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.     

Protoporphyrin IX–β‐Cyclodextrin Bimodal Conjugate: Nanosized Drug Transporter and Potent Phototoxin

Topical or systemic administration of 5‐aminolevulinic acid (ALA) and its esters results in increased production and accumulation of protoporphyrin IX (PpIX) in cancerous lesions allowing effective application of photodynamic therapy (PDT). The large concentrations of exogenous ALA practically required to bypass the negative feedback control exerted by heme on enzymatic ALA synthesis and the strong dimerization propensity of ALA are shortcomings of the otherwise attractive PpIX biosynthesis. To circumvent these limitations and possibly enhance the phototoxicity of PpIX by adjuvant chemotherapy, covalent bonding of PpIX with a drug carrier, β‐cyclodextrin (βCD) was implemented. The resulting PpIX + βCD product had both carboxylic termini of PpIX connected to the CD. PpIX + βCD was water soluble, was found to preferentially localize in mitochondria rather than in lysosomes both in MCF7 and DU145 cell lines while its phototoxiciy was comparable to that of PpIX. Moreover, PpIX + βCD effectively solubilized the breast cancer drug tamoxifen metabolite N‐desmethyltamoxifen (NDMTAM) in water. The PpIX + βCD/NDMTAM complex was readily internalized by both cell lines employed. Furthermore, the multimodal action of PpIX + βCD was demonstrated in MCF7 cells: while it retains the phototoxic profile of PpIX and its fluorescence for imaging purposes, PpIX + βCD can efficiently transport tamoxifen citrate intracellularly and confer cell death through a synergy of photo‐ and chemotoxicity.     

Combination of Oral Vitamin D3 with Photodynamic Therapy Enhances Tumor Cell Death in a Murine Model of Cutaneous Squamous Cell Carcinoma

Photodynamic therapy (PDT), in which 5‐ALA (a precursor for protoporphyrin IX, PpIX) is administered prior to exposure to light, is a nonscarring treatment for skin cancers. However, for deep tumors, ALA‐PDT is not always effective due to inadequate production of PpIX. We previously developed and reported a combination approach in which the active form of vitamin D₃ (calcitriol) is given systemically prior to PDT to improve PpIX accumulation and to enhance PDT‐induced tumor cell death; calcitriol, however, poses a risk of hypercalcemia. Here, we tested a possible strategy to circumvent the problem of hypercalcemia by substituting natural dietary vitamin D₃ (cholecalciferol; D₃) for calcitriol. Oral D₃ supplementation (10 days of a 10‐fold elevated D₃ diet) enhanced PpIX levels 3‐ to 4‐fold, and PDT‐mediated cell death 20‐fold, in subcutaneous A431 tumors. PpIX levels and cell viability in normal tissues were not affected. Hydroxylated metabolic forms of D₃ were only modestly elevated in serum, indicating minimal hypercalcemic risk. These results show that brief oral administration of cholecalciferol can serve as a safe neoadjuvant to ALA‐PDT. We suggest a clinical study, using oral vitamin D₃ prior to PDT, should be considered to evaluate this promising new approach to treating human skin cancer.     

Comparative Study of the Effects of Ferrochelatase‐siRNA Transfection Mediated by Ultrasound Microbubbles and Polyethyleneimine in Combination with Low‐dose ALA to Enhance PpIX Accumulation in Human Endometrial Cancer Xenograft Nude Mice Models

Comparison of the fluorescence intensity caused by the accumulation of PpIX in endometrial cancer xenografts in nude mice after low‐dose 5‐Aminolevulinic acid (ALA) injection combined with siRNA transfection was mediated by ultrasound microbubbles and polyethyleneimine (PEI) to explore the feasibility of the ultrasound microbubble technique as transfection agents. Knockdown of ferrochelatase (FECH) in human endometrial cancer xenografts in nude mice was performed by transfection with FECH‐siRNA mediated by PEI and ultrasound microbubbles alone or in combination; then, low‐dose ALA was injected. Subsequently, an in vivo animal imaging system was employed to detect the fluorescence intensity in xenografts. Red fluorescence was observed in xenografts given more than 6.25 mg kg^?1 of ALA. When the dose of ALA was greater than 50 mg kg^?1, there was a significant difference in the fluorescence between tumor and other tissues. After the nude mice were transfected with siRNA and treated with low‐dose ALA (1.0 mg kg^?1), apparent PpIX fluorescence of the xenografts was observed, and the fluorescence intensity was PEI+ ultrasound microbubbles > PEI > ultrasound microbubbles. Ultrasound microbubbles in combination with PEI could generate a higher fluorescence intensity of PpIX than that obtained with ultrasound microbubbles or PEI alone, and ultrasound microbubbles could wholly or partially replace PEI under certain conditions.     

