta roots exhibited high photodynamic antifungal activity against C. albicans [Minimal fungicide concentrations (MFCs)= 7.8 µg/ml] under UVA light irradiation. Chemical analysis of active extracts (Hex and DCM from roots) revealed the presence of photoactive thiophenes. Both extracts generate reactive oxygen species through type I and II mechanisms. These extracts, at sub-inhibitory concentrations, under light conditions decreased the adherence of C. albicans to Buccal Epithelial Cells (BEC), inhibited germ tube formation and reduced esterase production. Finally, they demonstrated activity against preformed biofilms submitted to irradiation (MFCs= 3.91 µg/ml and 15.63 µg/ml for Hex and DCM extracts, respectively).ConclusionTaking together, results demonstrated the strong photodynamic effects of T. pentachaeta root extracts under UVA irradiation, making them valuable alternatives to the already established antifungal drugs against C. albicans.     

Role of cell surface hydrophobicity in Candida albicans biofilm

Overall cell surface hydrophobicity (CSH) is predicted to play an important role during biofilm formation in Candida albicans but is the result of many expressed proteins. This study compares the CSH status and CSH1 gene expression in C. albicans planktonic cells, sessile biofilm, and dispersal cells. Greater percentages of hydrophobic cells were found in non-adhered (1.5 h) and dispersal forms (24 or 48 h) (41.34±4.17% and 39.52±7.45%, respectively), compared with overnight planktonic cultures (21.69±3.60%). Results from quantitative real-time PCR confirmed greater up-regulation of the CSH1 gene in sessile biofilm compared with both planktonic culture and dispersal cells. Up-regulation was also greater in dispersal cells compared with planktonic culture. The markedly increased CSH found both in C. albicans biofilm, and in cells released during biofilm formation could provide an advantage to dispersing cells building new biofilm.     

Use of Merocyanine 540 for Photodynamic Inactivation of Staphylococcus aureus Planktonic and Biofilm Cells

Photodynamic inactivation of Staphylococcus aureus planktonic and biofilm cells by a phtotosensitizer, merocyanine 540 (MC 540), was investigated. For the planktonic experiments, MC 540 binding efficiency to bacterial cells was found to increase with both increasing MC 540 concentration and increasing incubation time, but the binding became saturated following 10 min of incubation. The antimicrobial activity was enhanced with an increasing light dose, but an increase in the light dose could not further improve the antimicrobial activity if the maximum excitation level attainable was less than the necessary minimum threshold level. Complete inactivation was achieved when the excitation level of MC 540 was somewhere above the threshold level. The relationship between antimicrobial activity and the excitation level of MC 540 revealed that the more MC 540 was excited, the more S. aureus cells were killed. For the biofilm experiments, the antimicrobial activity was enhanced with an increase in the light dose. No viable cells were detected when organisms were exposed to 15 μg of MC 540 per ml and a light dose of 600 J/cm² or to 20 μg of MC 540 per ml and a light dose of 450 J/cm². A quantitative analysis of MC 540 bound to biofilms was also performed, and the images from confocal laser scanning microscopy provided direct evidence that revealed the difference between the MC 540 remaining in the biofilms prior to irradiation and the MC 540 remaining in the biofilms after irradiation. The results of both the planktonic and biofilm experiments suggest that the antimicrobial activity of photodynamic inactivation of S. aureus is closely related to the excitation level of MC 540.     

Luminol as the light source for in situ photodynamic therapy

Presently, the light sources used in photodynamic therapy are high intensity lasers or light emitting diodes, making it unsuitable for large-volume tumors and those located deep inside the body. To overcome this limitation, we propose an in situ light source to excite the photosensitizer to generate toxic singlet oxygen and kill tumor cells directly. In this research, luminol served as the in situ light source in 5-aminolevulinic acid-mediated photodynamic treatment of Caco-2 cell cultures. 72 h after luminol excitation the viability of the treated cells significantly decreased compared to the control cells in assays including cell viability, cytotoxicity, flow cytometry and fluorescence confocal microscopy. According to the results, we suggested luminol could be used as an in situ light source for 5-aminolevulinic acid-mediated photodynamic therapy. This method would have great potential to extend the application of photodynamic therapy to tumors located deep inside the body.     

Sensitization of Candida albicans biofilms to various antifungal drugs by cyclosporine A

Background

Biofilms formed by Candida albicans are resistant towards most of the available antifungal drugs. Therefore, infections associated with Candida biofilms are considered as a threat to immunocompromised patients. Combinatorial drug therapy may be a good strategy to combat C. albicans biofilms.

