FosPeg® PDT alters the EBV miRNAs and LMP1 protein expression in EBV positive nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) is one of the top ten cancers highly prevalent in Hong Kong and South China. Epstein-Barr virus (EBV) infection contributes to the tumorigenesis of NPC through the expression of different viral proteins. Among these, Latent Membrane Protein 1(LMP1) is the major oncoprotein expressed by EBV. Foscan® (Biolitec AG), m-tetrahydroxyphenylchlorin (mTHPC)-based photosensitizing drug, has been used in the photodynamic therapy (PDT) for head and neck cancers. FosPeg® (Biolitec AG) is a new formulation of mTHPC contained in PEGylated liposomes with optimized distribution properties. In this in vitro study, the potential of FosPeg®-PDT on human EBV positive NPC cell (c666-1) and EBV negative cells (HK1 and CNE2) were investigated. Effects of FosPeg®-PDT on the expression of EBV BART miRNAs (EBV miRNA BART 1-5p, BART 16, and BART 17-5p), LMP1 mRNA and proteins on c666-1 cells were also elucidated. The killing efficacy of FosPeg®-PDT on NPC cells were determined by MTT assay after LED activation. Effects of FosPeg®-PDT on the expression of LMP1 mRNA and protein were examined by real time PCR and western blot analysis. FosPeg®-PDT demonstrated its antitumor effect on c666-1 cells in a drug and light dose dependent manner. LD30, LD50 and LD70 were achieved by applying LED activation (3J/cm(2)) at 4h post incubated cells with 0.05µg/ml, 0.07µg/ml and 0.3µg/ml FosPeg®, respectively. Up-regulation of both LMP1 mRNA and protein were observed after FosPeg®-PDT in a dose dependent manner. FosPeg®-PDT exerted antitumor effect on c666-1 cells through up-regulation of LMP1 protein. Understanding the mechanism of FosPeg®-PDT may help to develop better strategies for the treatment of NPC.

Modulation of telomerase and signal transduction proteins by hexyl-ALA-photodynamic therapy (PDT) in human doxorubicin resistant cancer cell models.

AIMS: This study employed a doxorubicin resistant (MES-SA-Dx5) human uterine sarcoma cell line and its counterpart (MES-SA), to elucidate the efficacy of aminolevulinic acid-hexylester (hexyl-ALA) mediated PDT at molecular and transcriptional levels. METHODS: Hexyl-ALA generated protoporphyrin IX in both cells were determined by molecular probes using Confocal Laser Scanning Microscopy. The hexyl-ALA-PDT induced signal transduction proteins and mode of cell death were quantitated by CASE ELISA assays and DAPI staining. The modulation of hTERT mRNA expression and telomerase activity were investigated by TaqMan real-time PCR and ELISA respectively. Hexyl-ALA-PDT mediated cell migratory effect was determined by wound-healing assay. RESULTS: The results demonstrated that mitochondria were the major target of hexyl-ALA. At LD(30), hexyl-ALA-PDT significantly provoked an up-regulation of phosphorylated p38MAPK and JNK proteins in both cells. Hexyl-ALA-PDT down-regulated hTERT (a catalytic subunit of telomerase) mRNA expression and showed a strong correlation with diminished telomerase activity in both cells (MES-SA: r(2) = 0.9932; MES-SA-Dx5: r(2) = 0.9775). The suppression of cell migratory effect in both cells was obtained after hexyl-ALA-PDT. Further, 50% and 30% of apoptotic cells were attained at LD(50), for wild-type and drug resistant cells respectively. Unlike the wild-type, a higher PDT dose was crucial to induce apoptosis in the drug resistant cells. CONCLUSIONS: Our study provides the first evidence that p38MAPK and JNK kinases played a vital role in triggering hexyl-ALA-PDT-induced apoptosis, down-regulated hTERT mRNA expression and telomerase activity in both proposed cells. In vivo studies are worth examining for the benefit of clinical applications in drug resistant cancers and PDT development.

Hypericin-mediated photodynamic antimicrobial effect on clinically isolated pathogens.

