A Computational Framework for the Administration of 5-Aminovulinic Acid Before Glioblastoma Surgery.

5-Aminolevulinic Acid (5-ALA) is the only fluorophore approved by the FDA as an intraoperative optical imaging agent for fluorescence-guided surgery in patients with glioblastoma. The dosing regimen is based on rodent tests where a maximum signal occurs around 6 h after drug administration. Here, we construct a computational framework to simulate the transport of 5-ALA through the stomach, blood, and brain, and the subsequent conversion to the fluorescent agent protoporphyrin IX at the tumor site. The framework combines compartmental models with spatially-resolved partial differential equations, enabling one to address questions regarding quantity and timing of 5-ALA administration before surgery. Numerical tests in two spatial dimensions indicate that, for tumors exceeding the detection threshold, the time to peak fluorescent concentration is 2-7 h, broadly consistent with the current surgical guidelines. Moreover, the framework enables one to examine the specific effects of tumor size and location on the required dose and timing of 5-ALA administration before glioblastoma surgery.

Nanoparticles drug delivery for 5-aminolevulinic acid (5-ALA) in photodynamic therapy (PDT) for multiple cancer treatment: a critical review on biosynthesis, detection, and therapeutic applications.

Photodynamic therapy (PDT) is a promising cancer treatment that kills cancer cells selectively by stimulating reactive oxygen species generation with photosensitizers exposed to specific light wavelengths. 5-aminolevulinic acid (5-ALA) is a widely used photosensitizer. However, its limited tumour penetration and targeting reduce its therapeutic efficacy. Scholars have investigated nano-delivery techniques to improve 5-ALA administration and efficacy in PDT. This review summarises recent advances in biological host biosynthetic pathways and regulatory mechanisms for 5-ALA production. The review also highlights the potential therapeutic efficacy of various 5-ALA nano-delivery modalities, such as nanoparticles, liposomes, and gels, in treating various cancers. Although promising, 5-ALA nano-delivery methods face challenges that could impair targeting and efficacy. To determine their safety and biocompatibility, extensive preclinical and clinical studies are required. This study highlights the potential of 5-ALA-NDSs to improve PDT for cancer treatment, as well as the need for additional research to overcome barriers and improve medical outcomes.

Unleashing the potential of 5-Aminolevulinic acid: Unveiling a promising target for cancer diagnosis and treatment beyond photodynamic therapy.

The therapeutic properties of 5-aminolevulinic acid (5-ALA) have been extensively studied for cancer detection and treatment using photodynamic therapy (PDT). When administered externally, 5-ALA is converted to protoporphyrin IX (PpIX) in cancer cells, which generates reactive oxygen species (ROS) upon exposure to light. This process enables targeted cell death induction and cancer detection. Given the highly conserved nature of heme biosynthesis over billions of years, we hypothesized that natural mechanisms might exist to prevent excessive accumulation of PpIX or heme resulting from 5-ALA overload. Therefore, we anticipated alterations in protein expression profiles upon exogenous administration of 5-ALA. To understand cellular responses to 5-ALA, we investigated protein expression changes and identified OR1B1 as a promising target in bladder, prostate, lung, and cervical cancer cells. OR1B1 expression was observed only with 5-ALA and ferrous chloride, highlighting the central role of heme in this discovery. Immunofluorescence and electron microscopy confirmed OR1B1's sub-cellular localization. These findings suggest that 5-ALA transformation in cancer cells and OR1B1 expression have potential for enhancing cancer detection and developing alternative treatments, including immunotherapy. This approach overcomes the limitations of PDT and opens new avenues for effective and targeted cancer interventions.

Clinical assessment of a low-cost, hand-held, smartphone-attached intraoral imaging probe for 5-aminolevulinic acid photodynamic therapy monitoring and guidance.

