The Characteristic of Light Sources and Fluorescence in the 3-Dimensional Digital Exoscope "ORBEYE" for 5-Aminolevulinic Acid-Induced Fluorescence-Guided Surgery Compared with a Conventional Microscope.

BACKGROUND: The ORBEYE (ORB), an innovative 3-dimensional digital exoscope, is an equipped system for fluorescence-guided surgery with 5-aminolevulinic acid. Therefore, this study aimed to verify the characteristics of fluorescence-guided surgery with 5-aminolevulinic acid and excitation light source with ORB. METHODS: The same operative field of glioblastoma was recorded under blue light (BL) excitation using a conventional microscope (MS) and ORB. For in vitro studies, the energy of 405-nm wavelength light in white light and BL modes of each scope was examined in various focal lengths. To examine the degree of photobleaching with BL for each scope, protoporphyrin IX-soaked filter papers were continuously exposed with BL of an MS and ORB, and the video-recorded red fluorescence intensity was analyzed. RESULTS: The color tone of tumor-induced red fluorescence was remarkably different under each scope. Furthermore, nonfluorescent normal structures without red fluorescence were well recognized under ORB. The energy of 405-nm wavelength light in BL was significantly higher in ORB than that in an MS, especially in the short focal length. With continuous BL excitation to filter papers, the relative red fluorescence intensity of filter papers was significantly decreased over time in ORB than in an MS. In low protoporphyrin IX concentration, the difference was more significant. CONCLUSIONS: With ORB, the good visibility due to BL energy as compared with an MS might improve the surgical manipulation even in BL mode. However, the weak fluorescent tissue and short focal length should be carefully considered because photobleaching might be critical for FGS.

Modified 5-aminolevulinic acid photodynamic therapy (M-PDT) inhibits cutaneous squamous cell carcinoma cell proliferation via targeting PP2A/PP5-mediated MAPK signaling pathway.

BACKGROUND: We previously demonstrated that M-PDT is painless and effective in precancerous skin diseases treatment. However, whether M-PDT is effective in cSCC and the underlying inhibitory mechanism remains enigmatic. OBJECTIVE: We aims to unveil the effect of M-PDT on cSCC cell proliferation and the regulatory effect of M-PDT on MAPK signaling. METHODS: The proliferation and migration of cSCC cells were revealed by CCK8 assay, tumor sphere formation assay and scratch assay respectively. The expression of MAPKs was examined by western blot. The activity of PP2A and PP5 was regulated by inhibitor and recombinant adenoviruses. RESULTS: Here, we show that M-PDT inhibits cSCC cell proliferation by activating p-JNK, p-p38 and inhibiting p-Erk1/2, as well as activation of PP2A and inactivation of PP5. Furthermore, pharmacological inhibition of PP2A conferred resistance to M-PDT's suppression on p-Erk1/2 and attenuated inhibitory effects of M-PDT on cell proliferation whereas overexpression of wild-type PP2A showed the contrary results. Pharmacological inhibition of PP5 potentiated M-PDT's elevation on p-JNK and strengthened inhibitory effects of M-PDT on cell proliferation whereas overexpression of wild-type PP5 exhibited the contrary results. CONCLUSION: Our findings indicate that M-PDT inhibits cSCC cell proliferation via targeting PP2A/PP5-mediated MAPK signaling pathway.

