Topical and intratumoral photodynamic therapy with 5-aminolevulinic acid in a subcutaneous murine mammary adenocarcinoma.

One of the most promising substances used in photodynamic therapy (PDT) is 5-aminolevulinic acid (ALA), which induces endogenous synthesis and accumulation of porphyrins in malignant cells. In this paper we have shown that both topical and intratumoral administration of ALA in a subcutaneously implanted mammary carcinoma produced a significant synthesis of porphyrins and subsequent sensitization to laser light. Porphyrin accumulation was greater when ALA was administered intratumorally and tumour/normal skin porphyrin concentration ratios were higher compared with topical application. Irradiation was optimal between 2 and 3 h after topical application of 50 mg of a 20% ALA cream and 2-4 h after intratumoral administration of 30 mg ALA/cm3. The pattern of tumour response evaluated as the delay of tumour growth was similar following either route of drug administration. Applications of PDT were performed once, twice or three times in the study. The response to successive applications was constant for the same tumour, indicating that no resistance was acquired. Microscopic analysis showed both induction of foci of necrosis and haemorrhage, morphological features of apoptotic cells and total absence of cellular immune response. This paper reports on PDT with topical ALA in a subcutaneous carcinoma leading to tumour growth delay. These findings may have great relevance in the treatment of cutaneous metastasis of mammary carcinomas.     

5-aminolevulinic acid photodynamic therapy for the treatment of high-grade gliomas

Journal of Neuro-Oncology - In the original article, the author names were published incorrectly. The names are correct in this publication.     

5-Aminolevulinic Acid -Mediated Photodynamic Therapy of Human Glioma Cells In Vitro

OBJECTIVE To investigate the effect of 5-aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) on U251 human glioma cells in vitro.treated with ALA, a typelioma cells were routinely cultured and then of photosensitizer, at various concentrationsfollowed by light irradiation. The PDT-induced phototoxicity of the cells was determined by a MTT assay. In addition, cells were treated with ALA at a fixed concentration and subjected to various doses of light irradiation.RESULTS With the same light dosage (25.0 J/cm^2), the cell survival rates were 70.16% 5.02%, 50.19% 4.79%, 34.97% 5.34%, 27.04% 4.34%, and 24.26% 2.76% at ALA concentrations of 0.25, 0.5, 1.0, 2.0, and 4.0 mM,respectively (F =279.88, P =0.0000). But the survival rates of the cells incubated with 2.0 mM ALA compared to those with 4.0 mM ALA (27.04% 4.34% vs 24.26% 2.76%) showed no significant difference (P=0.611). At a single ALA concentration, the cell survival rates were 83.48% 6.79%,68.09% 6.02%, 33.75% 6.70%, 23.34% 5.08% and 15.14% 3.60% for light doses of 6.25, 12.5, 25.0, 50.0, and 100 J/cm2, respectively (F=422.03,P=0.0000). Without exposure to light, however, the cell survival rates were 96.64% 6.56%, 97.71% 5.48%, 98.10% 6.25%, 99.44% 7.02%, and 95.86% 7.80% for ALA concentrations at 0.25, 0.5, 1.0, 2.0, and 4.0 mM,respectively (F =0.68, P=0.6085). Without ALA in the medium, the cells urvival rates were 98.74% 6.20%, 96.49% 7.13%, 97.60% 5.94%,95.70% 4.86%, 98.08% 6.26% for light doses of 6.25, 12.5, 25.0, 50.0, and 100 J/cm2, respectively (F=0.6400, P=0.6368).CONCLUSION The PDT damage to the U251 cells increased with ALAconcentration within a relative lower range, but then plateaued at higherconcentrations. PDT damage was proportional to the doses of irradiatedlight. Without ALA, the light alone caused no photodynamic damage andALA itself was nontoxic. The ALA-induced PDT appears to be a promisingtherapy for glioma.     

Comparisons of 5-aminolevulinic acid photodynamic therapy and after-loading radiotherapy in vivo in cervical cancer

Objective

To compare the differences between 5-aminolevulinic acid photodynamic therapy (5-ALA-PDT) with traditional after-loading radiotherapy in aspects of efficacies and side effects.

Materials and methods

MTT assay was adopted to detect the inhibitive effects of 5-ALA-PDT on Hela cells proliferation. Flow cytometry was used to analyze cell apoptosis. After establishment of human cervical cancer xenograft model, the comparisons between 5-ALA-PDT with radiotherapy were performed with respect to treatment efficacies (survival rate, body weight, and tumor volume) and side effects (appearance and behavior, ovarian endocrine functions, and skin lesion around the tumor).