Significance: India has one of the highest rates of oral squamous cell carcinoma (OSCC) in the world, with an incidence of 15 per 100,000 and more than 70,000 deaths per year. The problem is exacerbated by a lack of medical infrastructure and routine screening, especially in rural areas. New technologies for oral cancer detection and timely treatment at the point of care are urgently needed. Aim: Our study aimed to use a hand-held smartphone-coupled intraoral imaging device, previously investigated for autofluorescence (auto-FL) diagnostics adapted here for treatment guidance and monitoring photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence (FL). Approach: A total of 12 patients with 14 buccal mucosal lesions having moderately/well-differentiated micro-invasive OSCC lesions (<2 cm diameter and <5 mm depth) were systemically (in oral solution) administered three doses of 20 mg/kg ALA (total 60 mg/kg). Lesion site PpIX and auto-FL were imaged using the multichannel FL and polarized white-light oral cancer imaging probe before/after ALA administration and after light delivery (fractionated, total 100 J/cm2 of 635 nm red LED light). Results: The handheld device was conducive for access to lesion site images in the oral cavity. Segmentation of ratiometric images in which PpIX FL is mapped relative to auto-FL enabled improved demarcation of lesion boundaries relative to PpIX alone. A relative FL (R-value) threshold of 1.4 was found to segment lesion site PpIX production among the patients with mild to severe dysplasia malignancy. The segmented lesion size is well correlated with ultrasound findings. Lesions for which R-value was >1.65 at the time of treatment were associated with successful outcomes. Conclusion: These results indicate the utility of a low-cost, handheld intraoral imaging probe for image-guided PDT and treatment monitoring while also laying the groundwork for an integrated approach, combining cancer screening and treatment with the same hardware.

An Efficient 5-Aminolevulinic Acid Photodynamic Therapy Treatment for Human Hepatocellular Carcinoma.

Photodynamic therapy (PDT) is a two-stage treatment relying on cytotoxicity induced by photoexcitation of a nontoxic dye, called photosensitizer (PS). Using 5-aminolevulinic acid (5-ALA), the pro-drug of PS protoporphyrin IX, we investigated the impact of PDT on hepatocellular carcinoma (HCC). Optimal 5-ALA PDT dose was determined on three HCC cell lines by analyzing cell death after treatment with varying doses. HCC-patient-derived tumor hepatocytes and healthy donor liver myofibroblasts were treated with optimal 5-ALA PDT doses. The proliferation of cancer cells and healthy donor immune cells cultured with 5-ALA-PDT-treated conditioned media was analyzed. Finally, therapy efficacy on humanized SCID mice model of HCC was investigated. 5-ALA PDT induced a dose-dependent decrease in viability, with an up-to-four-fold reduction in viability of patient tumor hepatocytes. The 5-ALA PDT treated conditioned media induced immune cell clonal expansion. 5-ALA PDT has no impact on myofibroblasts in terms of viability, while their activation decreased cancer cell proliferation and reduced the tumor growth rate of the in vivo model. For the first time, 5-ALA PDT has been validated on primary patient tumor hepatocytes and donor healthy liver myofibroblasts. 5-ALA PDT may be an effective anti-HCC therapy, which might induce an anti-tumor immune response.

Multi-charged nanoemulsion for photodynamic treatment of glioblastoma cell line in 2D and 3D in vitro models.