5-aminolevulinic acid combined with sodium ferrous citrate ameliorated lupus nephritis in a mouse chronic graft-versus-host disease model.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the abnormal activation of immune cells and hypersecretion of autoantibodies and causes irreversible chronic damage, such as lupus nephritis. Chronic graft-versus-host-disease (cGvHD) in mice induced by the injection of parental mouse lymphocytes into F1 hybrids leads to a disease similar to SLE. 5-aminolevulinic acid (5-ALA) is a key progenitor of heme, and its combination with sodium ferrous citrate (SFC) can up-regulate the heme oxygenase (HO-1) expression, resulting in an anti-inflammatory effect. While HO-1 had been reported to be involved in T cell activation and can limit immune-based tissue damage through Treg suppression, which promotes effector response. Thus, we hypothesized that treatment with 5-ALA/SFC could ameliorate lupus nephritis in a mouse cGvHD model. Our results showed that 5-ALA/SFC-treatment significantly decreased the anti-double-stranded DNA (ds-DNA) autoantibodies, blood urea nitrogen (BUN) and creatinine (Cre) levels, reduced kidney inflammatory dendritic cells (DCs) and B cell activation, and increased the regulatory T cells (Tregs) at nine weeks. Furthermore, 5-ALA/SFC suppressed mRNA expression of TNF-α, IL-1ß, IFN-γ and markers on DCs. In addition, we also found that 5-ALA/SFC treatment increased the HO-1 expression on donor-derived DCs and Tregs concurrently, increased the number of Tregs, and reduced the population of activated DCs, B cells and CD8+ T cells at three weeks (early stage of the disease). We thus identified a novel role of 5-ALA/SFC for therapeutically improving the symptoms of lupus nephritis in a mouse cGvHD model and expanded the current understanding of how this immunoregulatory agent can be used to generate beneficial immune responses and treat autoimmune disease.

5-Aminolevulinic Acid-Shedding Light on Where to Focus.

BACKGROUND: Surgical management of gliomas is predicated on "safe maximal resection" across all histopathologic grades because progression-free survival and overall survival are positively affected by the increasing extent of resection. Administration of the prodrug 5-aminolevulinic acid (5-ALA) induces tumor fluorescence with high specificity and sensitivity for malignant high-grade glioma (HGG). Fluorescence-guided surgery (FGS) using 5-ALA improves the extent of resection in the contrast-enhancing and nonenhancing tumor components in HGG. It has also shown preliminary usefulness in other central nervous system tumors, but with certain limitations. METHODS: We review and discuss the state of 5-ALA FGS for central nervous system tumors and identify the limitations in its use as a guide for future clinical optimization. RESULTS: 5-ALA FGS provides maximum clinical benefits in the treatment of newly diagnosed glioblastoma. 5-ALA fluorescence specificity is limited in low-grade glioma, recurrent HGG, and non-glial tumors. Several promising intraoperative adjuncts to 5-ALA FGS have been developed to expand its indications and improve the clinical efficacy and usefulness of 5-ALA FGS. CONCLUSIONS: 5-ALA FGS improves the clinical outcomes in HGG. However, further optimization of the diagnostic performance and clinical use of 5-ALA FGS is necessary for low-grade glioma and recurrent HGG tumors. Neurosurgical oncology will benefit from the novel use of advanced technologies and intraoperative visualization techniques outlined in this review, such as machine learning, hand-held fibe-optic probes, augmented reality, and three-dimensional exoscope assistance, to optimize the clinical usefulness and operative outcomes of 5-ALA FGS.

5-amino levulinic acid inhibits SARS-CoV-2 infection in vitro.

The current COVID-19 pandemic requires urgent development of effective therapeutics. 5-amino levulinic acid (5-ALA) is a naturally synthesized amino acid and has been used for multiple purposes including as an anticancer therapy and as a dietary supplement due to its high bioavailability. In this study, we demonstrated that 5-ALA treatment potently inhibited infection of SARS-CoV-2, a causative agent of COVID-19, in cell culture. The antiviral effects could be detected in both human and non-human cells, without significant cytotoxicity. Therefore, 5-ALA is worth to be further investigated as an antiviral drug candidate for COVID-19.

5-Aminolevulinic acid combined with sodium ferrous citrate (5-ALA/SFC) ameliorated liver injury in a murine acute graft-versus-host disease model by reducing inflammation responses through PGC1-α activation.