Results

5-Aminolevulinic acid photodynamic therapy exerted killing effects on cervical cancer cells. Morphological changes and flow cytometric analyses indicated apoptosis to be one of the mechanisms for tumor growth suppression. Both proliferation inhibition and cell apoptosis showed dependency on photosensitizer concentration and irradiation intensity. Repeated photodynamic therapy presented stronger inhibitive effects on tumor growth compared to after-loading radiotherapy, while producing milder impairment of ovarian endocrine functions and skin lesions around the tumors.

Conclusions

5-Aminolevulinic acid photodynamic therapy has great potential to be an alternative treatment modality for cervical cancer.     

Targeting tumour energy metabolism potentiates the cytotoxicity of 5-aminolevulinic acid photodynamic therapy

Background:

Cancerous cells usually exhibit increased aerobic glycolysis, compared with normal tissue (the Warburg effect), making this pathway an attractive therapeutic target.

Methods:

Cell viability, cell number, clonogenic assay, reactive oxygen (ROS), ATP, and apoptosis were assayed in MCF-7 tumour cells and corresponding primary human mammary epithelial cells (HMEC).

Results:

Combining the glycolysis inhibitors 2-deoxyglucose (2DG; 180 mM) or lonidamine (300 μM) with 10 J cm⁻² 5-aminolevulinic acid (ALA) photodynamic therapy (PDT) increases MCF-7 cytotoxicity (by 3.5-fold to 70% death after 24 h, and by 10-fold in 9-day clonogenic assays). However, glycolysis inhibition only slightly increases HMEC PDT cytotoxicity (between two-fold and three-fold to a maximum of 9% death after 24 h). The potentiation of PDT cytotoxicity only occurred if the glycolysis inhibitors were added after ALA incubation, as they inhibited intracellular accumulation of photosensitiser if coincubated with ALA.

Conclusion:

As 2DG and lonidamine are already used as cancer chemotherapeutic agents, our results are directly translatable to combination therapies with existing topical PDT.     

Repetitive 5-aminolevulinic acid-mediated photodynamic therapy on human glioma spheroids

The response of human glioma spheroids to repetitive 5-aminolevulinic acid-mediated photodynamic therapy (PDT) was investigated. In all cases, light fluences were kept below toxic thresholds to simulate conditions typically found at 1–2cm depths in brain adjacent to tumor. Significant inhibition of spheroid growth was observed following multiple PDT treatments at sub-threshold light fluences. The effect appears to be insensitive to the treatment intervals investigated (weekly or bi-monthly). In all cases, suppression of growth was observed for the duration of treatment. Low fluence rates (5mWcm^–2) appear to be more effective than high fluence rates (25mWcm^–2). No evidence of PDT resistance was found in this investigation.     

Acute morphological sequelae of photodynamic therapy with 5-aminolevulinic acid in the C6 spheroid model

Objective Aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) may represent a treatment option for malignant brain tumors. We used a three-dimensional cell culture system, the C6 glioma spheroid model, to study acute effects of PDT and how they might be influenced by treatment conditions. Methods Spheroids were incubated for 4 h in 100 μg/ml ALA in 5% CO₂ in room air or 95% O₂ with subsequent irradiation using a diode laser (λ = 635 nm, 40 mW/cm², total fluence 25 J/cm²). Control groups were “laser only”, “ALA only”, and “no drug no light”. Annexin V-FITC, a marker used for detection of apoptosis, propidium iodide (PI), a marker for necrotic cells and H 33342, a chromatin stain, were used for morphological characterization of PDT effects by confocal laser scanning and fluorescence microscopy. Hematoxylin–eosin staining and TdT-FragEL (TUNEL) assay were used on cryosections. Growth kinetics were followed for 8 days after PDT. Results PDT after incubation in 5% CO₂ provided incomplete cell death and growth delay in spheroids of >350 μm diameter. However, complete cell death and growth arrest occurred in smaller spheroids (<350 μm). Incubation in 95% O₂ with subsequent PDT resulted in complete cell death and growth arrest regardless of spheroid size. In incompletely damaged spheroids viable cells were restricted to spheroid centers. The rate of cell death in all control groups was negligible. Cell death was accompanied by annexin/PI costaining, but there was also evidence for annexin V-FITC staining without PI uptake. Conclusions PDT of experimental glioma results in rapid and significant cell death that could be verified as acute necrosis immediately after irradiation. This effect depended on O₂ concentration and spheroid size.     