The aim of this study was to determine the photodynamic antimicrobial effect of hypericin on clinically isolated Staphylococcus aureus and Escherichia coli cells. Bacterial cells (10(8) cells per mL) were incubated with hypericin (0-40 µM) for 30 min and followed by light irradiation of 600-800 nm at 5-30 J cm(-2). Cell survival was determined by colony counting, cellular hypericin uptake examined by flow cytometer, and cell membrane damage examined by scanning electron microscopy and leakage assay. The effectiveness of hypericin-mediated photodynamic killing was strongly affected by cellular structure and photosensitizer uptake. The combination of hypericin and light irradiation could induce significant killing of Gram positive methicillin-sensitive and -resistant S. aureus cells (>6 log reduction), but was not effective on Gram negative E. coli cells (<0.2 log reduction). The difference was caused by different cell wall/membrane structures that directly affected cellular uptake of hypericin.

Hypocrellin B-encapsulated nanoparticle-mediated rev-caspase-3 gene transfection and photodynamic therapy on tumor cells.

Gene therapy and photodynamic therapy are two kinds of important therapeutic strategies for treating malignant tumors. In order to explore the combined effects of gene therapy and PDT on tumor cells, rev-caspase-3 gene was transfected into the tumor model CNE2 cells using hypocrellin B-encapsulated nanoparticle (nano-HB) as a carrier. The transfected CNE2 cells were then irradiated by light from a LED source and the survival rate was investigated 18 h after PDT. Apoptosis was analyzed by a flow cytometer with propidium iodine (PI) staining and the active caspase-3 expression was measured using flow cytometry with phycoerythrin (PE)-conjugated anti-active caspase-3 antibody. The result from the flow cytometer showed that the level of the activated caspase-3 significantly increased up to 63.10% in the transfected CNE2 cells. The survival rate 18 h after gene transfection alone and nano-HB-mediated PDT was 96.6±2.07%, 72.6±4.15%, respectively. However, the survival rate of the transfected CNE2 cells 18 h after LED exposure significantly decreased to 50.6±5.98% under the light energy of 4 J/cm(2). Apoptotic rate 18 h after the combination of gene transfection and PDT increased up to 24.65%. Our findings demonstrated that nano-HB could significantly enhance the transfection efficiency of rev-caspase-3 gene in the CNE2 cells. LED irradiation could effectively kill the treated CNE2 cells and induce apoptosis, suggesting hypocrellin B-encapsulated nanoparticle as an efficient gene carrier and a novel photosensitizer. The combination of gene therapy and PDT using nanoparticle as a mediator can be developed for treating nasopharyngeal carcinoma.

Induction of early apoptosis in human nasopharyngeal carcinoma cells by mTHPC-mediated photocytotoxicity.

OBJECTIVES: In this study, the early apoptotic events elicited by mTHPC-mediated photo-cytotoxicity were explored in a human nasopharyngeal carcinoma cell line (NPC/HK1). METHODS AND MATERIALS: NPC/HK1 cells (5 x 10(3)) were incubated with photosensitizer mTHPC (0.8 microg/ml) in chamber slides for 20h and subjected to light irradiation at 2J/cm(2) (LD(80)). Morphologic changes of treated cells were examined under light microscopy and confocal microscopy at 0-4h after the light irradiation. The early stage of apoptosis was detected by fluorescein-conjugated Annexin V (Annexin V-FITC) assay. Mitochondrial membrane damage and cytochrome c release were determined by flowcytometric analysis. Bcl-2 expression was measured by Western blot analysis. RESULTS: One hour after mTHPC-mediated photodynamic therapy (PDT), microscopic examination showed membrane blebbing and cell shrinkage. Annexin V-FITC assay showed that a considerable number of NPC/HK1 cells became apoptotic. Flowcytometric analysis showed that the cytochrome c was released at 1h after PDT. Bcl-2 expression also declined significantly compared to control groups. CONCLUSIONS: mTHPC-mediated photo-cytotoxicity can effectively induce early apoptotic responses in NPC/HK1 cells which might be modulated by mitochondrial damages and Bcl-2 inhibition.