Acute graft-versus-host disease (aGvHD) remains lethal as a life-threatening complication after allogeneic hematopoietic stem cell transplantation (HSCT). Inflammatory responses play an important role in aGvHD. 5-Aminolevulinic acid combined with sodium ferrous citrate (5-ALA/SFC) has been widely reported to have a major effect on the anti-inflammatory response; however, these effects in aGvHD models have never been reported. In this study, a murine aGvHD model was developed by transferring spleen cells from donor B6/N (H-2kb) mice into recipient B6D2F1 (H-2kb/d) mice. In addition to evaluating manifestations in aGvHD mice, we analyzed the serum ALT/AST levels, liver pathological changes, infiltrating cells and mRNA expression of inflammation-related cytokines and chemokines. 5-ALA/SFC treatment significantly ameliorated liver injury due to aGvHD and decreased the population of liver-infiltrating T cells, resulting in a reduced expression of pro-inflammatory cytokines and chemokines. Furthermore, the mRNA expression proliferator-activated receptor-γcoactivator (PGC-1α) was enhanced, which might explain why 5-ALA/SFC treatment downregulates inflammatory signaling pathways. Our results indicated that 5-ALA/SFC can ameliorate liver injury induced by aGvHD through the activation of PGC-1α and modulation of the liver mRNA expression of inflammatory-related cytokines and chemokines. This may be a novel strategy for treating this disease.

Glutathione-Responsive Multifunctional "Trojan Horse" Nanogel as a Nanotheranostic for Combined Chemotherapy and Photodynamic Anticancer Therapy.

It remains a great challenge to design a multifunctional and robust nanoplatform for stimuli-responsive drug delivery toward a lesion, which tactfully integrates multiple molecules with therapeutic and diagnostic characteristics. Herein, we reported a facile and ingenious cross-linked nanogel (DSA) based on the chemical cross-link of drugs as a straightforward strategy to overcome the instability of the assembly. In DSA, doxorubicin (DOX) and 5-aminolevulinic acid (ALA) were cross-linked with a disulfide linker for realizing synergistic anticancer therapy. The stability of DSA was adjusted via balancing the hydrophobic/hydrophilic property with hydrophilic NH2-PEG1k. After regulating the coordination of the DOX part and ALA moiety, the drug-loaded nanogel exhibited superior chemotherapeutic efficacies. Additionally, the DSA could selectively biosynthesize fluorescent protoporphyrin IX (PpIX) in tumor cells, which could be applied for a real-time imaging probe of accurate cancer diagnosis. Besides, the in situ synthesized PpIX in mitochondria could serve as a photosensitizer to convert oxygen into toxic reactive oxygen species under a near infrared ray at 660 nm irradiation, leading to an excellent tumor-killing efficacy. This work proposed a unique strategy for designing a series of prodrug nanogels as a universal drug delivery platform for realizing precise disease therapy and diagnostics.

5-Aminolevulinic acid-mediated photodynamic activity in patient-derived cholangiocarcinoma organoids.

BACKGROUND: Accurate diagnosis of the disease extension of cholangiocarcinoma (CCA) is often difficult in clinical practice. The diagnostic yield of conventional pre-operative imaging or endoscopic procedures is sometimes insufficient for the evaluation of longitudinal spreading of CCA. Here we investigated the usefulness of 5-aminolevulinic acid (5-ALA) for the pre- or intra-operative diagnosis of CCA, using patient-derived organoids. METHODS: Four CCA- and two adjacent tissue-derived organoids were established. After 5-ALA treatment, we assessed their photodynamic activity using fluorescence microscopy. RESULTS: CCA organoids established from different patients showed diverse morphology in contrast to monolayer structures of non-tumor organoids, and had the ability to form subcutaneous tumors in immunodeficient mice. CCA organoids demonstrated remarkably high photodynamic activity based on higher accumulation of protoporphyrin IX as a metabolite of 5-ALA compared to non-tumor organoids (40-71% vs. < 4%, respectively). Importantly, cancer cell-specific high photodynamic activity distinguished the organoids originated from biliary stenotic lesions from those of non-stenotic lesions in a CCA patient. The high photodynamic activity did not depend on the expression profile of heme biosynthesis genes. CONCLUSIONS: Distinct 5-ALA-based photodynamic activity could have diagnostic potential for the discrimination of CCA from non-tumor tissues.