Ferrochelatase Deficiency Abrogated the Enhancement of Aminolevulinic Acid‐mediated Protoporphyrin IX by Iron Chelator Deferoxamine

Aminolevulinic acid (ALA) is a prodrug that is metabolized in the heme biosynthesis pathway to produce protoporphyrin IX (PpIX) for tumor fluorescence detection and photodynamic therapy (PDT). The iron chelator deferoxamine (DFO) has been widely used to enhance PpIX accumulation by inhibiting the iron‐dependent bioconversion of PpIX to heme, a reaction catalyzed by ferrochelatase (FECH). Tumor response to DFO treatment is known to be highly variable, and some tumors even show no response. Given the fact that tumors often exhibit reduced FECH expression/enzymatic activity, we examined how reducing FECH level affected the DFO enhancement effect. Our results showed that reducing FECH level by silencing FECH in SkBr3 breast cancer cells completely abrogated the enhancement effect of DFO. Although DFO enhanced ALA‐PpIX fluorescence and PDT response in SkBr3 vector control cells, it caused a similar increase in MCF10A breast epithelial cells, resulting in no net gain in the selectivity toward tumor cells. We also found that DFO treatment induced less increase in ALA‐PpIX fluorescence in tumor cells with lower FECH activity (MDA‐MB‐231, Hs 578T) than in tumor cells with higher FECH activity (MDA‐MB‐453). Our study demonstrates that FECH activity is an important determinant of tumor response to DFO treatment.     

Noninvasive Optical Imaging of UV‐Induced Squamous Cell Carcinoma in Murine Skin: Studies of Early Tumor Development and Vitamin D Enhancement of Protoporphyrin IX Production

Better noninvasive techniques are needed to monitor protoporphyrin IX (PpIX) levels before and during photodynamic therapy (PDT) of squamous cell carcinoma (SCC) of the skin. Our aim was to evaluate (1) multispectral fluorescent imaging of ultraviolet light (UV)‐induced cancer and precancer in a mouse model of SCC and (2) multispectral imaging and probe‐based fluorescence detection as a tool to study vitamin D (VD) effects on aminolevulinic acid (ALA)‐induced PpIX synthesis. Dorsal skin of hairless mice was imaged weekly during a 24‐week UV carcinogenesis protocol. Hot spots of PpIX fluorescence were detectable by multispectral imaging beginning at 14 weeks of UV exposure. Many hot spots disappeared after cessation of UV at week 20, but others persisted or became visible after week 20, and corresponded to tumors that eventually became visible by eye. In SCC‐bearing mice pretreated with topical VD before ALA application, our optical techniques confirmed that VD preconditioning induces a tumor‐selective increase in PpIX levels. Fluorescence‐based optical imaging of PpIX is a promising tool for detecting early SCC lesions of the skin. Pretreatment with VD can increase the ability to detect early tumors, providing a potential new way to improve efficacy of ALA‐PDT.     

Protein 4.1R is Involved in the Transport of 5‐Aminolevulinic Acid by Interaction with GATs in MEF Cells