Multi-charged nanoemulsions (NE) were designed to deliver Cannabidiol (CBD), Indocyanine green (ICG), and Protoporphyrin (PpIX) to treat glioblastoma (GBM) through Photodynamic Therapy (PDT). The phase-inversion temperature (PIT) method resulted in a highly stable NE that can be scaled easily, with a six-month shelf-life. We observed the quasi-spherical morphology of the nanoemulsions without any unencapsulated material and that 89% (± 5.5%) of the material was encapsulated. All physicochemical properties were within the expected range for a nanostructured drug delivery system, making these multi-charged nanoemulsions promising for further research and development. NE-PIC (NE-Protoporphyrin + Indocyanine + CBD) was easily internalized on GBM cells after three hours of incubation. Nanoemulsion (NE and NE-PIC) did not result in significant cytotoxicity, even for GBM or non-tumorigenic cell lines (NHF). Phototoxicity was significantly higher for the U87MG cell than the T98G cell when exposed to: visible (430 nm) and infrared (810 nm) laser light, with a difference of about 20%. From 50 mJ.cm-2, the viability of GBM cell lines decreases significantly, ranging from 65% to 85%. The NE-PIC was also effective for inhibiting cell proliferation into a 3D spheroidal GBM cell model, which is promising for mimicking the tumor cell environment. Irradiation at 810 nm was more effective in treating spheroid due to its deeper penetration in complex structures. NE-PIC has the potential as a drug delivery system for photoinactivation and photo diagnostic of GBM cell lines, taking advantage of the versatility of its active components.

Cell senescence-associated porphyrin metabolism affects the efficacy of aminolevulinic acid-photodynamic diagnosis in bladder cancer.

Aminolevulinic acid-photodynamic diagnosis (ALA-PDD) is a promising alternative method to detect cancer cells because of its high specificity and low rate of side effects. Exogenous ALA is administered and accumulates as protoporphyrin IX (PpIX) in cancer cells, which then emit red fluorescence following light irradiation to enable surgeons to accurately identify and remove cancerous tissue. Recent reports suggested that PpIX failed to accumulate in some patients who underwent ALA-PDD. We hypothesized that cell senescence, which is a relatively inactive state, affects porphyrin accumulation in bladder cancer cells. In this study, we evaluated the relationship between cell senescence and porphyrin accumulation in affecting the efficacy of ALA-PDD. First, we utilized three bladder cancer cell lines to evaluate senescence-related indicators and establish a cell senescence model. Then, we identified the differences in porphyrin production and the proteins involved in porphyrin accumulation between old and young cells. We found that compared with young cells, old cells possessed higher concentration of PpIX and had lower ABCG2 expression. The increase in PpIX levels following ABCG2 inhibition is three times higher in old cells than in young cells, suggesting that cell senescence was closely related with porphyrin accumulation in cancer. In conclusion, we found that the efficacy of ALA-PDD and porphyrin accumulation was relatively high in senescent cancer cells and that inhibition of ABCG2 could improve the efficacy of ALA-PDD in young bladder cancer cells.

Continuous Spatiotemporal Therapy of A Full-API Nanodrug via Multi-Step Tandem Endogenous Biosynthesis.

Nanomedicine holds great promise to enhance cancer therapy. However, low active pharmaceutical ingredient (API) loading content, unpredictable drug release, and potential toxicity from excipients limit their translational capability. We herein report a full-API nanodrug composed of FDA-approved 5-aminolevulinic acid (ALA), human essential element Fe3+, and natural bioactive compound curcumin with an ideal API content and pH-responsive release profile for continuous spatiotemporal cancer therapy achieved by multi-step tandem endogenous biosynthesis. First, ALA enzymatically converts into photosensitizer protoporphyrin IX (PpIX). Afterward, multiple downstream products including carbon monoxide (CO), Fe2+, biliverdin (BV), and bilirubin (BR) are individually biosynthesized through the PpIX-heme-CO/Fe2+/BV-BR metabolic pathway, further cooperating with released Fe3+ and curcumin, ultimately eliciting mitochondria damage, membrane disruption, and intracytoplasmic injury. This work not only provides a paradigm for exploiting diversified metabolites for tumor suppression, but also presents a safe and efficient full-API nanodrug, facilitating the practical translation of nanodrugs.

Analysis of Renal Cell Carcinoma Cell Response to the Enhancement of 5-aminolevulinic Acid-mediated Protoporphyrin IX Fluorescence by Iron Chelator Deferoxamine†.

