ALA-mediated Photodynamic Therapy Research Articles

5-aminolevulinic acid photodynamic therapy for chronic wound infection in rats with diabetes

Recent research indicated that ulcers and peripheral vascular disease resulting from drug-resistant bacterial infections are the main causes of delayed healing in chronic diabetic wounds. 5-Aminolevulinic acid (ALA) is a second-generation endogenous photosensitizer. The therapeutic effect and mechanism of ALA-mediated photodynamic therapy (ALA-PDT) on methicillin-resistant Staphylococcus aureus (MRSA)-infected wounds in diabetic rats were investigated in this study. The results revealed the promising antibacterial effects of ALA-PDT MRSA in vitro, with a minimum inhibitory concentration and minimum bactericidal concentration of 250 and 500 μM, respectively. ALA-PDT also changed the permeability and structural integrity of bacterial cell membranes by producing reactive oxygen species. Meanwhile, ALA-PDT accelerated wound healing in MRSA-infected diabetic rats, with 5 % ALA-PDT achieving complete sterilization in 14 days and wound closure in 21 days. Treatment with 5 % ALA-PDT additionally improved the histopathological appearance of skin tissue, as well as fibrosis, inflammatory cytokine release, and angiogenesis-related protein expression. These findings indicated that ALA-PDT significantly promoted the healing of MRSA-infected wounds in diabetic rats by eliminating bacteria, inhibiting inflammation, generating granulation tissues, promoting neovascularization, and restoring damaged nerves. In addition, the healing mechanism was related to the activation of inflammatory and angiogenesis pathways through the regulation of tumor necrosis factor-alpha and interleukin-6 expression and upregulation of CD206, CD31, and VEGF. These findings underscored the potential role of ALA-PDT in promoting the healing of chronic diabetic wounds.

Treatment of Reticular Oral Lichen Planus with Photodynamic Therapy: A Case Series.

The aim of the study was to clinically evaluate the efficacy of photodynamic therapy in treatment of the reticular form of oral lichen planus (OLP). Twenty patients aged 40-76, with 40 confirmed OLP lesions in total, underwent photodynamic therapy (PDT) following the authors' own protocol, which used 5% 5-aminolevulinic acid as a photosensitizer applied two hours prior to illumination with a diode lamp emitting light at 630 nm and 300 mW. The therapy comprised of 10 weekly illumination sessions and was clinically evaluated between its completion and the end of a 12-month follow-up. While the baseline mean size of all 40 lesions was 2.74 ± 3.03 cm2, it was 2.97 ± 3.4 cm2 for the 30 lesions on the buccal mucosa and 2.02 ± 1.32 cm2 for the remaining 10 on the gingiva and tongue. On completion of the therapy, 37 sites improved, including 14 showing complete remission. From that point, the mean size reduction of 56.2% (1.2 ± 1.4 cm2) rose to 67.88% (0.88 ± 1.3 cm2) 12 months later. The results suggest that ALA-mediated photodynamic therapy was effective for the reticular form of OLP and may become an optional or complementary treatment.

Application of 5-aminolevulinic acid-mediated Waterlase-assisted photodynamic therapy in the treatment of oral leukoplakia

Photodynamic therapy (PDT) is an alternative microinvasive approach with satisfying results in the treatment of oral leukoplakia (OL). PDT combined with laser irradiation shows promise, safety and efficacy in treating OL. The efficacy of waterlase (YSGG) combined with PDT was studied by brush and tissue biopsy. Seventy-one patients with histologically diagnosed OL were enrolled, including patients with mild to moderate dysplasia, severe dysplasia and various dysplastic tissues. Patients were evaluated at baseline (t0), the end of treatment (t1) and 1 year later (t2). At t1, PDT showed a significant therapeutic effect on OL with mild to moderate dysplasia. Clinical and histological examinations revealed 60 cases (84.51%) of complete remission and 11 cases (15.49%) of partial remission. On brush biopsy, all PDT-treated patients showed reduced aneuploidy and normal histological findings. Unfortunately, at t2, 9 patients relapsed with OL, which may be related to continued smoking and betel nut chewing. At t2, 5 patients developed new severe epithelial dysplasia and even carcinoma in situ in other areas, mostly the tongue. ALA-mediated PDT combined with YSGG is effective in treating OL, particularly that with mild to moderate dysplasia. However, severe dysplasia may present undesirable effects, and the mechanism remains to be further investigated. ALA-mediated PDT combined with YSGG provides a new method for OL treatment.