5‐aminolevulinic acid (5‐ALA )‐based photodynamic therapy (PDT ) has been successfully used in the treatment of cancers. However, the mechanism of 5‐ALA transportation into cancer cells is still not fully elucidated. Previous studies have confirmed that the efficiency of 5‐ALA‐PDT could be affected by the membrane skeleton protein 4.1R. In this study, we investigated the role of 4.1R in the transport of 5‐ALA into cells. Wild‐type (4.1R^+/+) and 4.1R gene knockout (4.1R^−/−) mouse embryonic fibroblast (MEF ) cells were incubated with 1 mm 5‐ALA and different concentrations of specific inhibitors of GABA transporters GAT (1‐3). Our results showed that the inhibition of GAT 1 and GAT 2 in particular markedly attenuated the intracellular PpIX production, reactive oxygen species (ROS ) level and 5‐ALA ‐induced photodamage. However, the inhibition of GAT 3 did not show such effects. Further research showed that 4.1R^−/− MEF cells had a lower expression of GAT 1 and GAT 2 than 4.1R^+/+ MEF cells. Additionally, 4.1R directly bound to GAT 1 and GAT 2. Taken together, GAT 1 and GAT 2 transporters are involved in the uptake of 5‐ALA in MEF cells. 4.1R plays an important role in transporting 5‐ALA into cells via at least partly interaction with GAT 1 and GAT 2 transporters in 5‐ALA ‐PDT .     

Improved In vitro and In vivo Cutaneous Delivery of Protoporphyrin IX from PLGA‐based Nanoparticles

We report the development of d , l lactic co‐glycolic acid) (PLGA)‐based nanoparticles (NPs) for topical delivery of protoporphyrin IX (PpIX), a photosensitizer (PS), in treatments like photodynamic therapy (PDT) of skin cancers. PpIX‐NPs were obtained in ~75.0% yield, encapsulation efficiency of 67.7%, drug content of 50.3 μg mg^?1, average diameter of 290 nm maintained up to 30 days and a zeta potential of 32.3 mV. Sustained in vitro release of PpIX through artificial membranes following Higuchi kinetics was kept up to 10 days. In vitro retentions of PpIX both in stratum corneum (SC) and epidermis + dermis ([EP + D]) were higher from NPs (23.0 and 10.0 times, respectively) compared to control solutions at all times. Quantification of PpIX by extraction, after in vivo skin application of NPs‐PpIX on hairless mice, showed higher retention of the PS both in SC and in [EP + D] (3.0 and 2.0 times, respectively) compared to control solutions. Taken together, the results indicate that NPs are suitable for PpIX encapsulation showing minimal permeation through the skin and a localized effect, characteristics of a potential and promising delivery system for PDT‐associated treatments of skin cancers, photodiagnosis and their off‐label uses.     

A Self‐Delivery Chimeric Peptide for Photodynamic Therapy Amplified Immunotherapy

In this paper, a self‐delivery chimeric peptide PpIX‐PEG₈‐KVPRNQDWL is designed for photodynamic therapy (PDT) amplified immunotherapy against malignant melanoma. After self‐assembly into nanoparticles (designated as PPMA), this self‐delivery system shows high drug loading rate, good dispersion, and stability as well as an excellent capability in producing reactive oxygen species (ROS). After cellular uptake, the ROS generated under light irradiation could induce the apoptosis and/or necrosis of tumor cells, which would subsequently stimulate the anti‐tumor immune response. On the other hand, the melanoma specific antigen (KVPRNQDWL) peptide could also activate the specific cytotoxic T cells for anti‐tumor immunity. Compared to immunotherapy alone, the combined photodynamic immunotherapy exhibits significantly enhanced inhibition of melanoma growth. Both in vitro and in vivo investigations confirm that PDT of PPMA has a positive effect on anti‐tumor immune response. This self‐delivery system demonstrates a great potential of this PDT amplified immunotherapy strategy for advanced or metastatic tumor treatment.     

Topical Photodynamic Therapy Using Different Porphyrin Precursors Leads to Differences in Vascular Photosensitization and Vascular Damage in Normal Mouse Skin

Different distributions of hexyl aminolevulinate (HAL), aminolevulinic acid (ALA) and methyl aminolevulinate (MAL) in the superficial vasculature are not well studied but they are hypothesized to play an important role in topical photodynamic therapy (PDT). The colocalization of fluorescent CD31 and protoporphyrin IX (PpIX) was calculated using confocal microscopy of mouse skin sections to investigate the vascular distribution after topical application. Vascular damage leads to disruption of the normal endothelial adherens junction complex, of which CD144 is an integral component. Therefore, normal CD31 combined with loss of normal fluorescent CD144 staining was visually scored to assess vascular damage. Both the vascular PpIX concentration and the vascular damage were highest for HAL, then ALA and then MAL. Vascular damage in MAL was not different from normal contralateral control skin. This pattern is consistent with literature data on vasoconstriction after PDT, and with the hypothesis that the vasculature plays a role in light fractionation that increases efficacy for HAL and ALA‐PDT but not for MAL. These findings indicate that endothelial cells of superficial blood vessels synthesize biologically relevant PpIX concentrations, leading to vascular damage. Such vascular effects are expected to influence the oxygenation of tissue after PDT which can be important for treatment efficacy.     