Effects of photoactivated 5-aminolevulinic acid hexyl ester on MDR1 over-expressing human uterine sarcoma cells.

The role of multi-drug resistance (MDR1) and its product, P-glycoprotein (P-gp) on 5-aminolevulinic acid hexyl ester (Hexyl-ALA) mediated phototoxicity was determined with human uterine sarcoma cells, MES-SA control and MDR1 expressing MES-SA-Dx5. MDR1 expression reduced intracellular levels of the Hexyl-ALA metabolite, protoporphyrin IX (PpIX) to a limited degree and could be reversed with a P-gp inhibitor, verapamil. P-gp expression also reduced Hexyl-ALA photosensitivity. More importantly, photoactivated Hexyl-ALA reduced at the mRNA and protein levels without altering housekeeping GAPDH mRNA. These findings suggest that Hexyl-ALA could be used to selectively reduce P-gp expression in overcoming resistance to chemotherapy agents such as doxorubicin and paclitaxel.

Combination of photodynamic therapy and immunomodulation: current status and future trends.

Photodynamic therapy (PDT) has been used for the treatment of nonmalignant and malignant diseases from head to toe. Over the last decade its clinical application has gained increasing acceptance around the world. Pre-clinical studies demonstrate that, in addition to the direct local cytotoxicity and vascular effects, PDT can induce various host immune responses. Recent clinical data also show that improved clinical outcomes are obtained through the combination of PDT and immunomodulation. This review will summarize and discuss recent progress in developing innovative regimen of PDT combined with immunomodulation for the treatment of both nonmalignant and malignant diseases.

A comparative in vitro photoinactivation study of clinical isolates of multidrug-resistant pathogens.

Photodynamic inactivation (PDI) has been investigated to cope with the increasing incidence of multidrug-resistant (MDR) pathogens. In Hong Kong, methicillin-resistant Staphylococcus aureus (MRSA) and extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli are the two commonest MDR pathogens. Here, we studied the photodynamic inactivation (PDI) mediated by poly-L-lysine chlorin(e6) conjugate (pL-ce6) and toluidine blue O (TBO) in clinical MRSA and ESBL producing E. coli, together with their corresponding American Type Culture Collection (ATCC) strains. Both pL-ce6 and TBO mediated a light- and drug dose-dependent efficacy for the four pathogens. pL-ce6 was more effective. pL-ce6 at 8 microM, 30 Jcm(-2), attained 5 log killing for ESBL-producing E. coli and E. coli (ATCC 25922); 4 log killing for MRSA, and 3 log killing for S. aureus (ATCC 25923). TBO at 80 microM, 30 Jcm(-2), only exhibited 3 log killing in MRSA and 2 log killing in S. aureus (ATCC 25923). TBO (400 microM, 30 Jcm(-2)) induced equal killing for ESBL-producing E. coli and E. coli (ATCC 25922). Our studied MRSA isolate responded better than S. aureus (ATCC 25923). Thus, pL-ce6-mediated PDI in other MRSA isolates deserves further investigation.

Assessment of membrane protection by traditional Chinese medicines using a flow cytometric technique: preliminary findings.

In this preliminary study, we used a 'living cell' flow-cytometric approach to membrane protection by four traditional Chinese medicines (TCMs). Cells were incubated, separately, for 30 min with aqueous extracts (1.5% w/v) of lingzhi (Ganoderma lucidum), ginger (Zingiber officianale), ginseng (Panax ginseng), and green tea (Camellia sinensis). Membranes were labelled with a fluorescent probe, cells were then incubated with cumene hydroperoxide, and site-specific oxidation induced by iron/ascorbate. Oxidation of membrane lipids quenches fluorescence. Forward-scatter fluorescence was measured at timed intervals after initiation of oxidation. Results indicate that lingzhi and ginger contain antioxidant component(s) that act within the cell membrane and slow lipid peroxidation in situ. Results demonstrate also that this living cell model is a useful biomonitoring tool to help determine molecular aspects of putative health effects of TCMs.