025 ALA-PDT inhibits skin squamous cell carcinoma (cSCC) via regulating formation of tertiary lymphoid structures

Topical ALA-mediated PDT, ALA-PDT, is a novel therapeutic modality widely used to treat actinic keratosis, Bowen’s Disease, superficial skin SCC, and other cancerous and precancerous skin diseases. Several studies have proved that ALA-PDT can inhibit SCC growth. Subsequent research suggested that ALA-PDT not only directly induced tumor cells apoptosis, but also improve tumor microenvironment through regulation of immune cells. However, the anti-tumor immune function of ALA-PDT is still need to be elucidated. Here, we found that tertiary lymphoid structures (TLSs), which are ectopic lymphoid organs that develop in non-lymphoid tissues at sites of chronic inflammation including tumors, play a pivotal role in anti-tumor immune function of ALA-PDT. We analyzed 77 samples of cSCC patient in our hospital. TLS was observed in 79% of the patient samples, and the density of TLS is negatively correlation with the Brodes classification of cSCC. Intriguingly, immunohistochemistry showed that ALA-PDT could promote the formation of TLS in vivo. We also found that promotion effect of ALA-PDT on TLS is mediated by M1 macrophage, which function as lymphoid tissue inducer cells in TLS formation and recruited by ALA-PDT. Further investigation substantiated that PDT-secreted exosomes were involved in M1 macrophage polarization. Our study elucidated a mechanism that ALA-PDT influence M1 macrophage polarization via PDT-secreted exosomes, which could facilitate the formation of TLS. Thus, our research may contribute to in-depth molecular understanding of the ALA-PDT on anti-tumor immune function.

Determination of the distribution of drug concentration and tissue optical properties for ALA-mediated anal photodynamic therapy.

PDT efficacy depends on the availability and dynamic interactions of photosensitizer, light, and oxygen. Tissue optical properties influence the delivered light dose and impact PDT outcome. In-vivo measurements of tissue optical properties and photosensitizer concentration enable determination of explicit and implicit dose factors affecting PDT and helps to understand the underlying biophysical mechanism of PDT. In this study, we measure tissue optical properties (absorption μa (λ) and scattering μs' (λ) coefficients) and PpIX concentration in tissue simulating liquid phantoms with a geometry that resembles anal canal. In-vivo light fluence rate and photosensitizer fluorescence of 405nm excitation light source were acquired using a dual-motor continuous wave transmittance spectroscopy system. We characterized the tissue optical properties correction factor of fluorescence signal using a series of tissue simulating phantoms with known PpIX concentrations and with absorption coefficient between 0.1 - 0.9 cm-1 and reduced scattering coefficient between 5 - 40 cm-1. The results demonstrated that our spectroscopy system could determine the distribution of tissue optical properties and PPIX concentration during anal PDT.

Antimicrobial and immunomodulatory responses of photodynamic therapy in Galleria mellonella model

BackgroundNew therapeutics are urgently needed for infectious diseases, especially for the fungal infection like Fonsecaea monophora. Photodynamic therapy has been showing antimicrobial activity on some pathogens. The combination of antimicrobial medicines and photodynamic therapy (PDT) might be a practical approach. However, whether the treatment of PDT could do benefits to the host immunity remains poorly documented.ResultsIn this study, Galleria mellonella larvae were employed as a model organism to evaluate the activity of PDT, and also to investigate the regulation of humoral immunity by PDT. Photosensitizer 5-aminolevulinic acid (ALA) was applied to the G. mellonella infection model. It was found that ALA-mediated PDT was non-toxic to G. mellonella, and could extend the median survival of infected larvae from 3 days to 5.5 days. We observed that larval hemocytes inhibited the growth of Candida albicans and Staphylococcus aureus, without any contribution by ALA-PDT. Furthermore, the application of ALA-PDT demonstrated the immunomodulation of larval innate immunity as increased hemocyte density counting, cell morphological transformation, and sensitivity to pathogens.ConclusionsG. mellonella could be considered as a useful model to study the immunoregulation of PDT. This model revealed that ALA-PDT positively defense against infections through inducing humoral immune responses of larvae.