Methods

Combinations of five antifungal drugs- fluconazole (FLC), voriconazole (VOR), caspofungin (CSP), amphotericin B (AmB) and nystatin (NYT) with cyclosporine A (CSA) were tested in vitro against planktonic and biofilm growth of C. albicans. Standard broth micro dilution method was used to study planktonic growth, while biofilms were studied in an in vitro biofilm model. A chequerboard format was used to determine fractional inhibitory concentration indices (FICI) of combination effects. Biofilm growth was analyzed using XTT-metabolic assay.

Results

MICs of various antifungal drugs for planktonic growth of C. albicans were lowered in combination with CSA by 2 to 16 fold. Activity against biofilm development with FIC indices of 0.26, 0.28, 0.31 and 0.25 indicated synergistic interactions between FLC-CSA, VOR-CSA, CSP-CSA and AmB-CSA, respectively. Increase in efficacy of the drugs FLC, VOR and CSP against mature biofilms after addition of 62.5 μg/ml of CSA was evident with FIC indices 0.06, 0.14 and 0.37, respectively.

Conclusions

The combinations with CSA resulted in increased susceptibility of biofilms to antifungal drugs. Combination of antifungal drugs with CSA would be an effective prophylactic and therapeutic strategy against biofilm associated C. albicans infections.     

Porphyrin derivatives as photosensitizers for the inactivation of Bacillus cereus endospores

Aims:  In this study, we propose (i) to study the photodynamic inactivation (PDI) efficiency of neutral and cationic porphyrin derivatives, (ii) to characterize the kinetics of the inactivation process using Bacillus cereus as a model endospore-producing bacterium and (iii) to conclude on the applicability of porphyrin derivatives in the inactivation of bacterial endospores.
Methods and Results:  The study of PDI of Bacillus cereus endospores, taken as model-endospores, using porphyrin derivatives differing in the number of positive charges and in the meso-substituent groups, showed that neutral, monocationic and dicationic porphyrins are quite ineffective, in contrast with the tri- and tetra-cationic molecules. The most effective porphyrin is a tricationic porphyrin with a meso-pentafluorophenyl group. With this photosensitizer (PS), at 0·5  μ mol l⁻¹, a reduction of 3·5 log units occurs after only 4 min of irradiation. None of the porphyrin derivatives showed toxicity in the absence of light.
Conclusions:  Some porphyrin derivatives are efficient PSs for the inactivation of bacterial endospores and should be considered in further studies. Small modifications in the substituent groups, in addition to charge, significantly improve the effectiveness of the molecule as a PS for endospore inactivation.
Significance and Impact of the Study:  Tetrapyrrolic macrocycles should be regarded as worthy to explore for the PDI of spore-producing gram-positive bacteria. The development of molecules, more selective and effective, emerges as a new objective.     

In vitro activity of eugenol against <Emphasis Type="Italic">Candida albicans</Emphasis> biofilms

Most manifestations of candidiasis are associated with biofilm formation occurring on the surfaces of host tissues and medical devices. Candida albicans is the most frequently isolated causative pathogen of candidiasis, and the biofilms display significantly increased levels of resistance to the conventional antifungal agents. Eugenol, the major phenolic component of clove essential oil, possesses potent antifungal activity. The aim of this study was to investigate the effects of eugenol on preformed biofilms, adherent cells, subsequent biofilm formation and cell morphogenesis of C. albicans. Eugenol displayed in vitro activity against C. albicans cells within biofilms, when MIC₅₀ for sessile cells was 500 mg/L. C. albicans adherent cell populations (after 0, 1, 2 and 4 h of adherence) were treated with various concentrations of eugenol (0, 20, 200 and 2,000 mg/L). The extent of subsequent biofilm formation were then assessed with the tetrazolium salt reduction assay. Effect of eugenol on morphogenesis of C. albicans cells was observed by scanning electron microscopy (SEM). The results indicated that the effect of eugenol on adherent cells and subsequent biofilm formation was dependent on the initial adherence time and the concentration of this compound, and that eugenol can inhibit filamentous growth of C. albicans cells. In addition, using human erythrocytes, eugenol showed low hemolytic activity. These results indicated that eugenol displayed potent activity against C. albicans biofilms in vitro with low cytotoxicity and therefore has potential therapeutic implication for biofilm-associated candidal infections.     