As a tumor photodiagnostic agent, 5-aminolevulinic acid (ALA) is metabolized in the heme biosynthesis pathway to produce protoporphyrin IX (PpIX) with fluorescence. ALA-PpIX fluorescence was evaluated in human renal cell carcinoma (RCC) cell lines and non-tumor HK-2 cell lines. We found that extracellular PpIX level was correlated with ABCG2 activity, illustrating its importance as a PpIX efflux transporter. Extracellular PpIX was also related to the Km of ferrochelatase (FECH) that chelates PpIX with ferrous iron to form heme. The Vmax of FECH was higher in all RCC cell lines tested than in the HK-2 cell line. TCGA dataset analysis indicates a positive correlation between FECH expression and RCC patient survival. These findings suggest FECH as an important biomarker in RCC. Effects of iron chelator deferoxamine (DFO) on the enhancement of PpIX fluorescence were assessed. DFO increased intracellular PpIX in both tumor and non-tumor cells, resulting in no gain in tumor/non-tumor fluorescence ratios. DFO appeared to increase ALA-PpIX more at 1-h than at 4-h treatment. There was an inverse correlation between ALA-PpIX fluorescence and the enhancement effect of DFO. These results suggest that enhancement of ALA-PpIX by DFO may be limited by the availability of ferrous iron in mitochondria following ALA administration.

C5α secreted by tumor mesenchymal stem-like cells mediates resistance to 5-aminolevulinic acid-based photodynamic therapy against glioblastoma tumorspheres.

PURPOSE: Advancements in photodynamic diagnosis (PDD) and photodynamic therapy (PDT) as a standard care in cancer therapy have been limited. This study is aimed to investigate the clinical availability of 5-aminolevulinic acid (5-ALA)-based PDD and PDT in glioblastoma (GBM) patient-derived tumorspheres (TSs) and mouse orthotopic xenograft model. METHODS: PDT was performed using a 635 nm light-emitting diode (LED). Transcriptome profiles were obtained from microarray data. For knockdown of C5α, siRNA was transfected into tumor mesenchymal stem-like cells (tMSLCs). The invasiveness of TSs was quantified using collagen-based 3D invasion assays. RESULTS: Treatment with 1 mM 5 ALA induced distinct protoporphyrin IX (PpIX) fluorescence in GBM TSs, but not in non-tumor cells or tissues, including tMSLCs. These observations were negatively correlated with the expression levels of FECH, which catalyzes the conversion of accumulated PpIX to heme. Furthermore, the 5-ALA-treated GBM TSs were sensitive to PDT, thereby significantly decreasing cell viability and invasiveness. Notably, the effects of PDT were abolished by culturing TSs with tMSLC-conditioned media. Transcriptome analysis revealed diverse tMSLC-secreted chemokines, including C5α, and their correlations with the expression of stemness- or mesenchymal transition-associated genes. By adding or inhibiting C5α, we confirmed that acquired resistance to PDT was induced via tMSLC-secreted C5α. CONCLUSIONS: Our results show substantial therapeutic effects of 5-ALA-based PDT on GBM TSs, suggesting C5α as a key molecule responsible for PDT resistance. These findings could trigger PDT as a standard clinical modality for the treatment of GBM.

Ablation efficacy of 5-aminolevulinic acid-mediated photodynamic therapy on human glioma stem cells.