Metronomic Photodynamic Therapy as a New Paradigm for Photodynamic Therapy: Rationale and Preclinical Evaluation of Technical Feasibility for Treating Malignant Brain Tumors¶

The concept of metronomic photodynamic therapy (mPDT) is presented, in which both the photosensitizer and light are delivered continuously at low rates for extended periods of time to increase selective tumor cell kill through apoptosis. The focus of the present preclinical study is on mPDT treatment of malignant brain tumors, in which selectivity tumor cell killing versus damage to normal brain is critical. Previous studies have shown that low‐dose PDT using 5‐aminolevulinic acid (ALA)‐induced protoporphyrin IX(PpIX) can induce apoptosis in tumor cells without causing necrosis in either tumor or normal brain tissue or apoptosis in the latter. On the basis of the levels of apoptosis achieved and model calculations of brain tumor growth rates, metronomic delivery or multiple PDT treatments, such as hyperfractionation, are likely required to produce enough tumor cell kill to be an effective therapy. In vitro studies confirm that ALA‐mPDT induces a higher incidence of apoptotic (terminal deoxynucleotidyl transferase‐mediated 2′‐deoxyuridine 5′‐triphosphate, sodium salt nick‐end labeling positive) cells as compared with an acute, high‐dose regimen (ALA‐αPDT). In vivo, mPDT poses two substantial technical challenges: extended delivery of ALA and implantation of devices for extended light delivery while allowing unencumbered movement. In rat models, ALA administration via the drinking water has been accomplished at very high doses (up to 10 times therapeutic dose) for up to 10 days, and ex vivo spectro‐fluorimetry of tumor (9L gliosarcoma) and normal brain demonstrates a 3–4 fold increase in the tumor‐to‐brain ratio of PpIX concentration, without evidence of toxicity. After mPDT treatment, histological staining reveals extensive apoptosis within the tumor periphery and surrounding microinvading colonies that is not evident in normal brain or tumor before treatment. Prototype light sources and delivery devices were found to be practical, either using a laser diode or light‐emitting diode (LED) coupled to an implanted optical fiber in the rat model or a directly implanted LED using a rabbit model. The combined delivery of both drug and light during an extended period, without compromising survival of the animals, is demonstrated. Preliminary evidence of selective apoptosis of tumor under these conditions is presented.     

Detection of Early Stages of Carcinogenesis in Adenomas of Murine Lung by 5-Aminolevulinic Acid-Induced Protoporphyrin IX Fluorescence

Abstract— Administration of the heme precursor 5-aminolevulinic acid (ALA) leads to the selective accumulation of the photosensitizer protoporphyrin IX (PpIX) in certain types of normal and abnormal tissues. This phenomenon has been exploited clinically for detection and treatment of a variety of malignant and nonmalignant lesions. The present preclinical study examined the specificity of ALA-induced porphyrin fluorescence in chemically induced murine lung tumors in vivo. During the early stages of tumorigenesis, ALA-induced PpIX fluorescence developed in hyperplastic tissues in the lung and later in early lung tumor foci. In early tumor foci, maximum PpIX fluorescence occurred 2 h after the administration of ALA and returned to background levels after 4 h. There was approximately a 20-fold difference in PpIX fluorescence intensity between tumor foci and the adjacent normal tissue. The specificity of ALA-induced fluorescence for hyperplastic tissues and benign tumors in lung during tumorigenesis suggests a possible use for this fluorochrome in the detection of premalignant alterations in the lung by fluorescence endoscopy. Two non-small cell lung cancer cell lines developed ALA-induced PpIX fluorescence in vitro . These lines exhibited a light-dose-dependent phototoxic response to ALA photodynamic therapy (PDT) in vitro . Because PpIX is a clinically effective photosensitizer for a wide variety of malignancies, these results support the possible use of ALA-induced PpIX PDT for lung cancer.     