617 Skin squamous cell carcinoma (SCC)-derived exosomes after ALA-PDT treatment deliver microRNA-3473 to induce M1 macrophage polarization via NF-κB pathway

Topical ALA-mediated PDT, ALA-PDT, is a novel therapeutic modality widely used to treat actinic keratosis, Bowen’s Disease, superficial skin SCC, and other cancerous and precancerous skin diseases. Several studies have proved that ALA-PDT can inhibit SCC growth and reduce tumor volume. Subsequent research suggested that ALA-PDT not only directly induced tumor cells apoptosis, but also improve tumor microenvironment through regulation of immune cells. However, the anti-tumor immune function of ALA-PDT is still need to be elucidated.

Clinical uses of 5-aminolaevulinic acid in photodynamic treatment and photodetection of cancer: A review.

ALA-mediated Photodynamic Therapy (ALA-PDT) is one of the most promising fields in Photodynamic therapy (PDT) research for cancer treatment. 5-aminolaevulinic acid (ALA) is the prodrug of the photosensitiser Protoporphyrin IX (PpIX). After ALA administration, cells generate PpIX through the haem biosynthetic pathway. Although the exact reasons for ALA/PpIX selectivity are unknown, it is believed that due to the special regulation of haem enzymes, PpIX is accumulated in the tumours. Both ALA and its derivative ALA Methyl ester, are mainly used in dermatology. Besides, ALA-PDT has been employed for palliative and even curative treatment of endoscopically accessible tumours. Lung, oesophagus, gastric and bladder carcinomas, and also oral premalignant lesions, gynaecological intraepithelial neoplasias and Barrett's oesophagus are the conditions mostly treated with ALA-PDT. However, due to the limited penetration of ALA and light, non-dermatologic uses of ALA-PDT have not moved beyond phase I clinical trials. On the other hand, ALA-induced PpIX fluorescence is successfully employed for the Photodynamic Diagnosis (PDD) or assistance in cytoreductive surgery (Fluorescence-guided Resection, FGR). ALA has been approved for the FGR of high-grade gliomas and ALA Hexyl ester, for fluorescence cystoscopy in the diagnosis of bladder cancer. ALA-FGR is currently applied in brain, bladder, lung, colon cancers, etc. and ALA-PDD for oral premalignancies, gynaecological intraepithelial lesions and peritoneal metastases, among others. Besides, PDT can be applied concomitantly in the same diagnostic procedure. This review aimed to analyse the state of the art of clinical uses of ALA in the areas of treatment and detection in the non-dermatologic oncology fields.

A clinical evaluation of efficacy of photodynamic therapy in treatment of reticular oral lichen planus: A case series.

Background The aim of the study was to clinically evaluate the efficacy of photodynamic therapy in treatment of reticular oral lichen planus (OLP). Methods Fifty patients aged 26-84, with 124 OLP lesions in total, underwent photodynamic therapy (PDT) mediated with topically applied 5% 5-aminolevulinic acid. ALA was activated by a custom-made diode lamp with a high-power LED emitting light at 630 nm and 300 mW delivered through an optical fiber probe. A light exposure dose was 150 J/cm2. The therapy comprised of 10 weekly illumination sessions. The lesions' response was macroscopically measured in millimeters with a periodontal probe and clinically evaluated at each session, then on completion of the series and throughout the 12-month follow-up. Results The baseline mean size of lesions was 3.99 cm2±3.73. The lesions on the buccal mucosa and lips (lining mucosa) were larger than those on the gingiva and tongue (masticatory mucosa) - 4.58 cm2±4.01 and 2.93 cm2±2.91 respectively. On completion of the therapy 109 sites improved, including 46 in complete remission. The mean reduction in size was 62.91% (p = 0.000000). 12-month after therapy mean reduction of the lesions was 78.7% (p = 0.000000), specifically 79.48% (p = 0.000000) within the lining mucosa and 76.11% on the masticatory mucosa. Conclusions The results proved that ALA-mediated photodynamic therapy with a 630 nm light was effective and as such it can be used as an optional treatment for symptomatic OLP.