BACKGROUND: Cancer cells with stem cell-like features are generally more resistant to chemotherapy and radiotherapy than differentiated tumor cells. Thus, these cells tend to increase the propensity for tumor recurrence and metastasis. This study investigated the efficacy of 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) in destructing glioma stem cells (GSCs), including the mesenchymal subtype (MES-GSCs) demonstrated to have the lowest radio- and chemosensitivity. METHODS: Five high-grade glioma (HGG) GSC lines and derived differentiated glioma cell (DGC) lines were examined for protoporphyrin-IX (PpIX) expression using fluorescence-activated cell sorting (FACS) and then assessed for ALA-PDT sensitivity using cell viability assays. MES-GSCs surviving ALA-PDT were then isolated and evaluated for stem cell and mesenchymal marker expression levels (CD44, ALDH1A3, KLF4, nestin) by qRT-PCR. The ability of these surviving cells to form tumors was then examined using colony forming and by xenograft tumor assays in athymic mice. Finally, the relationship between PpIX expression level (high versus low) and ALA-PDT sensitivity was examined by FACS and colony forming assays. RESULTS: ALA-PDT was effective against all GSC lines including MES-GSCs. MES-GSC lines exhibited higher PpIX expression than derived DGCs. Surviving MES-GSCs demonstrated lower stem cell marker expression and tumor forming potential than naive MES-GSCs. Higher PpIX production capacity by MES-GSCs was associated with greater colony forming ability, and ALA-PDT was more effective against MES-GSCs with greater PpIX accumulation. CONCLUSION: ALA-PDT may be clinically effective against HGG by targeting GSCs, including MES-GSCs.

Suppression of resistance to aminolevulinic acid-based photodynamic therapy in esophageal cell lines by administration of iron chelators in collagen type I matrices.

PURPOSE: Photodynamic therapy (PDT) utilizes visible light to activate the cytotoxic effects of photosensitizing drugs. PDT protocols require optimization to overcome treatment resistance and induce a beneficial anti-tumor immune response. The aim of this study was to examine the possibility to suppress the resistance of esophageal cell lines to aminolevulinic acid (ALA)-PDT by administration of iron chelators to induce sufficient cell cytotoxicity under pathophysiologically relevant conditions, mimicking the advanced stages of cancer. MATERIALS AND METHODS: Effects of ALA-PDT in combination with iron chelators were compared in three esophageal cell lines in conventional monolayers and in 3 D cultures based on collagen type I. Modified colony assay and fluorescence-based live cell imaging, respectively were applied. The latter was used also to test the capability of pre-polarized macrophages to interact with cancer cells subjected to ALA-PDT with or without iron chelators. RESULTS: Iron chelators were effective in the enhancement of ALA-PDT in all cell lines under both culture conditions. Fluorescence evaluation of cell viability in 3 D cultures indicated the contribution of apoptotic cell death after ALA-PDT, both with and without iron chelators. Engulfment of remnants of dead cancer cells by macrophages in 2 D cultures was indicated, however, the interaction between macrophages and cancer cells in 3 D cultures subjected to ALA-PDT with or without iron chelators was not present. CONCLUSIONS: The potential of iron chelators to enhance ALA-PDT was maintained in 3 D collagen matrices. Although PDT dose (ALA concentration, light exposure time) required modification in a cell line-dependent manner to achieve a comparable effect of PDT alone in conventional monolayers and in collagen matrices, the potential of iron chelators to suppress the resistance of esophageal cells to ALA-PDT was not influenced by a fibrillar collagen matrix.

Enlarging the Scope of 5-Aminolevulinic Acid-Mediated Photodiagnosis towards Breast Cancers.

Today, most research on treating cancers targets one single cancer, often because of the very specific operation principle of the therapy. For instance, immunotherapies require the expression of a particular antigen, which might not be expressed in all cancers or in all patients. What about metastases? Combination therapies are promising but require treatment personalization and are an expensive approach that many health systems are not willing to pay for. Resection of cancerous tissues may be conducted beforehand. However, the precise location and removal of tumors are in most cases, hurdles that require margins to prevent recurrence. Herein, we further demonstrate the wide application of aminolevulinate-based photodynamic diagnosis and therapy toward breast cancers. By selecting four breast cancer cell lines that represent the main breast tumor subtypes, we investigated their ability to accumulate the fluorescent protoporphyrin IX upon treatment with the marketed 5-aminolevulinic acid hexyl ester (ALA-Hex) or our new and more stable derivative PSI-ALA-Hex. We found that all cell lines were able to accumulate PpIX under a few hours independent of their hormonal status with both treatments. Additionally, this accumulation was less dose-dependent with PSI-ALA-Hex and induced similar or higher fluorescence intensity than ALA-Hex in three out of four cell lines. The toxicity of the two molecules was not different up to 0.33 mM. However, PSI-ALA-Hex was more toxic at 1 mM, even though lower concentrations of PSI-ALA-Hex led to the same PpIX accumulation level. Additional illumination with blue light to induce cell death by generating reactive oxygen species was also considered. The treatments led to a dramatic death of the BT-474 cells under all conditions. In SK-BR-3 and MCF-7, ALA-Hex was also very efficient at all concentrations. However, increasing doses of PSI-ALA-Hex (0.33 and 1 mM) surprisingly led to a higher viability rate. In contrast, the triple-negative breast cancer cells MDA-MB-231 showed a higher death induction with higher concentrations of ALA-Hex or PSI-ALA-Hex. Derivatives of ALA seem promising as fluorescence-guided resection tools and may enable subsequent completion of cancer cell destruction by blue light irradiation.