Metal-Organic Layer Delivers 5-Aminolevulinic Acid and Porphyrin for Dual-Organelle-Targeted Photodynamic Therapy

The efficacy of photodynamic therapy (PDT) depends on the subcellular localization of photosensitizers. Herein, we report a dual-organelle-targeted nanoparticle platform for enhanced PDT of cancer. By grafting 5-aminolevulinic acid (ALA) to a Hf₁₂-based nanoscale metal-organic layer (Hf-MOL) via carboxylate coordination, ALA/Hf-MOL enhanced ALA delivery and protoporphyrin IX (PpIX) synthesis in mitochondria, and trapped the Hf-MOL comprising 5,15-di-p-benzoatoporphyrin (DBP) photosensitizers in lysosomes. Light irradiation at 630 nm simultaneously excited PpIX and DBP to generate singlet oxygen and rapidly damage both mitochondria and lysosomes, leading to synergistic enhancement of the PDT efficacy. The dual-organelle-targeted ALA/Hf-MOL outperformed Hf-MOL in preclinical PDT studies, with a 2.7-fold lower half maximal inhibitory concentration in cytotoxicity assays in vitro and a 3-fold higher cure rate in a colon cancer model in vivo.     

The Hydroxypyridinone Iron Chelator CP94 Can Enhance PpIX-induced PDT of Cultured Human Glioma Cells

Photodynamic therapy (PDT) with the pro-drugs 5-aminolevulinic acid (ALA) or methyl aminolevulinate (MAL) utilizes the combined interaction of a photosensitizer, light and molecular oxygen to ablate tumor tissue. To potentially increase accumulation of the photosensitizer, protoporphyrin IX (PpIX), within tumor cells an iron chelator can be employed. This study analyzed the effects of ALA/MAL-induced PDT combined with the iron chelator 1, 2-diethyl-3-hydroxypyridin-4-one hydrochloride (CP94) on the accumulation of PpIX in human glioma cells in vitro. Cells were incubated for 0, 3 and 6 h with various concentrations of ALA/MAL with or without CP94 and the resulting accumulations of PpIX, which naturally fluoresces, were quantified prior to and following light irradiation. In addition, counts of viable cells were recorded. The use of CP94 in combination with ALA/MAL produced significant enhancements of PpIX fluorescence in human glioma cells. At the highest concentrations of each prodrug, CP94 enhanced PpIX fluorescence significantly at 3 h for ALA and by more than 50% at 6 h for MAL. Cells subsequently treated with ALA/MAL-induced PDT in combination with CP94 produced the greatest cytotoxicity. It is therefore concluded that with further study CP94 may be a useful adjuvant to photodiagnosis and/or PpIX-induced PDT treatment of glioma.     

Biodistribution of Photofrin II® and 5-Aminolevulinic Acid-Induced Protoporphyrin IX in Normal Rat Bladder and Bladder Tumor Models: Implications for Photodynamic Therapy

Photodynamic therapy (PDT) has been considered as a potential therapy for superficial bladder carcinomas. Cutaneous photosensitivity and reduction of bladder capacity are the two well-known complications following systemic administration of the commonly used photosensitizer, Photofrin II® (PII). The objective of the present study was to evaluate whether intravesical. (i.b.) instillation of photosensitizers for PDT of bladder cancer might be a more suitable treatment method. Female Fischer rats were utilized to develop orthotopic and heterotopic bladder tumor models. Rats bearing orthotopic bladder tumors were treated either intravesically or intravenously with graded doses of 5-aminolevulinic acid (ALA) or PII. Normal rats received the same doses of ALA or PII. As well, rats bearing heterotopic tumor were studied for comparison. The biodistribution times (times allowed for tissue uptake and bioconversion following drug administration) were 2, 4 or 6 h. Porphyrin fluorescence intensities within tumor, urothelium, submucosa, bladder muscularis and abdominal muscle were quantitated by confocal laser scanning microscopy. Following intravenous (i.v.) injection of ALA, tumor protoporphyrin IX (PpIX) levels peaked at 4 h and diminished by 6 h. The PpIX ratios of tumor-to-bladder mucosa, submucosa and muscle layers were 3:1, 5:1 and 8:1, respectively, 4 h following 1000 mg/kg ALA injection. After ALA instillation, the optimal biodistribution time appeared to be 4 h. Bladder instillation provided comparable tumor labeling with the i.v. route, but lost selectivity of PpIX accumulation between tumor and normal urothelium. The PpIX ratio of tumor-to-bladder muscularis was 5:1. After i.b. instillation of PII, porphyrin fluorescence was detected only within tumor and urothelium, while porphyrin fluorescence was mainly located in bladder submucosa following i.v. injection. Intravesical administration of ALA or PII might be feasible for PDT of superficial bladder cancers.     