Polymeric iron chelators for enhancing 5-aminolevulinic acid-induced photodynamic therapy

5-Aminolevulinic acid (5-ALA) is an amino acid that can be metabolized into a photosensitizer, protoporphyrin IX (PpIX) selectively in a tumor cell, permitting minimally invasive photodynamic diagnosis/therapy. However, some malignant tumor cells have excess intracellular labile iron and facilitate the conversion of PpIX into heme, which compromises the therapeutic potency of 5-ALA. Here, we examined the potential of chelation of such unfavorable intratumoral labile iron in photodynamic therapy (PDT) with 5-ALA hydrochloride, using polymeric iron chelators that we recently developed. The polymeric iron chelator efficiently inactivated the intracellular labile iron in cultured cancer cells and importantly enhanced the accumulation of PpIX, thereby improving the cytotoxicity upon photoirradiation. Even in in vivo study with subcutaneous tumor models, the polymeric iron chelator augmented the intratumoral accumulation of PpIX and the PDT effect. This study suggests that our polymeric iron chelator could be a tool for boosting the effect of 5-ALA-induced PDT by modulating tumor microenvironment.     

Silencing of ferrochelatase enhances 5-aminolevulinic acid-based fluorescence and photodynamic therapy efficacy

Background:

Recurrence of glioma frequently occurs within the marginal area of the surgical cavity due to invading residual cells. 5-Aminolevulinic acid (5-ALA) fluorescence-guided resection has been used as effective therapeutic modalities to improve discrimination of brain tumour margins and patient prognosis. However, the marginal areas of glioma usually show vague fluorescence, which makes tumour identification difficult, and the applicability of 5-ALA-based photodynamic therapy (PDT) is hampered by insufficient therapeutic efficacy in glioma tissues.

Methods:

To overcome these issues, we assessed the expression of ferrochelatase (FECH) gene, which encodes a key enzyme that catalyses the conversion of protoporphyrin IX (PpIX) to heme, in glioma surgical specimens and manipulated FECH in human glioma cell lines.

Results:

Prominent downregulation of FECH mRNA expression was found in glioblastoma tissues compared with normal brain tissues, suggesting that FECH is responsible for PpIX accumulation in glioblastoma cells. Depletion of FECH by small interference RNA enhanced PpIX fluorescence after exposure to 5-ALA concomitant with increased intracellular PpIX accumulation in glioma cells. Silencing of FECH caused marked growth inhibition and apoptosis induction by PDT in glioma cells.

Conclusion:

These results suggest that knockdown of FECH is a potential approach to enhance PpIX fluorescent quality for optimising the subjective discrimination of vague fluorescence and improving the effect of 5-ALA-PDT.     

5-氨基乙酰丙酸介导的光动力对CNE2和CNE2/DDP细胞体外作用的观察

目的:观察5-氨基乙酰丙酸(5-ALA)介导的光动力对CNE2和CNE2/DDP的生物作用。方法:荧光分光光度计检测PpⅨ荧光强度;MTT法测定细胞存活率。结果:两种细胞内PpⅨ荧光强度与孵育时间(CNE2:F=65.56,P=0.000;CNE2/DDP:F=28.07,P=0.000)和5-ALA浓度(CNE2:F=6292.36,P=0.000;CNE2/DDP:F=914.36,P=0.000)成正相关,在相同孵育时间(t=17.28,P=0.01)及5-ALA浓度(t=36.07,P=0.000)条件下,CNE2胞内PpⅨ荧光强度均显著高于CNE2/DDP。CNE2或CNE2/DDP细胞存活率与孵育时间(CNE2:F=2245.98,P=0.000;CNE2/DDP:F=236.92,P=0.000)、5-ALA浓度(CNE2:F=55.26,P=0.000;CNE2/DDP:F=10.68,P=0.000)和激光能量(CNE2:F=3294.21,P=0.000;CNE2/DDP:F=285.65,P=0.000)成负相关,相同孵育时间(t=31.35,P=0.000)、5-ALA浓度(t=37.16,P=0.001)及激光能量(t=12.84,P=0.001)条件下,CNE2细胞存活率显著高于CNE2/DDP。结论:5-ALA-PDT对CNE2和CNE2/DDP细胞均具有杀伤作用,但在相同条件下,5-ALA-PDT对CNE2的杀伤作用大于CNE2/DDP。     