Influence of ALA-mediated photodynamic therapy on secretion of interleukins 6, 8 and 10 by colon cancer cells in vitro.

Photodynamic therapy has apart from a direct cytotoxic effect also immunomodulatory properties. The aim of our study was to investigate how photodynamic therapy with 5-aminolevulinic acid (ALA-PDT) in sublethal doses influences the secretion of interleukins 6, 8 and 10 from colon cancer cells in vitro. We used two human colon cancer cell lines SW480 and SW620 of different malignancies which were treated with a sublethal PDT protocol. Determination of interleukins was carried out using the Bio- Plex Assay Pro™ kit on the Bio- Plex Suspension Array System. Sublethal ALA-PDT did not affect IL-6 secretion by SW480 cells, but caused a 40% decrease of IL-6 release by the SW620 cell line. It increased IL-8 secretion in both, the SW480 and SW620 cell lines, by 23% and 46%, respectively, and decreased the production of IL-10 (25% in SW480 and 32% in SW620 cells). ALA-PDT in sublethal doses might influence colon cancer cell's progression and invasion by reducing the secretion of IL-6, IL-10 and increasing the IL-8 concentration with higher values in the more malignant cell line.

Doxycycline potentiates antitumor effect of 5-aminolevulinic acid-mediated photodynamic therapy in malignant peripheral nerve sheath tumor cells.

Neurofibromatosis type 1 (NF1) is one of the most common neurocutaneous disorders. Some NF1 patients develop benign large plexiform neurofibroma(s) at birth, which can then transform into a malignant peripheral nerve sheath tumor (MPNST). There is no curative treatment for this rapidly progressive and easily metastatic neurofibrosarcoma. Photodynamic therapy (PDT) has been developed as an anti-cancer treatment, and 5-aminolevulinic (ALA) mediated PDT (ALA-PDT) has been used to treat cutaneous skin and oral neoplasms. Doxycycline, a tetracycline derivative, can substantially reduce the tumor burden in human and animal models, in addition to its antimicrobial effects. The purpose of this study was to evaluate the effect and to investigate the mechanism of action of combined doxycycline and ALA-PDT treatment of MPNST cells. An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the combination of ALA-PDT and doxycycline significantly reduce MPNST survival rate, compared to cells treated with each therapy alone. Isobologram analysis showed that the combined treatment had a synergistic effect. The increased cytotoxic activity could be seen by an increase in cellular protoporphyrin IX (PpIX) accumulation. Furthermore, we found that the higher retention of PpIX was mainly due to increasing ALA uptake, rather than activity changes of the enzymes porphobilinogen deaminase and ferrochelatase. The combined treatment inhibited tumor growth in different tumor cell lines, but not in normal human Schwann cells or fibroblasts. Similarly, a synergistic interaction was also found in cells treated with ALA-PDT combined with minocycline, but not tetracycline. In summary, doxycycline can potentiate the effect of ALA-PDT to kill tumor cells. This increased potency allows for a dose reduction of doxycycline and photodynamic radiation, reducing the occurrence of toxic side effects in vivo.

The influence of ala-mediated photodynamic therapy on secretion of selected growth factors and S100 protein (S100) by colon cancer cells in vitro

The influence of ala-mediated photodynamic therapy on secretion of selected growth factors and S100 protein (S100) by colon cancer cells in vitro

Aminolevulinic Acid-Mediated Photodynamic Therapy Causes Cell Death in MG-63 Human Osteosarcoma Cells.