Effects of photodynamic therapy with topical application of 5-aminolevulinic acid on normal skin of hairless guinea pigs.

Photodynamic therapy (PDT) is a relatively new approach to the treatment of neoplasms which involves the use of photoactivatable compounds to selectively destroy tumors. 5-Aminolevulinic acid (ALA) is an endogenous substance which is converted to protoporphyrin IX (PpIX) in the synthetic pathway to heme. PpIX is a very effective photosensitizer. The goal of this study was to evaluate the effect of PDT using topical ALA on normal guinea pig (g.p.) skin and g.p. skin in which the stratum corneum was removed by being tape-stripped (TS). Evaluation consisted of gross examination, PpIX fluorescence detection, reflectance spectroscopy, and histology. There was no effect from the application of light or ALA alone. Normal non-TS g.p. skin treated with ALA and light was unaffected unless high light and ALA doses were used. Skin from which the stratum corneum was removed was highly sensitive to treatment with ALA and light: 24 h after treatment, the epidermis showed full thickness necrosis, followed by complete repair within 7 d. Time-dependent fluorescence excitation and emission spectra were determined to characterize the chromophore and to demonstrate a build-up of the porphyrin in the skin. These data support the view that PDT with topical ALA is a promising approach for the treatment of epidermal cutaneous disorders.     

5-Aminolevulinic Acid-induced Protoporphyrin IX Levels in Tissue of Human Malignant Brain Tumors

Protoporphyrin IX (PpIX) produced from exogenous, orally administered 5-aminolevulinic acid (ALA) displays high tumor-selective uptake and is being successfully employed for fluorescence-guided resection (FGR) of human malignant gliomas. Furthermore, the phototoxicity of PpIX can be utilized for photodynamic therapy (PDT) of brain tumors, which has been shown previously. Here, the absolute PpIX concentration in human brain tissue was investigated following oral ALA administration (20 mg kg⁻¹ b.w.). An extraction procedure was used to quantify PpIX in macroscopic tissue samples, weighing 0.013–0.214 g, obtained during FGR. The PpIX concentration was significantly higher in vital grade IV tumors (5.8 ± 4.8 μm , mean ± SD, range 0–28.2 μm , n = 8) as compared with grade III tumors (0.2 ± 0.4 μm , mean ± SD, range 0–0.9 μm , n = 4). There was also a large heterogeneity within grade IV tumors with PpIX displaying significantly lower levels in infiltration zones and necrotic regions as compared with vital tumor parts. The average PpIX concentration in vital grade IV tumor parts was in the range previously shown sufficient for PDT-induced tissue damage following irradiation. However, the feasibility of PDT for grade III brain tumors and for grade IV brain tumors displaying mainly necrotic tissue areas without solid tumor parts needs to be further investigated.     

Fractionated illumination in oesophageal ALA-PDT: effect on ferrochelatase activity

