In vitro evaluation of the efficacy of photodynamic therapy using 5-ALA on homologous feline mammary tumors in 2D and 3D culture conditions and a mouse subcutaneous model with 3D cultured cells.

BACKGROUND: Numerous studies have shown that photodynamic therapy (PDT) has a therapeutic effect on mammary tumor cells, with 5-aminolevulinic acid (5-ALA-HCL) being a commonly used photosensitizer for PDT. Feline mammary tumors (FMTs) are relatively common. However, the cytotoxic and antitumor effects of 5-ALA-PDT on FMTs have not been clarified. To this end, we evaluated the therapeutic effect of 5-ALA-PDT on FMTs through in vitro experiments using an FMT FKR cell line established for this study. METHODS: We performed 5-ALA-PDT in 2D-cultured FKR-A (adherent cells) and 3D-cultured FKR-S (spheroid cells) cells and performed a series of studies to evaluate the cell viability and determine the protoporphyrin IX (PpIX) content in the cells as well as the expression levels of mRNAs associated with PpIX production and release. An in vivo study was performed to assess the effectiveness of 5-ALA-PDT. RESULTS: There was a significant difference in the concentration of PpIX in FMT cells under different incubation culture modes (2D versus 3D culture). The concentration of PpIX in FMT cells was correlated with the differences in cell culture (2D and 3D) as well as the expression levels of genes such as PEPT1, PEPT2, FECH, and HO-1. CONCLUSIONS: In the in vitro study, 5-ALA-PDT had a stronger inhibitory effect on 3D-cultured FKR-S cells, which resemble the internal environment of organisms more closely. We also observed a significant inhibitory effect of 5-ALA-PDT on FMT cells in vivo. To our knowledge, this is the first study on 5-ALA-PDT for FMTs under both 2D and 3D conditions.

Suppression of angiotensin converting enzyme 2, a host receptor for SARS-CoV-2 infection, using 5-aminolevulinic acid in vitro.

Angiotensin converting enzyme 2 (ACE2), an entry receptor found on the surface of host cells, is believed to be detrimental to the infectious capability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Scientists have been working on finding a cure since its outbreak with limited success. In this study, we evaluated the potential of 5-aminolevulinic acid hydrochloride (ALA) in suppressing ACE2 expression of host cells. ACE2 expression and the production of intracellular porphyrins following ALA administration were carried out. We observed the reduction of ACE2 expression and intracellular porphyrins following ALA administration. ALA suppressed the ACE2 expression in host cells which might prevent binding of SARS-CoV-2 to host cells. Co-administration of ALA and sodium ferrous citrate (SFC) resulted in a further decrease in ACE2 expression and increase in intracellular heme level. This suggests that the suppression of ACE2 expression by ALA might occur through heme production. We found that the inhibition of heme oxygenase-1 (HO-1), which is involved in heme degradation, also resulted in decrease in ACE2 expression, suggesting a potential role of HO-1 in suppressing ACE2 as well. In conclusion, we speculate that ALA, together with SFC administration, might serve as a potential therapeutic approach in reducing SARS-CoV-2 infectivity through suppression of ACE2 expression.

Mechanism of Differential Susceptibility of Two (Canine Lung Adenocarcinoma) Cell Lines to 5-Aminolevulinic Acid-Mediated Photodynamic Therapy.

Photodynamic therapy (PDT) is a clinically approved, minimally invasive treatment for malignant tumors. Protoporphyrin IX (PpIX), derived from 5-aminolevulinic acid (5-ALA) as the prodrug, is one of the photosensitizers used in PDT. Recently, we reported a significant difference in response to 5-ALA-mediated PDT treatment in two canine primary lung adenocarcinoma cell lines (sensitive to PDT: HDC cells, resistant to PDT: LuBi cells). This study aimed to examine the difference in cytotoxicity of 5-ALA-mediated PDT in these cells. Although intracellular PpIX levels before irradiation were similar between HDC and LuBi cells, the percentage of ROS-positive cells and apoptotic cells in LuBi cells treated with 5-ALA-mediated PDT was significantly lower than that in HDC cells treated with 5-ALA-mediated PDT. A high dosage of the NO donor, DETA NONOate, significantly increased the cytotoxicity of 5-ALA-mediated PDT against LuBi cells. These results suggest that the sensitivity of 5-ALA-mediated PDT might be correlated with NO.