A Thiosemicarbazone Derivative as a Booster in Photodynamic Therapy-A Way to Improve the Therapeutic Effect.

Photodynamic therapy is one of the most patient friendly and promising anticancer therapies. The active ingredient is irradiated protoporphyrin IX, which is produced in the body that transfers energy to the oxygen-triggering phototoxic reaction. This effect could be enhanced by using iron chelators, which inhibit the final step of heme biosynthesis, thereby increasing the protoporphyrin IX concentration. In the presented work, we studied thiosemicarbazone derivative, which is a universal enhancer of the phototoxic effect. We examined several genes that are involved in the transport of the heme substrates and heme itself. The results indicate that despite an elevated level of ABCG2, which is responsible for the PpIX efflux, its concentration in a cell is sufficient to trigger a photodynamic reaction. This effect was not observed for 5-ALA alone. The analyzed cell lines differed in the scale of the effect and a correlation with the PpIX accumulation was observed. Additionally, an increased activation of the iron transporter MFNR1 was also detected, which indicated that the regulation of iron transport is essential in PDT.

Protoporphyrin IX delayed fluorescence imaging: a modality for hypoxia-based surgical guidance.

Significance: Hypoxia imaging for surgical guidance has never been possible, yet it is well known that most tumors have microregional chronic and/or cycling hypoxia present as well as chaotic blood flow. The ability to image oxygen partial pressure (pO2) is therefore a unique control of tissue metabolism and can be used in a range of disease applications to understand the complex biochemistry of oxygen supply and consumption. Aim: Delayed fluorescence (DF) from the endogenous molecule protoporphyrin IX (PpIX) has been shown to be a truly unique reporter of the local oxygen partial pressure in tissue. PpIX is endogenously synthesized by mitochondria in most tissues, and the particular property of DF emission is directly related to low microenvironmental oxygen concentration. Here, it is shown that PpIX has a unique emission in hypoxic tumor tissue regions, which is measured as a DF signal in the red to near-infrared spectrum. Approach: A time-gated imaging system was used for PpIX DF for wide field direct mapping of pO2 changes. Acquiring both prompt and DF in a rapid sequential cycle allowed for imaging oxygenation in a way that was insensitive to the PpIX concentration. By choosing adequate parameters, the video rate acquisition of pO2 images could be achieved, providing real-time tissue metabolic information. Results: In this report, we show the first demonstration of imaging hypoxia signals from PpIX in a pancreatic cancer model, exhibiting >5X contrast relative to surrounding normal oxygenated tissues. Additionally, tissue palpation amplifies the signal and provides intuitive temporal contrast based upon neoangiogenic blood flow differences. Conclusions: PpIX DF provides a mechanism for tumor contrast that could easily be translated to human use as an intrinsic contrast mechanism for oncologic surgical guidance.

Synthetic biology-instructed transdermal microneedle patch for traceable photodynamic therapy.