Neuroprotective effects of 5-aminolevulinic acid against neurodegeneration in rat models of Parkinson's disease and stroke.

Oxidative stress is associated with the progression of the neurodegenerative diseases Parkinson's disease (PD) and cerebral ischemia. Recently, 5-aminolevulinic acid (5-ALA), an intermediate in the porphyrin synthesis pathway, was reported to exert antioxidative effects on macrophages and cardiomyocytes. Here, we demonstrated the neuroprotective effects of 5-ALA using rat models of PD and ischemia as well as in vitro in SH-SY5Y cells. 5-ALA partially prevented neurodegeneration in each condition. These results suggest that 5-ALA has a potential for promising therapeutic agent to protect against neurodegeneration exacerbated by oxidative stress.

Amylase-Protected Ag Nanodots for in vivo Fluorescence Imaging and Photodynamic Therapy of Tumors.

BACKGROUND: Fluorescent metallic nanodots (NDs) have become a promising nanoprobe for a wide range of biomedical applications. Because Ag NDs have a high tendency to be oxidized, their synthesis and storage are a big challenge. Thus, the method for preparing stable Ag NDs is urgently needed. Surface modification and functionalization can enrich the capability of Ag NDs. METHODS: In this work, fluorescent Ag NDs were synthesized in deoxygenated water by using porcine pancreatic α-amylase (PPA) as the stabilizing/capping agent. The absorption and fluorescence of PPA-protected Ag NDs (PPA@AgNDs) were measured with a spectrophotometer and a spectrofluorometer, respectively. The morphology of PPA@AgNDs was characterized by high-angle annular dark-field (HAADF) scanning transmission electron microscopy (STEM). The biocompatibility of PPA@AgNDs was evaluated by tetrazolium (MTT)-based assay. PolyLys-Cys-SH (sequence: KKKKKKC) peptides were conjugated to PPA@AgNDs via heterobifunctional crosslinkers. PolyLys-Cys-linked PPA@AgNDs absorbed 5-aminolevulinic acid (ALA) by electrostatic interaction at physiological pH. The capability of tumor targeting was evaluated by intravenously injecting PPA@AgND-ALA into 4T1 breast cancer xenograft mouse models. Photodynamic therapy (PDT) against tumors was performed under 635 nm laser irradiation. RESULTS: PPA@AgNDs emitted at 640 nm with quantum yield of 2.1%. The Ag NDs exhibited strong photostability over a long period and a fluorescence lifetime of 5.1 ns. PPA@AgNDs easily entered the cells to stain the nuclei, showing the capabilities of living cell imaging with negligible cytotoxicity. ALA-loaded PPA@AgNDs (PPA@AgND-ALA) presented the superiority of passive tumor targeting via the enhanced permeability and retention (EPR) effect. Tumors were visualized in the near-infrared (NIR) region with reduced background noise. ALA molecules released from PPA@AgND-ALA was converted into the photosensitizer (PS) of protoporphyrin IX (PpIX) intracellularly and intratumorally, which greatly improved the PDT efficacy. CONCLUSION: Our approach opens a new way to design a novel theranostic nanoplatform of PPA@AgND-ALA for effective tumor targeting and fluorescence image-guided PDT.

Enhanced anti-tumor efficacy of 5-aminolevulinic acid-gold nanoparticles-mediated photodynamic therapy in cutaneous squamous cell carcinoma cells.