The aim of this study was to test the efficacy of aminolevulinic acid-mediated photodynamic therapy (PDT) against the human osteosarcoma cell line MG-63. Osteosarcoma is the most common type of primary malignant bone tumor diagnosed in the United States among adolescents and children. Treatments for osteosarcoma often result in diminished limb use or amputation. Because ALA-mediated PDT exhibits dual specificity in the context of tumor killing, this therapy could represent a less invasive, but effective, treatment for this disease. To assess ALA dark toxicity in MG-63 cells, cells were incubated with varying concentrations of ALA, and cell viability was determined by crystal violet assay. Protoporphyrin IX (PpIX) accumulation was assessed subsequent to ALA incubation at various concentrations using spectrofluorometry. Cell death subsequent to ALA-PDT was determined by illuminating cells at a wavelength of 635 nm at various light intensities subsequent to ALA incubation. Cell viability was assessed using the MTT assay. ALA dark toxicity was observed only at the highest concentrations of 2, 5, and 10 mM. Maximal PpIX concentration was observed at 0.5 and 1 mM ALA, subsequent to a 24-h incubation. Maximal cell death with minimal light toxicity was observed at 0.5 and 1 mM ALA after illumination with 0.6 and 3 J/cm(2) light. Collectively, our data indicate that ALA-PDT can result in the death of MG-64 human osteosarcoma cells in vitro.

Her2 oncogene transformation enhances 5-aminolevulinic acid-mediated protoporphyrin IX production and photodynamic therapy response.

Enhanced protoporphyrin IX (PpIX) production in tumors derived from the administration of 5-aminolevulinic acid (ALA) enables the use of ALA as a prodrug for photodynamic therapy (PDT) and fluorescence-guided tumor resection. Although ALA has been successfully used in the clinic, the mechanism underlying enhanced ALA-induced PpIX production in tumors is not well understood. Human epidermal growth receptor 2 (Her2, Neu, ErbB2) is a driver oncogene in human cancers, particularly breast cancers. Here we showed that, in addition to activating Her2/Neu cell signaling, inducing epithelial-mesenchymal transition and upregulating glycolytic enzymes, transfection of NeuT (a mutated Her2/Neu) oncogene in MCF10A human breast epithelial cells significantly enhanced ALA-induced PpIX fluorescence by elevating some enzymes involved in PpIX biosynthesis. Furthermore, NeuT-transformed and vector control cells exhibited drastic differences in the intracellular localization of PpIX, either produced endogenously from ALA or applied exogenously. In vector control cells, PpIX displayed a cell contact-dependent membrane localization at high cell densities and increased mitochondrial localization at low cell densities. In contrast, no predominant membrane localization of PpIX was observed in NeuT cells and ALA-induced PpIX showed a consistent mitochondrial localization regardless of cell density. PDT with ALA caused significantly more decrease in cell viability in NeuT cells than in vector cells. Our data demonstrate that NeuT oncogene transformation enhanced ALA-induced PpIX production and altered PpIX intracellular localization, rendering NeuT-transformed cells increased response to ALA-mediated PDT. These results support the use of ALA for imaging and photodynamic targeting Her2/Neu-positive tumors.

In-vitro study of photodynamic therapy of antibiotic-resistant staphylococcus from patients with chronic rhinosinusitis

To evaluate the photodynamic therapy (PDT) against multi-antibiotic-resistant Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S.epidermidis) obtained from patients with chronic rhinosinusitis (CRS). Forty-five CRS patients who had been given medical treatment but still needed endoscopic surgery were included in this study. The mucus from middle meatus was collected from these patients during surgery, followed by separation of S. aureus and S. epidermidis and drug sensitive test. The strains which could form biofilm were selected. Light emitting diode (LED) array with a major wavelength of (633±10) nm was used as light source and 5-Aminolevulinic acid (ALA) was used as photosensitizer in this PDT experiment. The safe range of LED dose and ALA concentration which were not toxic to bacteria by themselves were confirmed, and then did PDT experiment on S. aureus and S. epidermidis. The data of bacterial colony forming unit were transformed to lgCFU before statistical analysis.The Graph Pad Prism 5 software was used to analyzed the data. Thirteen S. aureus and 16 S. epidermidis were included in this experiment(from 45 patients), all of them were multi-antibiotic-resistant bacteria, and four of S. aureus and five of S. epidermidis could form biofilm in each group. In planktonic S. aureus experiment, the mean lgCFU was 8.32±0.31 in control group whereas the experiment group was 6.47±0.67 (t=9.01, P<0.01), and in planktonic S. epidermidis experiment the final data was 8.34±0.20 (control group) and 6.97±0.59 (experiment group) (t=8.84, P<0.01). In biofilm S. aureus experiment, the mean lgCFU was 8.68±0.05 (control group), 6.90±0.96(experiment group) (t=3.68, P<0.05); and in biofilm S. epidermidis experiment the data was 8.67±0.05 (control group), 7.29±0.61 (experiment group, t=5.07, P<0.01). Our results demonstrated that ALA-mediated PDT on multi-antibiotic-resistant S. aureus and S. epidermidis from CRS patients was effective in vitro. Additional work defining if the PDT treatment would damage the nasal mucosa and further checking the effectiveness of PDT in vivo is still needed.

