Ellie Shihng Meir Chu

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 (108 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.

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.

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.

Photodynamic action of LED-activated nanoscale photosensitizer in nasopharyngeal carcinoma cells

BackgroundPhotodynamic therapy has been confirmed to an efficient therapeutic modality of malignant tumors. The aim of the present study was to explore the photodynamic action of LED-activated nanoscale photosensitizer-loading hypocrellin in nasopharyngeal carcinoma cells.Material/MethodsNasopharyngeal carcinoma cell line CNE2 cells were subjected to photodynamic therapy with hypocrellinloaded nanophotosensitizer. The uptake of the nanophotosensitizer in the CNE-2 cells was measured using a spectrophotometer and photodynamic toxicity was investigated 18 h after LED radiation treatment. Apoptosis was determined using flow cytometry with propidum iodine staining, and nuclear staining with Hoechst 33258. Active caspase-3 in the CNE2 cells was evaluated using flow cytometry with phycoerythrin (PE)-conjugated anti-active caspase-3 antibodies.ResultsThe cellular uptake of the nanophotosensitizer in the CNE-2 cells reached optimal at 6 h. LED-activated nanophotosensitizer resulted in doseand light-dependent phototoxicity. Apoptotic rate 18 h after PDT increased to 34.32 ± 1.94% under the light energy of 1 J/cm2. Hoechest 33258 staining reinforced the findings above. Condensation of chromatin and nuclear fragmentations was found in many PDT-treated cells. The activated caspase-3 in the CNE2 cells significantly increased up to 43.90% when the CNE2 cells were exposed to the nanophotosensitizer for 6 h and then 1 J/cm2 irradiation.ConclusionLED-activated nanophotosensitizer significantly killed the CNE2 cells and enhanced apoptosis and activated caspase-3 in the CNE2 cells. The hypocrellin-loaded nanophotosensitizer might be efficient photosensitizer and LED-activated nanophotosensitizer can be developed for treating nasopharyngeal carcinoma.

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.

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.

The effect of aloe‑emodin‑induced photodynamic activity on the apoptosis of human gastric cancer cells: A pilot study

The aim of the present study was to explore the effect of aloe-emodin (AE)-induced photodynamic activity in human gastric cancer cells. AE was used as a photosensitizer to explore the effect of photodynamic therapy (PDT) in human gastric cancer cells (SGC-7901). An MTT assay was used to detect the effect of AE-induced PDT in optimal concentrations and illumination energy densities in human gastric cancer cells. Following AE-induced PDT, morphological changes of the cells and the rate of cell death were evaluated by TUNEL assay and flow cytometry, respectively. The expression levels of caspase-9 and caspase-3 were determined by western blot analysis. The AE and AE-induced PDT demonstrated a significant inhibitive effect on the proliferation of human gastric cancer cells in dose-dependent and energy-dependent manners. For subsequent experiments, 10 µM AE and 12.8 J/cm2 illumination energy density were used. Typical morphological changes of apoptosis were observed in the cells using a TUNEL assay 12 h subsequent to AE-induced PDT. The percentage of apoptotic cells treated with AE-induced PDT significantly increased when compared with the control group, the 10 µM AE group and the illumination group (P<0.05). Upregulation of caspase-9 and caspase-3 protein levels was also observed following AE-induced PDT. The present study revealed that 10 µM AE-induced PDT had an inhibitory effect on human gastric cancer cells, and it may induce cell apoptosis by upregulating caspase-9 and caspase-3, which indicated that the mitochondrial pathway may be involved. AE-induced PDT has the potential to be a novel therapy for the treatment of human gastric cancer. However, further investigations are required.

An in vitro investigation of photodynamic efficacy of FosPegⓇ on human colon cancer cells

Photodynamic therapy (PDT) is a novel therapeutic approach for combating various cancers. PDT involves the administration of a photosensitizer which generates singlet oxygen after light activation. FosPegⓇ is the liposomal formulation of mTHPC. In this in vitro study, the photodynamic efficacy of FosPegⓇ on a human colon cancer cell line (HT29) was investigated via studying the cellular uptake of FosPegⓇ, FosPegⓇ PDT mediated photocytotoxicity and the cell death mechanism were triggered. FosPegⓇ PDT demonstrated its antitumor effect in a drug and light dose-dependent manner in HT-29 cells. Lethal dose (LD50) was achieved with 0.4 μg/mL of drug and 3 J/cm-2 of light dose. FosPegⓇ PDT triggered apoptotic cell death via activating caspase cascade and regulating cell cycle progression. In conclusion, FosPegⓇ-PDT is an effective measure to combat human colon cancer cells.

Modulation of COX2 and hTERT expression by photodynamic therapy in human colon cancer cells

Photodynamic therapy (PDT) was employed as a cancer therapy with photosensitizer (PS)-loaded cancer cells, eradicated by the reactive oxygen species after light activation. Cyclo-oxygenase 2 (COX2) is an enzyme expressed in 80% of colon adenocarcinoma and is one of the targets for effective cancer treatment. There is also uprising evidence that the human telomerase reverse transcriptase (hTERT), a catalytic component of telomerase, is reported as a promising indicator for monitoring cancer treatment. In this study, NPe6 mediated PDT on COX2 induced apoptosis in HT-29 was investigated. The cell cycle changes was analysed by flow cytometry and the hTERT expression at pre and post PDT was evaluated at transcription level by Taqman real time PCR. NPe6-PDT in HT-29 cells demonstrated anti-proliferating effect in a drug and light dose dependent manner. LD50 was achieved at 16μg/mL and 2J/cm2 at 4 hour-post treatment with a significant down-regulation of COX2 expression at LD30 and LD50 by immunohistochemical staining (IHC) (p<0.05, One-Way ANOVA). Membrane blebbing was detected in over 60% of cells. 35.2% of treated cells arrested in S-phase at LD50 after 24 hours by flow cytometry. A 0.25- and 0.6-fold down-regulation of hTERT mRNA expression was achieved at LD30 and LD50 respectively by TaqMan real-time PCR. To summarize, NPe6 mediated PDT down-regulated COX2 expression and triggered cell apoptosis. The hTERT can serve as an indicative marker for monitoring NPe6-PDT cancer treatment efficacy.