The objective of this study was to investigate whether the conjugation of gold nanoparticles (GNPs) to 5-aminolevulinic acid (5-ALA) could enhance the anti-tumor efficiency of photodynamic therapy (PDT) in epidermoid carcinoma cells. The mRNA and protein expression levels were determined by quantitative real-time PCR and western blot, respectively. Cell viability, apoptosis, invasion, and migration were determined by MTT assay, flow cytometry, transwell invasion assay, and migration assay, respectively. Singlet oxygen generation was detected by the singlet oxygen sensor green reagent assay. Our results showed that PDT with 5-ALA and GNPs-conjugated 5-ALA (5-ALA-GNPs) significantly suppressed cell viability, increased cell apoptosis and singlet oxygen generation in both HaCat and A431 cells, and PDT with 5-ALA and 5-ALA-GNPs had more profound effects in A431 cells than that in HaCat cells. More importantly, 5-ALA-GNPs treatment potentiated the effects of PDT on cell viability, cell apoptosis, and singlet oxygen generation in A431 cells compared to 5-ALA treatment. Further in vitro assays showed that PDT with 5-ALA-GNPs significantly decreased expression of STAT3 and Bcl-2 and increased expression of Bax in A431 cells compared with PDT with 5-ALA. In addition, 5-ALA-GNPs treatment enhanced the inhibitory effects of PDT on cell invasion and migration and Wnt/ß-catenin signaling activities in A431 cells compared to 5-ALA treatment. In conclusion, our results suggested that GNPs conjugated to 5-ALA significantly enhanced the anti-tumor efficacy of PDT in A431 cells, which may represent a better strategy to improve the outcomes of patients with cutaneous squamous cell carcinoma.

Enhancing selective photosensitizer accumulation and oxygen supply for high-efficacy photodynamic therapy toward glioma by 5-aminolevulinic acid loaded nanoplatform.

The complex biology of glioma compromises therapeutic efficacy and results in poor prognosis. Photodynamic therapy (PDT) has emerged as a promising modality for localized tumor ablation with limited damage to healthy brain tissues. However, low photosensitizer concentration and hypoxic microenvironment in glioma tissue hamper the practical applications of PDT. To address the challenges, biocompatible periodic mesoporous organosilica coated Prussian blue nanoparticles (PB@PMOs) are constructed to load a biosafe prodrug 5-aminolevulinic acid (5-ALA), which is pronouncedly converted to protoporphyrin IX (PpIX) in malignant cells. PB@PMO-5-ALA induces a higher accumulation of PpIX in glioma cells compared to free 5-ALA. Meanwhile, the PB@PMOs, with a mean edge length of 81 nm and good biocompatibility, effectively decompose hydrogen peroxide to oxygen in a temperature-responsive manner. Oxygen supply further contributes to the promotion of 5-ALA-PDT. Thus, the photodynamic effect of PB@PMO-5-ALA is significantly improved, imposing augmented cytotoxicity to glioma U87MG cells. Furthermore, ex vivo fluorescence imaging elucidates the tumor PpIX increases by 75% in PB@PMO-5-ALA treated mice than that in 5-ALA treated ones post 12 h injection. Magnetic resonance imaging (MRI) and iron staining strongly demonstrate the accumulation of PB@PMO-5-ALA in glioma tissues with negative contrast enhancement and blue staining deposits, respectively. The nanoparticle accumulation and high PpIX level collaboratively enhance PDT efficacy through PB@PMO-5-ALA, which efficiently suppresses tumor growth, providing a promising option with safety for local glioma ablation.

Newly formulated 5% 5-aminolevulinic acid photodynamic therapy on Candida albicans.

BACKGROUND: A large number of systemic diseases can be linked to oral candida pathogenicity. The global trend of invasive candidiasis has increased progressively and is often accentuated by increasing Candida albicans resistance to the most common antifungal medications. Photodynamic therapy (PDT) is a promising therapeutic approach for oral microbial infections. A new formulation of 5-aminolevulinic acid (5%ALA) in a thermosetting gel (t) (5%ALA-PTt) was patented and recently has become available on the market. However, its antimicrobial properties, whether mediated or not by PDT, are not yet known. In this work we characterised them. METHODS: We isolated a strain of C. albicans from plaques on the oral mucus membrane of an infected patient. Colonies of this strain were exposed for 1 24 h, to 5%ALA-PTt, 5%ALA-PTt buffered to pH 6.5 (the pH of the oral mucosa) (5%ALA-PTtb) or not exposed (control). The 1 h-exposed samples were also irradiated at a wavelength of 630 nm with 0.14 watts (W) and 0.37 W/cm2 for 7 min at a distance of <1 mm. RESULTS AND CONCLUSION: The 5% ALA-PTt preparation was shown to be effective in reducing the growth of biofilm and inoculum of C. albicans. This effect seems to be linked to the intrinsic characteristics of 5%ALA-TPt, such acidic pH and the induction of free radical production. This outcome was significantly enhanced by the effect of PDT at relatively short incubation and irradiation times, which resulted in growth inhibition of both treated biofilm and inoculum by ∼80% and ∼95%, respectively.