Efficacy of CO2 fractional laser combined with 5-aminolevulinic acid photodynamic therapy on refractory verruca plana

Objective To investigate the safety and efficacy of CO2 fractional laser combined with 5-aminolevulinic acid (ALA)-mediated photodynamic therapy (ALA-PDT) for the treatment of refractory flat wart. Methods The 80 patients with facial flat wart were randomly divided into observation group (40 cases) and control group (40 cases). The observation group was treated with CO2 fractional laser combined with 5-ALA-PDT, and the control group was treated with 5-ALA-PDT only. Those with residual lesions after the first treatment were treated with the second or third times. The interval duration between treatments was 2 weeks. The efficacy and the adverse reaction were evaluated after the 2-week, 4-week and 6-month after the last treatment. Results Compared with prior treatment, the facial lesions achieved complete response and improvement. The total effective rate of the observation group were 90.0% (36/40) and 72.5% (29/40) for the control group, the difference was statistically significant (P<0.05). There were 2 cases of pigmentation occurred in observation group and 3 cases in control group. The complications including infection and hypertrophic scar were seldom occurred in the two groups. Conclusions The safety and efficacy of CO2 fractional laser combined with ALA-PDT are better than that with ALA-PDT only in the treatment of refractory flat wart. Key words: Flat wart; CO2 fractional laser; 5-aminolevulinic acid (ALA)-mediated photodynamic therapy

The influence of ALA-mediated photodynamic therapy on secretion of selected growth factors by colon cancer cells in hypoxia-like environment in vitro

BackgroundPhotodynamic therapy (PDT) affects the immune system and tumor cells’ secretory activity. Hypoxia may limit PDT effects. The aim of this study was to determine the influence of PDT with aminolevulinic acid (ALA-PDT) in a hypoxic-like microenvironment on the secretion of growth factors: GM-CSF (granulocyte and macrophage colony stimulating factor), G-CSF (granulocyte colony-stimulating factor) and FGF (fibroblast growth factor) by experimental models of colon cancer cells in vitro. MethodsSublethal doses of ALA-PDT (ALA of 1000μM, light fluence 10J/cm2, power density rate of 1.5mW/cm2, wavelength 600–720nm) were administered to two colorectal cancer cell lines varying in malignancy potential: SW480 (local malice) and SW620 (high metastatic activity) under normoxia and hypoxia-like environment. Hypoxia-mimic conditions were achieved by adding environment cobalt (II) chloride. Concentrations of growth factors were analyzed with Bio-Plex Pro™ Assay. ResultsALA-PDT amplified the secretion of GM-CSF by both cell lines. The decrease in secretion of G-CSF and FGF was noticed in the SW620. SW620 line cells secreted higher levels of FGF and G-CSF, while SW480 cells more actively released GM-CSF. Compared to normoxic condition, no differences in the secretion of these factors in a hypoxic-like environment were found. ConclusionsALA-PDT increased GM-CSF secretion, which stimulates antitumor defense and decreased secretion of FGF and G-CSF—factors responsible for tumor progression. No differences in the effects of ALA-PDT in the hypoxic-like environment suggests that the beneficial results of PDT are also continued in the later stages of the reaction.