5-Aminolevulinic acid-based photodynamic therapy heavily depends on the biological transformation efficiency of 5-aminolevulinic acid to protoporphyrin IX, while the lack of an effective delivery system and imaging navigation are major hurdles in improving the accumulation of protoporphyrin IX and optimizing therapeutic parameters. Herein, we leverage a synthetic biology approach to construct a transdermal theranostic microneedle patch integrated with 5-aminolevulinic acid and catalase co-loaded tumor acidity-responsive copper-doped calcium phosphate nanoparticles for efficient 5-aminolevulinic acid-based photodynamic therapy by maximizing the enrichment of intratumoral protoporphyrin IX. We show that continuous oxygen generation by catalase in vivo reverses tumor hypoxia, enhances protoporphyrin IX accumulation by blocking protoporphyrin IX efflux (downregulating hypoxia-inducible factor-1α and ferrochelatase) and upregulates protoporphyrin IX biosynthesis (providing exogenous 5-aminolevulinic acid and upregulating ALA-synthetase). In vivo fluorescence/photoacoustic duplex imaging can monitor intratumoral oxygen saturation and protoporphyrin IX metabolic kinetics simultaneously. This approach thus facilitates the optimization of therapeutic parameters for different cancers to realize Ca2+/Cu2+-interferences-enhanced repeatable photodynamic therapy, making this theranostic patch promising for clinical practice.

Effects of 5-ALA mediated photodynamic therapy in oral cancer stem cells.

The aim of the present study was to investigate the effects of PDT using the photosensitizer 5-aminoulevulinic acid (5-ALA) in oral squamous cell carcinoma (OSCC) behavior, mainly regarding its role on the cancer stem cell (CSC) phenotypes and in maintenance of the stem cell properties. Two OSCC cell lines were used and divided in the groups: Control, 5-ALA, LED 6 J/cm2 and PDT. MTT and Neutral red assays were used to access cellular viability, cell migration was evaluated by the wound healing assay. The stem cell phenotype was analyzed by flow cytometry to evaluate the CD44high/ESAhigh, CD44high/ESAlow and CD44low populations, by the clonogenic and tumor sphere formation assays as well as by RT-qPCR. The presence of Protoporphyrin IX in each CSC fraction was evaluated by flow cytometry. The OSCC cell lines showed a significant decrease in cell viability and migration after PDT. The percentage of CD44high/ESAhigh cells decreased after PDT, which was associated with an increase in the CD44low cells and with a functional decrease in the colony and sphere formation capacity. CD44high/ESAhigh cells showed increased PpIX, which contributed for their greater sensitivity to PDT. INV gene increased significantly after PDT, indicating cellular differentiation. Altogether, our results demonstrate that 5-ALA mediated PDT decreases not only the fraction of oral CSC but also their functional capabilities, inducing their differentiation.

In-Vitro Use of Verteporfin for Photodynamic Therapy in Glioblastoma.

BACKGROUND: Stummer et al. established fluorescence-guided surgery (FGS) for glioblastoma (GBM) using 5-aminolevulinic acid (5-ALA). Its metabolite, protoporphyrin IX (PPIX), is also a photosensitizer and can be used for photodynamic therapy (PDT) using a laser beam of 635 nm. The porphyrin derivate verteporfin (VP) was discovered to have properties to penetrate the brain, pharmacologically target glioma cells, and is approved for PDT of choroidal neovascularization in wet age-related macular degeneration at 689 nm. OBJECTIVE: To elucidate whether GBM cell lines are susceptible to PDT with second-generation photosensitizer VP. METHODS: Human glioma cell lines LN229, HSR-GBM1, and a low-passage patient-derived GBM cell line P1 were treated with variable concentrations of VP for 24 h, followed by PDT at 689 nm using a diode laser light. Cell viability was measured using the MTT assay and VP uptake was measured using a desktop cytometer. RESULTS: Significantly higher cell death following PDT with VP compared to VP treatment alone or no treatment was detected in all cell models (LN229, HSR-GBM1, P1). Flowcytometric measurements revealed a concentration-dependent cellular uptake of VP after 24 h incubation up to 99% at 10 µM (HSR-GBM1). CONCLUSION: This study demonstrates that PDT with VP causes cell death in GBM cells at marginal concentrations. Additionally, red spectrum fluorescence was detected at therapeutic concentrations in all cell lines, validating the cellular uptake of VP in GBM cells. VP, therefore, is not only a potential drug for targeting GBM pharmacologically but can be used as an optical imaging dye in surgery and photosensitizer to make GBM susceptible to PDT.