Differential in vitro sensitivity of oral precancerous and squamous cell carcinoma cell lines to 5-aminolevulinic acid-mediated photodynamic therapy.

OBJECTIVES: The clinical effect of 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) may be correlated with the degree of dysplasia of cancer tissues, but much is still unknown regarding the differences in its effectiveness, especially in oral cancer and precancerous lesions. The aim of this study is to compare the effects of ALA-PDT on a human oral precancerous cell line (DOK) and an oral squamous cell carcinoma cell line (CAL-27). METHODS: First, we explored the dose- and time-dependent responses of DOK and CAL-27 cells to ALA-PDT. DOK and CAL-27 cells were incubated with various concentrations of ALA (from 0.25 to 2 mM), followed by PDT using laser irradiation at 635 nm. The resulting photocytotoxicity was assessed in both cell lines using MTT assays. Further, apoptosis was assessed using flow cytometry, reactive oxygen species (ROS) generation was evaluated with 2,7-dichlorofluorescein diacetate (DCFH2-DA), and the response to treatment was examined via RT-qPCR and Western blotting to measure the mRNA and protein expression levels of matrix metallopeptidase 2 (MMP-2) and MMP-9. RESULTS: ALA-PDT inhibited the proliferation of DOK and CAL-27 cells in a dose- and time-dependent manner. Dose-effect and inhibition-time relationships were also found. The rates of DOK and CAL-27 cell apoptosis when the ALA dose was 1 mM were 30.66 ±â€¯3.10% and 75.40 ±â€¯1.29%, respectively (P < 0.01). Following PDT, compared with DOK cells, the ROS level in CAL-27 cells was significantly increased and was correlated with an increase in the ALA concentration. Mechanistically, both the mRNA and protein expression levels of MMP-2 and MMP-9 were found to be regulated in both cell types after ALA-PDT. CONCLUSION: ALA-PDT effectively killed DOK and CAL-27 cells in a dose- and time-dependent manner in vitro. However, under the same conditions, the susceptibilities of these cell lines to ALA-PDT were different. Further studies are necessary to confirm whether this difference is present in clinical oral cancer and precancerous lesions.

Combination of levulinic acid and sodium dodecyl sulfate on inactivation of foodborne microorganisms: A review.

The combination of levulinic acid and sodium dodecyl sulfate (SDS) in recent years has shown considerable promise as an antimicrobial intervention. Both ingredients have been designated by the U.S. Food and Drug Administration (FDA) as Generally Recognized as Safe (GRAS) for being used as a flavoring agent and multipurpose food additive, respectively. The use of levulinic acid and SDS alone has limited antimicrobial efficacy on tested microorganisms, and synergism between levulinic acid and SDS has been observed. The postulated mechanism of action of the synergistic effect is presented. The antimicrobial efficacy of levulinic acid plus SDS remains high even when organic materials are present. The other features, including penetration, foamability, and being readily soluble, extend its potential applications to disinfection of difficult-to-access areas and control of foodborne pathogens both in a planktonic state and in a biofilm. These features indicate that the levulinic acid plus SDS combination may have the potential to be applied within the food processing environment on a large scale.