Induction of apoptosis in HPV16 E7 transfected human keratinocyte by ALA-mediated photodynamic therapy

Background and objective5-Aminolevulinic acid (ALA)-mediated photodynamic therapy (ALA-PDT) is effective for the treatment of condyloma and intraepithelial neoplasia associated with human papillomaviruses (HPV). However, the mechanism of PDT on HPV-infected host cells is not fully understood. The aim of this in vitro study was to examine ALA-PDT induced apoptosis in a high-risk HPV E7 gene transfected cell line. Materials and methodsHPV16 E7 gene obtained from HPV16 positive human cervical carcinoma was transfected into a human keratinocyte cell line. PpIX generation and ALA-PDT-induced apoptosis and mitochondrial damage were investigated in vitro. ResultsFluorescence examination showed that HPV16E7 transfected cells could produce PpIX fairly quickly in the presence of 1mM ALA. Apoptosis occurred after ALA-PDT at dose levels of 4, 8, and 12J/cm2. Mitochondrial potential collapse occurred as early as 2h post PDT. ALA-PDT induced a relatively weak and shorter caspase-9 activation but a strong and long lasting caspase-3 activation. However, mRNA levels of caspase-9 and -3 genes in cells treated with ALA-PDT were unchanged. ConclusionThis in vitro study demonstrated that HPV16 E7 transfected keratinocytes could generate PpIX in the presence of ALA. Under light irradiation, ALA-PDT could induce mitochondrial-dependent apoptosis in HPV16 E7 transfected cells.

Effects of Silencing Heme Biosynthesis Enzymes on 5-Aminolevulinic Acid-mediated Protoporphyrin IX Fluorescence and Photodynamic Therapy.

Aminolevulinic acid (ALA)-mediated protoporphyrin IX (PpIX) production is being explored for tumor fluorescence imaging and photodynamic therapy (PDT). As a prodrug, ALA is converted in heme biosynthesis pathway to PpIX with fluorescent and photosensitizing properties. To better understand the role of heme biosynthesis enzymes in ALA-mediated PpIX fluorescence and PDT efficacy, we used lentiviral shRNA to silence the expression of porphobilinogen synthase (PBGS), porphobilinogen deaminase (PBGD) and ferrochelatase (FECH) in SkBr3 human breast cancer cells. PBGS and PBGD are the first two cytosolic enzymes involved in PpIX biosynthesis, and FECH is the enzyme responsible for converting PpIX to heme. PpIX fluorescence was examined by flow cytometry and confocal fluorescence microscopy. Cytotoxicity was assessed after ALA-mediated PDT. Silencing PBGS or PBGD significantly reduced ALA-stimulated PpIX fluorescence, whereas silencing FECH elevated basal and ALA-stimulated PpIX fluorescence. However, compared with vector control cells, the ratio of ALA-stimulated fluorescence to basal fluorescence without ALA was significantly reduced in all knockdown cell lines. PBGS or PBGD knockdown cells exhibited significant resistance to ALA-PDT, while increased sensitivity to ALA-PDT was found in FECH knockdown cells. These results demonstrate the importance of PBGS, PBGD and FECH in ALA-mediated PpIX fluorescence and PDT efficacy.

Cisplatin enhances the efficacy of 5-Aminolevulinic acid mediated photodynamic therapy in human head and neck squamous cell carcinoma

Photodynamic therapy (PDT) has become a promising option for the treatment of head and neck, and other forms of cancer. 5-Aminolevulinic acid (ALA) is one of the popular photosensitizers used in PDT. It is a heme precursor and is converted to a photosensitizer protoporphyrin IX. In this present study, the combination of anticancer drug cisplatin (CDDP)- and ALA- mediated PDT was used to study the cytotoxicity in vitro as well as in vivo. Human head and neck cancer cells AMC-HN3 were treated with cisplatin- and ALA-mediated PDT individually, and also in combination. Several approaches like confocal microscopic study, cytotoxicity assay, etc have been performed to study the intracellular accumulation of protophorphyrin IX in cells and its effectiveness in PDT, when treated in combination with chemotherapy drug, cisplatin (CDDP). The combination of treatments efficacy was also studied in tumor xenograft model. Compared to the individual treatments, combination of CDDP and PDT was found to be more cytotoxic in AMC-HN3, and also more effective in reducing the tumor volume in mice xenograft. Thus, with the combined therapy, not only the efficacy of treatment can be enhanced, but the doses of the drugs can also be lowered. This in turn can reduce the side effects of the chemotherapy drugs. Therefore, this study may lead to a potential drug-PDT combination that may be a useful treatment modality for human head and neck cancer.