Morphogenesis control in Candida albicans and Candida dubliniensis through signaling molecules produced by planktonic and biofilm cells

Morphogenesis control by chemical signaling molecules is beginning to be highlighted in Candida biology. The present study focuses on morphogenic compounds produced in situ by Candida albicans and Candida dubliniensis during planktonic and biofilm growth that may at least partially substantiate the effect promoted by supernatants in morphogenesis. For both species, planktonic versus biofilm supernatants were analyzed by headspace-solid-phase microextraction and gas chromatography-mass spectrometry. Both planktonic cells and biofilm supernatants of C. albicans and C. dubliniensis contained isoamyl alcohol, 2-phenylethanol, 1-dodecanol, E-nerolidol, and E,E-farnesol. Alcohol secretion profiles were species, culture mode, and growth time specific. The addition of exogenous alcohols to the cultures of both species inhibited the morphological transition from the yeast to the filamentous form by up to 50%. The physiological role of these alcohols was put to evidence by comparing the effects of a 96-h cultured supernatant with synthetic mixtures containing isoamyl alcohol, 2-phenylethanol, E-nerolidol, and E,E-farnesol at concentrations determined herein. All synthetic mixtures elicited a morphological effect similar to that observed for the corresponding supernatants when used to treat C. albicans and C. dubliniensis cultures, except for the effect of the 96-h C. dubliniensis planktonic supernatant culture on C. albicans. Overall, these results reveal a group of alcohol extracellular signaling molecules that are biologically active with C. albicans and C. dubliniensis morphogenesis.     

Dispersion as an Important Step in the Candida albicans Biofilm Developmental Cycle

Biofilms are dynamic microbial communities in which transitions between planktonic and sessile modes of growth occur interchangeably in response to different environmental cues. In the last decade, early events associated with C. albicans biofilm formation have received considerable attention. However, very little is known about C. albicans biofilm dispersion or the mechanisms and signals that trigger it. This is important because it is precisely C. albicans cells dispersed from biofilms that are the main culprits associated with candidemia and establishment of disseminated invasive disease, two of the gravest forms of candidiasis. Using a simple flow biofilm model recently developed by our group, we have performed initial investigations into the phenomenon of C. albicans biofilm dispersion, as well as the phenotypic characteristics associated with dispersed cells. Our results indicate that C. albicans biofilm dispersion is dependent on growing conditions, including carbon source and pH of the media used for biofilm development. C. albicans dispersed cells are mostly in the yeast form and display distinct phenotypic properties compared to their planktonic counterparts, including enhanced adherence, filamentation, biofilm formation and, perhaps most importantly, increased pathogenicity in a murine model of hematogenously disseminated candidiasis, thus indicating that dispersed cells are armed with a complete arsenal of “virulence factors” important for seeding and establishing new foci of infection. In addition, utilizing genetically engineered strains of C. albicans (tetO-UME6 and tetO-PES1) we demonstrate that C. albicans biofilm dispersion can be regulated by manipulating levels of expression of these key genes, further supporting the evidence for a strong link between biofilms and morphogenetic conversions at different stages of the C. albicans biofilm developmental cycle. Overall, our results offer novel and important insight into the phenomenon of C. albicans biofilm dispersion, a key part of the biofilm developmental cycle, and provide the basis for its more detailed analysis.     

Photodynamic Therapy Using Systemic Administration of 5-Aminolevulinic Acid and a 410-nm Wavelength Light-Emitting Diode for Methicillin-Resistant Staphylococcus aureus-Infected Ulcers in Mice

Bacterial resistance to antibiotics has become a worldwide problem. One potential alternative for bacterial control is photodynamic therapy. 5-aminolevulinic acid is a natural precursor of the photosensitizer protoporphyrin IX. Relatively little is known about the antibacterial efficacy of photodynamic therapy using the systemic administration of 5-aminolevulinic acid; a few reports have shown that 5-aminolevulinic acid exerts photodynamic effects on methicillin-resistant Staphylococcus aureus (MRSA) in vitro. In this study, we evaluated the effectiveness of photodynamic therapy using 5-aminolevulinic acid and a 410-nm wavelength light-emitting diode in vitro and in vivo for the treatment of MRSA. We found that 5-aminolevulinic acid photodynamic therapy with the light-emitting diode had an in-vitro bactericidal effect on MRSA. In vivo, protoporphyrin IX successfully accumulated in MRSA on ulcer surfaces after intraperitoneal administration of 5-aminolevulinic acid to mice. Furthermore, 5-aminolevulinic acid photodynamic therapy accelerated wound healing and decreased bacterial counts on ulcer surfaces; in contrast, vancomycin treatment did not accelerate wound healing. Our findings indicate that 5-aminolevulinic acid photodynamic therapy may be a new treatment option for MRSA-infected wounds.     