Enhanced anti-tumor efficacy of 5-aminolevulinic acid-gold nanoparticles-mediated photodynamic therapy in cutaneous squamous cell carcinoma cells

The objective of this study was to investigate whether the conjugation of gold nanoparticles (GNPs) to 5-aminolevulinic acid (5-ALA) could enhance the anti-tumor efficiency of photodynamic therapy (PDT) in epidermoid carcinoma cells. The mRNA and protein expression levels were determined by quantitative real-time PCR and western blot, respectively. Cell viability, apoptosis, invasion, and migration were determined by MTT assay, flow cytometry, transwell invasion assay, and migration assay, respectively. Singlet oxygen generation was detected by the singlet oxygen sensor green reagent assay. Our results showed that PDT with 5-ALA and GNPs-conjugated 5-ALA (5-ALA-GNPs) significantly suppressed cell viability, increased cell apoptosis and singlet oxygen generation in both HaCat and A431 cells, and PDT with 5-ALA and 5-ALA-GNPs had more profound effects in A431 cells than that in HaCat cells. More importantly, 5-ALA-GNPs treatment potentiated the effects of PDT on cell viability, cell apoptosis, and singlet oxygen generation in A431 cells compared to 5-ALA treatment. Further in vitro assays showed that PDT with 5-ALA-GNPs significantly decreased expression of STAT3 and Bcl-2 and increased expression of Bax in A431 cells compared with PDT with 5-ALA. In addition, 5-ALA-GNPs treatment enhanced the inhibitory effects of PDT on cell invasion and migration and Wnt/β-catenin signaling activities in A431 cells compared to 5-ALA treatment. In conclusion, our results suggested that GNPs conjugated to 5-ALA significantly enhanced the anti-tumor efficacy of PDT in A431 cells, which may represent a better strategy to improve the outcomes of patients with cutaneous squamous cell carcinoma.

Antioxidant System and Biomolecules Alteration in Pisum sativum under Heavy Metal Stress and Possible Alleviation by 5-Aminolevulinic Acid.

Environmental pollution is the most serious problem that affects crop productivity worldwide. Pisum sativum is a leguminous plant that is cultivated on a large scale in the Nile Delta of Egypt as a winter crop, and many of the cultivated fields irrigated with drainage water that contained many pollutants including heavy metals. The present research aimed to investigate the impact of Cd and Ni on the biochemical and physiological processes in P. sativum and evaluate the potential alleviation of their toxicity by 5-aminolevulinic acid (ALA). Seedlings of P. sativum were grown in Hoagland solution treated with CdCl2 or NiCl2 for 72 h in the growth chamber. Hydrogen peroxide, lipid peroxidation, protein carbonylation, reduced glutathione, oxidized glutathione, proline, phenolics, antioxidant enzymes, as well as Cd and Ni concentrations were measured at 0, 12, 24, 36, 48, 72 h. An experiment of alleviation was conducted where ALA was added to the growth solution at a concentration of 200 µM coupled with 100 µM of either CdCl2 or NiCl2. Hydrogen peroxide, lipid peroxidation, protein carbonylation, reduced glutathione, oxidized glutathione, proline, and phenolics were induced due to the toxicity of Cd and Ni. The activities of antioxidant enzymes [NADH-oxidase (EC: 1.6.3.1), ascorbate peroxidase (EC: 1.11.1.11), glutathione reductase (EC: 1.6.4.2), superoxide dismutase (EC: 1.15.1.1), and catalase (EC: 1.11.1.6)] were induced under the treatments of both metals. On the other hand, the soluble protein decreased gradually depending upon the time of exposure to the heavy metals. The concentration of Cd and Ni in the leaves treated plants increased in time of exposure dependent manner, while their contents remained within the acceptable limits. The addition of ALA decreased the oxidative stress in treated P. sativum plants. The results revealed the significance of using ALA in the cultivation of P. sativum might improve its tolerance against heavy metal stress.

5-ALA/SFC enhances HO-1 expression through the MAPK/Nrf2 antioxidant pathway and attenuates murine tubular epithelial cell apoptosis.