BACKGROUND AND OBJECTIVE: Administration of 5-aminolevulinic acid (ALA) induces accumulation of the photosensitive compound protoporphyrin IX (PpIX) in certain tissues. PplX can be used as photosensitizer in photodynamic therapy (PDT). More selective or higher PpIX accumulation in the area to be treated could optimize the results of ALA-PDT. Porphobilinogen deaminase (PBGD) is rate-limiting in PpIX formation whereas ferrochelatase converts PpIX into haem by chelation of ferrous iron into PpIX. This results in a moment of close interaction (ferrochelatase binding to PpIX) during which ferrochelatase could selectively be destroyed resulting in an increased PpIX concentration. The aim of the present study was to investigate whether illumination before PDT can selectively destroy ferrochelatase. and whether this results in higher PpIX accumulation and thereby increases the PDT effect. Furthermore, the effect of a second ALA dose was tested. STUDY DESIGN/MATERIALS AND METHODS: Oesophageal tissue of 60 rats were allocated to 2 groups of 30 animals each. In one group, enzyme and PpIX measurements were performed after ALA administration (200 mg/kg orally, n=20), or a second dose of 200 mg/kg ALA at 4 h (n=10), half of each group with and without illumination at 1 h with 12.5 J/cm diffuser length. In the second group, PDT was performed. Ten animals were illuminated at 3 h after ALA administration with 20 (n=5) or 32.5 J/cm (n=5), 10 animals were illuminated at 1 h (12.5 J/cm) and received intra-oesophageal PDT treatment (20 J/cm) at 3 h (n=5) or 4 h (n=5) after ALA. Additionally, 10 animals received a second dose of 200 mg/kg ALA at 4 h and were illuminated (20 J/cm) at 7 h after the first dose of ALA with (n=5) or without (n=5) illumination at 4 h (12.5 J/cm). RESULTS: Illumination with 12.5 J/cm at 1 h after ALA administration caused inhibition of the activity of ferrochelatase at 3 and 4 h after ALA (P=0.02 and P<0.001, respectively), but not at 7 h (P=0.3). In animals sacrificed at 4 h the ratio PBGD:ferrochelatase was higher in animals illuminated at 1 h compared to non-illuminated animals (P<0.001). PpIX concentration was highest (42.7 +/- 3.2 pmol/mg protein) at 3 h after ALA administration and did not increase by illumination at 1 h. Administration of a second dose of ALA did not result in higher PpIX accumulation. After PDT, no difference in epithelial or muscular damage was found between the various groups. CONCLUSION: Illumination at 1 h after ALA administration can cause selective destruction of ferrochelatase, resulting in a higher ratio of PBGD:ferrochelatase. This does not result in accumulation of more porphyrins, even when a second dose of ALA is given. Therefore, under the conditions used in this study fractionated illumination does not enhance ALA-PDT-induced epithelial ablation of the rat oesophagus.     

Photodynamic Therapy Mediated by 5‐aminolevulinic Acid Promotes the Upregulation and Modifies the Intracellular Expression of Surveillance Proteins in Oral Squamous Cell Carcinoma

Expression of proteins related to cell surveillance has been described in tumors presenting resistance to photodynamic therapy (PDT). The aim of this study was to verify whether there was upregulation of proteins related to resistance in oral squamous cell carcinoma (OSCC) after PDT. OSCC was chemically induced in rats and treated after one cycle of PDT mediated by 5‐aminolevulinic acid (5‐ALA‐PDT). Immunolabeling of p‐NFκB, Bcl‐2, survivin, iNOS, p‐Akt, p‐mTOR and cyclin D1 was performed after the treatment. There was increased expression of Bcl‐2 (P = 0.008), iNOS (P = 0.020), p‐Akt (P = 0.020) and p‐mTOR (P = 0.010) by surviving neoplastic cells after PDT when compared to the control. In conclusion, after one cycle of 5‐ALA‐mediated PDT, Bcl‐2, p‐Akt, p‐mTOR and iNOS were upregulated in neoplastic cells of OSCC, suggesting an activation of antiapoptosis and cell proliferation pathways. This fact must be considered in the establishment of PDT protocols for OSCC treatment, mainly those in which PDT will be combined with chemotherapy drugs targeted at the studied proteins.     

Vitamin D and Other Differentiation-promoting Agents as Neoadjuvants for Photodynamic Therapy of Cancer

The efficacy of photodynamic therapy (PDT) using aminolevulinic acid (ALA), which is preferentially taken up by cancerous cells and converted to protoporphyrin IX (PpIX), can be substantially improved by pretreating the tumor cells with vitamin D (Vit D). Vit D is one of several “differentiation-promoting agents” that can promote the preferential accumulation of PpIX within the mitochondria of neoplastic cells, making them better targets for PDT. This article provides a historical overview of how the concept of using combination agents (“neoadjuvants”) for PDT evolved, from initial discoveries about neoadjuvant effects of methotrexate and fluorouracil to later studies to determine how vitamin D and other agents actually work to augment PDT efficacy. While this review focuses mainly on skin cancer, it includes a discussion about how these concepts may be applied more broadly toward improving PDT outcomes in other types of cancer.