ALA-PDT诱导白血病细胞株HL-60凋亡

背景与目的:基于5-氨基乙酰丙酸的光动力疗法(ALA-PDT)利用肿瘤细胞对ALA产生的内源性光敏剂原卟啉IX(PpIX)的优先摄取,使肿瘤细胞在受到一定的光照后被选择性地杀伤。到目前为止,ALA-PDT引起肿瘤细胞破坏的确切机制尚未完全阐明。本研究探讨ALA-PDT对白血病细胞株HL-60的凋亡诱导作用。方法:以白血病细胞株HL-60为实验模型。实验分为4组,对照组、单纯ALA组、单纯光照组及ALA PDT组。用MTT法检测细胞的存活率,瑞氏染色观察细胞形态学改变,用Annexin V-FITC/PI双染法检测细胞凋亡率,并用共聚焦激光显微镜(LSCM)观察凋亡细胞的特征。结果:ALA PDT组光照后细胞形态学可见凋亡改变;MTT法显示细胞存活率明显下降,24 h为(46±9)%,48 h为(26±8)%,两者比较差异有显著性(P<0.05);流式细胞仪检测显示光照后4、5和24 h细胞凋亡率分别为(26±9)%、(29±11)%和(51±6)%,与对照组相比差异有显著性(P<0.05);LSCM观察AnnexinV-FITC单阳性及AnnexinV-FITC/PI双阳性细胞均具有典型的凋亡特征,而单纯ALA组、单纯光照组及对照组则无上述改变。结论:ALA-PDT能杀伤白血病细胞株HL-60,主要通过诱导凋亡的方式实现的,并呈一定的时间依赖性。     

Comparison of photodynamic therapy for skin cancers and pre-cancers with δ-aminolevulinic acid

Objective  

The aim of the study was to compare the effects of photodynamic therapy (PDT) with δ-aminolevulinic acid (ALA) for patients with different kinds of skin cancers and pre-cancers.     

Successful treatment of oral verrucous hyperplasia with topical 5-aminolevulinic acid-mediated photodynamic therapy

Previous studies have shown a selective accumulation of 5-aminolevulinic acid (ALA)-derived protoporphrin IX (PpIX) in oral premalignant and malignant tissues. This provides a biologic rationale for the clinical use of ALA-mediated PDT (ALA-PDT) for oral premalignant and malignant lesions. In this study, five patients with oral verrucous hyperplasia (OVH) were treated with a new protocol of ALA-PDT composed of multiple 3-min irradiations with a light emitting diode (LED) red light at 635+/-5 nm separated with several 3-min rests for a total of 1000 s (fluence rate, 100 mW/cm(2); light exposure dose, 100 J/cm(2)) after topical application of 20% ALA for 1.5 or 2 h. Topical ALA-PDT was repeated once a week until the complete regression of the lesion. Complete regression of all OVH lesions was observed after 1-3 treatments (average, 2 treatments) of topical ALA-PDT. At an average follow-up of 5.6 months (range, 3-11 months), all the five OVH patients were free of tumor recurrence. We conclude that topical ALA-PDT with fractionated irradiations by an LED red light at 635+/-5 nm is an effective and successful treatment modality for OVH.     

Tumor vascular shutdown following photodynamic therapy based on polyhematoporphyrin or 5-aminolevulinic Acid

The effects of photodynamic therapy (PDT) based on polyhaematoporphyrin (PHP) or 5-aminolaevulinic acid (ALA) on the tumour vascular perfusion (TVP) of a rat fibrosarcoma were examined using an (RbCl)-Rb-86 extraction technique. Both forms of therapy induced large and highly significant reductions in TVP, countering the previous suggestion that ALA-PDT has no vascular effects. Ultrasensitive digital imaging fluorescence microscopy indicated that ALA-induced PpIX was distributed relatively evenly throughout the tumour. Use of Hoechst 33342 as a vascular marker revealed that PpIX was also present in cells lining the tumour microvascular spaces, contrary to previous findings.     

Enhancement of the effect of 5-aminolevulinic acid-based photodynamic therapy by simultaneous hyperthermia