Cyclosporin A (CsA) is a common immunosuppressant, but its use is limited as it can cause chronic kidney injury. Oxidative stress and apoptosis play a key role in CsA-induced nephrotoxicity. This study investigated the protective effect of 5-aminolevulinic acid and iron (5-ALA/SFC) on CsA-induced injury in murine proximal tubular epithelial cells (mProx24). 5-ALA/SFC significantly inhibited apoptosis in CsA-treated mProx24 cells with increases in heme oxygenase (HO)-1, nuclear factor E2-related factor 2 (Nrf2), and p38, and Erk-1/2 phosphorylation. Moreover, 5-ALA/SFC suppressed production of reactive oxygen species in CsA-exposed cells and inhibition of HO-1 suppressed the protective effects of 5-ALA/SFC. In summary, 5-ALA/SFC may have potential for development into a treatment for the anti-nephrotoxic/apoptotic effects of CsA.

Systemic MEK inhibition enhances the efficacy of 5-aminolevulinic acid-photodynamic therapy.

BACKGROUND: Protoporphyrin IX (PpIX) gets accumulated preferentially in 5-aminolevulinic acid (5-ALA)-treated cancer cells. Photodynamic therapy (PDT) utilises the accumulated PpIX to trigger cell death by light-induced generation of reactive oxygen species (ROS). We previously demonstrated that oncogenic Ras/MEK decreases PpIX accumulation in cancer cells. Here, we investigated whether combined therapy with a MEK inhibitor would improve 5-ALA-PDT efficacy. METHODS: Cancer cells and mice models of cancer were treated with 5-ALA-PDT, MEK inhibitor or both MEK inhibitor and 5-ALA-PDT, and treatment efficacies were evaluated. RESULTS: Ras/MEK negatively regulates the cellular sensitivity to 5-ALA-PDT as cancer cells pre-treated with a MEK inhibitor were killed more efficiently by 5-ALA-PDT. MEK inhibition promoted 5-ALA-PDT-induced ROS generation and programmed cell death. Furthermore, the combination of 5-ALA-PDT and a systemic MEK inhibitor significantly suppressed tumour growth compared with either monotherapy in mouse models of cancer. Remarkably, 44% of mice bearing human colon tumours showed a complete response with the combined treatment. CONCLUSION: We demonstrate a novel strategy to promote 5-ALA-PDT efficacy by targeting a cell signalling pathway regulating its sensitivity. This preclinical study provides a strong basis for utilising MEK inhibitors, which are approved for treating cancers, to enhance 5-ALA-PDT efficacy in the clinic.

A new GSH-responsive prodrug of 5-aminolevulinic acid for photodiagnosis and photodynamic therapy of tumors.

5-Aminolevulinic acid (5-ALA) and its two ester derivatives (5-ALA-OMe and 5-ALA-OHex) have been approved for photodiagnosis and photodynamic therapy (PDT) of tumors in the clinical. However, their pharmacological activities are limited by their instability under physiological conditions and lack of tumor selectivity. With the aim to overcome these shortcomings, a glutathione-responsive 5-ALA derivative (SA) was designed based on the fact that many types of tumor cells have higher intracellular glutathione level than normal cells. SA was synthesized by masking the 5-amion group of 5-ALA methyl ester (5-ALA-OMe) with a self-immolative disulfide linker. Compared with 5-ALA and 5-ALA-OMe, SA exhibited higher stability under physiological conditions, and it can efficiently release the parent compound 5-ALA-OMe in response to glutathione. In tumor cells, SA displayed excellent protoporphyrin IX (PpIX) production activity at low concentrations while 5-ALA and 5-ALA-OMe were ineffective at the same concentration. The SA-induced PpIX production was positively correlated with the intracellular glutathione level, and SA exhibited enhanced phototoxicity due to its excellent PpIX generation activity. This study indicates that modification of the amino group in 5-ALA derivatives with a self-immolative disulfide linker is an effective strategy to improve their chemical stability and pharmacological activities, and SA is a potential photosensitizer for photodiagnosis and PDT of tumors.