In Vitro Comparison of Hypericin and 5-Aminolevulinic Acid-Derived Protoporphyrin IX for Photodynamic Inactivation of Medulloblastoma Cells

Background

Hypericin (HYP) is a naturally occurring photosensitizer. Cellular uptake and photodynamic inactivation after incubation with this photosensitizer have neither been examined in medulloblastoma cells in vitro, nor compared with 5-aminolevulinic acid-derived protoporphyrin IX (5-ALA-derived PpIX).

Methods

In 3 medulloblastoma cell lines (D283 Med, Daoy, and D341 Med) the time- and concentration-dependent intracellular accumulation of HYP and 5-ALA-derived PpIX was analyzed by fluorescence microscopy (FM) and FACS. Photocytotoxicity was measured after illumination at 595 nm (HYP) and 635 nm (5-ALA-derived PpIX) in D283 Med cells and compared to U373 MG glioma cells.

Results

All medulloblastoma cell lines exhibited concentration- and time-dependent uptake of HYP. Incubation with HYP up to 10 µM resulted in a rapid increase in fluorescence intensity, which peaked between 2 and 4 hours. 5-ALA-derived PpIX accumulation increased in D283 Med cells by 22% over baseline after 5-ALA incubation up to 1.2 mM. Photocytotoxicity of 5-ALA-derived PpIX was higher in D283 Med medulloblastoma compared to U373MG glioma. The [lethal dose (light dose that is required to reduce cell survival to 50% of control)] of 5-ALA-derived PpIX was 3.8 J/cm² in D283 Med cells versus 5.7 J/cm2 in U373MG glioma cells. Photocytotoxicity of HYP in D283 Med cells was determined at 2.5 µM after an incubation time of 2 h and an illumination wavelength of 595 nm. The value was 0.47 J/cm².

Conclusion

By its 5-fold increase in fluorescence over autofluorescence levels HYP has excellent properties for tumor visualization in medulloblastomas. The high photocytotoxicity of HYP, compared to 5-ALA-derived PpIX, is convincingly demonstrated by its 8- to 13-fold lower . Therefore HYP might be a promising molecule for intraoperative visualization and photodynamic treatment of medulloblastomas.     

β-Actin-binding complementarity-determining region 2 of variable heavy chain from monoclonal antibody C7 induces apoptosis in several human tumor cells and is protective against metastatic melanoma

Complementarity-determining regions (CDRs) from monoclonal antibodies tested as synthetic peptides display anti-infective and antitumor activities, independent of the specificity of the native antibody. Previously, we have shown that the synthetic peptide C7H2, based on the heavy chain CDR 2 from monoclonal antibody C7, a mAb directed to a mannoprotein of Candida albicans, significantly reduced B16F10 melanoma growth and lung colony formation by triggering tumor apoptosis. The mechanism, however, by which C7H2 induced apoptosis in tumor cells remained unknown. Here, we demonstrate that C7H2 interacts with components of the tumor cells cytoskeleton, being rapidly internalized after binding to the tumor cell surface. Mass spectrometry analysis and in vitro validation revealed that β-actin is the receptor of C7H2 in the tumor cells. C7H2 induces β-actin polymerization and F-actin stabilization, linked with abundant generation of superoxide anions and apoptosis. Major phenotypes following peptide binding were chromatin condensation, DNA fragmentation, annexin V binding, lamin disruption, caspase 8 and 3 activation, and organelle alterations. Finally, we evaluated the cytotoxic efficacy of C7H2 in a panel of human tumor cell lines. All tumor cell lines studied were equally susceptible to C7H2 in vitro. The C7H2 amide without further derivatization significantly reduced lung metastasis of mice endovenously challenged with B16F10-Nex2 melanoma cells. No significant cytotoxicity was observed toward nontumorigenic cell lines on short incubation in vitro or in naïve mice injected with a high dose of the peptide. We believe that C7H2 is a promising peptide to be developed as an anticancer drug.