The effect of hypoxia on photodynamic therapy with 5-aminolevulinic acid in malignant gliomas.

BACKGROUND: Glioblastoma (GBM) is a high-grade, poor prognosis tumor that is resistant to standard treatment. The presence of a small number of glioma stem cells (GSCs) surviving in the harsh microenvironment is responsible for their refractoriness. This study aimed to investigate the effect of a hypoxic environment on the sensitivity of GSCs to photodynamic therapy with 5-aminolevulinic acid (ALA-PDT). MATERIALS AND METHODS: Six human GSC lines, Mesenchymal types HGG13, HGG30, HGG1123, and Proneural types HGG146, HGG157, HGG528, were divided into two groups: normoxia (O2 21%)-cultured cells (Normoxia-GSCs), and hypoxia (O2 5%)-cultured cells (Hypoxia-GSCs). To compare the effects of different oxygen partial pressures on photoporphyrin Ⅸ (PpⅨ) biosynthetic activity, PpⅨ biosynthetic enzyme and transporter expression levels were examined by qRT-PCR; the intracellular PpⅨ concentration was determined using flow cytometry. Additionally, the sensitivity of these two groups of cells to ALA-PDT was evaluated in vitro. RESULTS: Hypoxia-GSCs showed higher mRNA levels of FECH (ferrochelatase), which is required for iron synthesis to convert PpⅨ to heme, compared with Normoxia-GSCs. Flow cytometry revealed that the accumulation of PpⅨ in Hypoxia-GSCs reduced upon incubation with ALA. However, Hypoxia-GSCs showed less reduction in sensitivity to ALA-PDT than Normoxia-GSCs. CONCLUSION: Hypoxia-GSCs had lower intracellular PpⅨ accumulation than Normoxia-GSCs due to increased gene expression of FECH, and that their sensitivity to ALA-PDT was reduced less, despite accumulating lower concentrations of PpⅨ. ALA-PDT is a potentially effective therapy for hypoxia-tolerant GSCs that exist in hypoxia at 5% oxygen concentration.

5-aminolevulinic acid and sodium ferrous citrate decreased cell viability of gastric cancer cells by enhanced ROS generation through improving COX activity.

BACKGROUND: Mitochondrial dysfunctions are related to cancer development.. 5-aminolevulinic acid (ALA) is used for photodynamic therapy (PDT). In this PDT, protoporphyrin IX (PpIX), which is converted from ALA, can generate reactive oxygen species (ROS) that kill the cancer cell. ALA is also reported to promote cytochrome c oxidase (COX) activity, which can generate ROS itself. Therefore, this study focused on the effect of ALA during PDT. In addition, in the previous study, sodium ferrous citrate (SFC) is reported to increase COX activity. So, this study also aims to improve the COX activity by the addition of SFC that can promote ROS generation, which has a cytotoxic effect. METHODS: In this study, we used ALA and SFC, then evaluated the effects of the treatment on the human gastric cancer cell line MKN45, including the induction of cell death. RESULTS: This study showed that treatment with ALA and SFC increases intracellular heme and heme proteins. Moreover, COX activity was promoted, resulting in the production of intracellular reactive oxygen species (ROS), which eventually reduced the cell viability in human gastric cancer cell line MKN45. CONCLUSION: Our study can detect ROS generation with ALA and SFC. Furthermore, we found this generation of ROS has a cytotoxic effect. Therefore, this phenomenon contributes to the effect of PDT.