In the present study, we have investigated a combination of photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA) and simultaneous hyperthermia (HT) on osteosarcoma (HOSM-1) cells, squamous cell carcinoma (KB) cells and fibroblasts (HF), including an assessment of the differences in the sensitivity of these cells to such treatment in vitro. The intracellular accumulation of protoporphyrin IX (PPIX) formed by metabolism of ALA in mitochondria and the influence of the treatment on the mitochondrial membrane potential were evaluated using flow cytometry. The antitumor effects of HT, PDT using ALA (ALA-PDT) and ALA-PDT combined with HT (PDT+HT) were determined by an MTT assay. Western blot analysis of the expression of heat shock protein 60 (Hsp60) and Hsp70 was performed to evaluate the mitochondrial stress caused by each treatment. The intracellular PPIX accumulation in HOSM-1 cells was about 2-fold higher than that in KB cells. An antitumor effect of ALA-PDT and PDT+HT was obtained in each cell line, and indicated a synergistic interaction of the combination therapy in tumor cells. A marked degree of depolarization of the mitochondrial membrane was observed in both tumor cell lines, and there was no marked difference in the degree of depolarization between the cell lines. Marked expression of Hsp60 was observed in HOSM-1 cells treated with PDT+ HT and ALA-PDT, but not in KB cells. Slightly increased expression of Hsp70 was observed for all treatments in both tumor cell lines. These results suggest that the antitumor effect of ALA-PDT therapy against malignant tumor cells is enhanced by simultaneous HT. Furthermore, the differences in sensitivity to these therapies between the two cell types may have occurred because PPIX was not effectively utilized in HOSM-1 cells, compared to its utilization in KB cells.     

Effect of photodynamic therapy using 5-aminolevulinic acid on 4-nitroquinoline-1-oxide-induced premalignant and malignant lesions of mouse tongue

A new photosensitizing agent 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) has been demonstrated as a useful method for treatment of superficial neoplastic lesions. The aim of this study was to determine the effects of topically and systemically ALA-based PDT in neoplastic lesions of the oral cavity. Premalignant and malignant lesions of mouse tongue induced by 4-nitroquinoline-l-oxide (4NQO) were used in this study. At 1, 2 and 3 h after topical application of ALA or 3 h after systemic administration of ALA (250 or 1000 mg/kg), the lesions were irradiated with Nd: YAG dye laser at 630 nm (200 J/cm2). Both premalignant and early malignant lesions showed complete response to systemically ALA-based PDT. In an invasive nodular malignant lesion, however, only the superficial portion was affected. There was no apparent difference in the PDT effect between 250 and 1000 mg/kg doses of ALA. In contrast, topically ALA-based PDT had virtually no effect on most lesions. It was concluded that systemically ALA-based PDT is a useful method for treating premalignant and early malignant lesions in the oral cavity, while topically ALA-based PDT using this preparation may be unsuitable for treatment of oral lesions.     

Enhanced cytotoxic effects of 5-aminolevulinic acid-mediated photodynamic therapy by concurrent hyperthermia in glioma spheroids

During photodynamic therapy (PDT) both normal and pathological brain tissue, in close proximity to the light source, can experience significant temperature increases. The purpose of this study was to investigate the anti-tumor effects of concurrent 5-aminolevulinic acid (ALA)-mediated PDT and hyperthermia (HT) in human and rat glioma spheroids.Human or rat glioma spheroids were subjected to PDT, HT, or a combination of the two treatments. Therapies were given concurrently to simulate the conditions that will occur during patient PDT.Predictions of diffusion theory suggest that brain tissue immediately adjacent to a spherical light applicator may experience temperature increases approaching 8 °C for laser input powers of 2 W. In the in vitro model employed here, HT had no effect on spheroid survival at temperatures below 49 °C, while sub-threshold fluence PDT results in only modest decrease in survival. HT (40–46 °C) and PDT interact in a synergistic manner if the two treatments are given concurrently. The degree of synergism increases with increasing temperature and light fluence. Apoptosis is the primary mode of cell death following both low-fluence rate PDT and combined HT + PDT     

Improvement of systemic 5-aminolevulinic acid-based photodynamic therapy in vivo using light fractionation with a 75-minute interval

We have studied different single and fractionated illumination schemes after systemic administration of 5-aminolevulinic acid (ALA) to Improve the response of nodular tumors to ALA-mediated photodynamic therapy. Tumors transplanted on the thigh of female WAG/Rij rats were transdermally illuminated with red light (633 nm) after systemic ALA administration (200 mg/kg). The effectiveness of each treatment scheme was determined from the tumor volume doubling time. A single illumination (100 J/cm2 at 100 mW/cm2, 2.5 h after ALA administration) yielded a doubling time of 6.6+/-1.2 days. This was significantly different from the untreated control (doubling time, 1.7+/-0.1 days). The only treatment scheme that yielded a significant improvement compared to all other schemes studied was illumination at both 1 and 2.5 h after ALA administration (both 100 J/cm2 at 100 mW/cm2) and resulted in a tumor volume doubling time of 18.9+/-2.9 days. A possible mechanism to explain this phenomenon is that the protoporphyrin IX formed after administration of ALA is photodegraded by the first illumination. In the 75-min interval, new porphyrin is formed enhancing the effect of the second illumination.