Efficacy of 5-Aminolevulinic Acid in Photodynamic Detection and Photodynamic Therapy in Veterinary Medicine.

5-Aminolevulinic acid (5-ALA), a commonly used photosensitizer in photodynamic detection (PDD) and therapy (PDT), is converted in situ to the established photosensitizer protoporphyrin IX (PpIX) via the heme biosynthetic pathway. To extend 5-ALA-PDT application, we evaluated the PpIX fluorescence induced by exogenous 5-ALA in various veterinary tumors and treated canine and feline tumors. 5-ALA-PDD sensitivity and specificity in the whole sample group for dogs and cats combined were 89.5 and 50%, respectively. Notably, some small tumors disappeared upon 5-ALA-PDT. Although single PDT application was not curative, repeated PDT+/-chemotherapy achieved long-term tumor control. We analyzed the relationship between intracellular PpIX concentration and 5-ALA-PDT in vitro cytotoxicity using various primary tumor cells and determined the correlation between intracellular PpIX concentration and 5-ALA transporter and metabolic enzyme mRNA expression levels. 5-ALA-PDT cytotoxicity in vitro correlated with intracellular PpIX concentration in carcinomas. Ferrochelatase mRNA expression levels strongly negatively correlated with PpIX accumulation, representing the first report of a correlation between mRNA expression related to PpIX accumulation and PpIX concentration in canine tumor cells. Our findings suggested that the results of 5-ALA-PDD might be predictive for 5-ALA-PDT therapeutic effects for carcinomas, with 5-ALA-PDT plus chemotherapy potentially increasing the probability of tumor control in veterinary medicine.

Identification of two teleost homologs of the Drosophila sex determination factor, transformer-2 in medaka (Oryzias latipes).

Transformer-2 (Tra2), an RNA-binding protein, is an important regulator in Drosophila sex determination. In vertebrates, however, the role of Tra2 homologues is not known. We identified two teleost homologues of Tra2, which we named Tra2a and Tra2b, in medaka (Oryzias latipes). Furthermore, we demonstrated that both Tra2 mRNAs were predominantly expressed in germ cells of both sexes before the onset of sex differentiation, suggesting that both Tra2 homologues might be involved in the sex differentiation in medaka.

Controlled Growth and the Maintenance of Human Pluripotent Stem Cells by Cultivation with Defined Medium on Extracellular Matrix-Coated Micropatterned Dishes.

Here, we introduce a new serum-free defined medium (SPM) that supports the cultivation of human pluripotent stem cells (hPSCs) on recombinant human vitronectin-N (rhVNT-N)-coated dishes after seeding with either cell clumps or single cells. With this system, there was no need for an intervening sequential adaptation process after moving hPSCs from feeder layer-dependent conditions. We also introduce a micropatterned dish that was coated with extracellular matrix by photolithographic technology. This procedure allowed the cultivation of hPSCs on 199 individual rhVNT-N-coated small round spots (1 mm in diameter) on each 35-mm polystyrene dish (termed "patterned culture"), permitting the simultaneous formation of 199 uniform high-density small-sized colonies. This culture system supported controlled cell growth and maintenance of undifferentiated hPSCs better than dishes in which the entire surface was coated with rhVNT-N (termed "non-patterned cultures"). Non-patterned cultures produced variable, unrestricted cell proliferation with non-uniform cell growth and uneven densities in which we observed downregulated expression of some self-renewal-related markers. Comparative flow cytometric studies of the expression of pluripotency-related molecules SSEA-3 and TRA-1-60 in hPSCs from non-patterned cultures and patterned cultures supported this concept. Patterned cultures of hPSCs allowed sequential visual inspection of every hPSC colony, giving an address and number in patterned culture dishes. Several spots could be sampled for quality control tests of production batches, thereby permitting the monitoring of hPSCs in a single culture dish. Our new patterned culture system utilizing photolithography provides a robust, reproducible and controllable cell culture system and demonstrates technological advantages for the mass production of hPSCs with process quality control.