Methadone enhances the effectiveness of 5‐aminolevulinic acid‐based photodynamic therapy for squamous cell carcinoma and glioblastoma in vitro

Although having shown promising clinical outcomes, the effectiveness of 5‐aminolevulinic acid‐based photodynamic therapy (ALA‐PDT) for squamous cell carcinoma (SCC) and glioblastoma remains to be improved. The analgesic drug methadone is able to sensitize various tumors to chemotherapy. In this in vitro study, the influence of methadone to the effectiveness of ALA‐PDT for SCC (FADU) and glioblastoma (A172) was investigated on the protoporphyrin IX (PpIX) fluorescence, survival rates, apoptosis, and cell cycle phase, each with or without the presence of methadone. The production of PpIX was increased by methadone in FADU cells while it was decreased in A172 cells. The survival rates of both cell lines treated by ALA‐PDT were significantly reduced by the combination with methadone (P < .05). Methadone also significantly increased the percentage of apoptotic cells and improved the effect of ALA‐PDT on the cell cycle phase arrest in the G0/G1 phase (P < .05). This study demonstrates the potential of methadone to influence the cytotoxic effect of ALA‐PDT for both SCC and glioblastoma cell lines.      

Factors implicated in the assessment of aminolevulinic acid-induced protoporphyrin IX fluorescence

Background

Photodynamic therapy and photodiagnosis of cancer requires preferential accumulation of fluorescent photosensitizers in tumors. Clinical evidence documents feasibility of ALA-based photodiagnosis for tumor detection. However, false positive results and large variations in fluorescence intensities are also reported. Furthermore, selective accumulation of fluorescent species of photosensitizers in tumor cell lines, as compared to normal ones, when cultured in vitro, is not always observed. To understand this discrepancy we analyzed the impact of various factors on the intensity of detected PpIX fluorescence.

Methods

Impacts of cell type, mitochondrial potential, cell–cell interactions and relocalization of PpIX among different cell types in co-cultures of different cell lines were analyzed by confocal microscopy and flow cytometry. Fluorescence spectroscopy was used to estimate absolute amounts of ALA-induced PpIX in individual cell lines. Immunofluorescence staining was applied to evaluate the ability of cell lines to produce collagen.

Results

Higher ALA-induced PpIX fluorescence in cancer cell lines as compared to normal ones was not detected by all the methods used. Mitochondrial activity was heterogeneous throughout the cell monolayers and could not be clearly correlated with PpIX fluorescence. Positive collagen staining was detected in all cell lines tested.

Conclusions

Contrary to in vivo situation, ALA-induced PpIX production by cell lines in vitro may not result in higher PpIX fluorescence signals in tumor cells than in normal ones. We suggest that a combination of several properties of tumor tissue, instead of tumor cells only, is responsible for increased ALA-induced PpIX fluorescence in solid tumors.

General significance

Understanding the reasons of increased ALA-induced PpIX fluorescence in tumors is necessary for reliable ALA-based photodiagnosis, which is used in various oncological fields.     

A novel protein,sperm head and tail associated protein (SHTAP), interacts with cysteine‐rich secretory protein 2 (CRISP2) during spermatogenesis in the mouse

Background information. CRISP2 (cysteine‐rich secretory protein 2) is a sperm acrosome and tail protein with the ability to regulate Ca²⁺ flow through ryanodine receptors. Based on these properties, CRISP2 has a potential role in fertilization through the regulation of ion signalling in the acrosome reaction and sperm motility. The purpose of the present study was to determine the expression, subcellular localization and the role in spermatogenesis of a novel CRISP2‐binding partner, which we have designated SHTAP (sperm head and tail associated protein). Results. Using yeast two‐hybrid screens of an adult testis expression library, we identified SHTAP as a novel mouse CRISP2‐binding partner. Sequence analysis of all Shtap cDNA clones revealed that the mouse Shtap gene is embedded within a gene encoding the unrelated protein NSUN4 (NOL1/NOP2/Sun domain family member 4). Five orthologues of the Shtap gene have been annotated in public databases. SHTAP and its orthologues showed no significant sequence similarity to any known protein or functional motifs, including NSUN4. Using an SHTAP antiserum, multiple SHTAP isoforms (～20–87 kDa) were detected in the testis, sperm, and various somatic tissues. Interestingly, only the ～26 kDa isoform of SHTAP was able to interact with CRISP2. Furthermore, yeast two‐hybrid assays showed that both the CAP (CRISP/antigen 5/pathogenesis related‐1) and CRISP domains of CRISP2 were required for maximal binding to SHTAP. SHTAP protein was localized to the peri‐acrosomal region of round spermatids, and the head and tail of the elongated spermatids and sperm tail where it co‐localized with CRISP2. During sperm capacitation, SHTAP and the SHTAP—CRISP2 complex appeared to be redistributed within the head. Conclusions. The present study is the first report of the identification, annotation and expression analysis of the mouse Shtap gene. The redistribution observed during sperm capacitation raises the possibility that SHTAP and the SHTAP—CRISP2 complex play a role in the attainment of sperm functional competence.     

MicroRNA‐146b‐5p overexpression attenuates premature ovarian failure in mice by inhibiting the Dab2ip/Ask1/p38‐Mapk pathway and γH2A.X phosphorylation

ObjectiveTo examine the role of high‐fat and high‐sugar (HFHS) diet‐induced oxidative stress, which is a risk factor for various diseases, in premature ovarian failure (POF).Materials and methodsOvarian granulosa cells (OGCs) were isolated from mice and cultured in medium supplemented with HFHS and poly (lactic‐co‐glycolic acid) (PLGA)‐cross‐linked miR‐146b‐5p nanoparticles (miR‐146@PLGA). RNA and protein expression levels were examined using quantitative real‐time polymerase chain reaction and Western blotting, respectively. HFHS diet‐induced POF model mice were administered miR‐146@PLGA.ResultsThe ovarian tissue of mice fed a HFHS diet exhibited the typical pathological characteristics of POF. HFHS supplementation induced oxidative stress injury in the mouse OGCs, activation of the Dab2ip/Ask1/p38‐Mapk signalling pathway and phosphorylation of γH2A.X in vitro and in vivo. The results of the luciferase reporter assay revealed that miR‐146 specifically downregulated p38‐Mapk14 expression. Meanwhile, co‐immunoprecipitation and Western blot analyses revealed that HFHS supplementation upregulated nuclear p38‐Mapk14 expression and consequently enhanced γH2A.X (Ser139) phosphorylation. The HFHS diet‐induced POF mouse model treated with miR‐146@PLGA exhibited downregulated p38‐Mapk14 expression in the OGCs, mitigated OGC ageing and alleviated the symptoms of POF.ConclusionsThis study demonstrated that HFHS supplementation activates the Dab2ip/Ask1/p38‐Mapk signalling pathway and promotes γH2A.X phosphorylation by inhibiting the expression of endogenous miR‐146b‐5p, which results in OGC ageing and POF development.     

Wnt5a regulates the cell proliferation and adipogenesis via MAPK‐independent pathway in early stage of obesity

The early stage of obesity is an important stage in the development of obesity. However, there are few studies which explored the property or changes in obesity at early stage especially involving Wnt5a. The associated gene expression of Wnt5a on cell regeneration and the effect of Wnt5a on rat adipose‐derived stem cell (rASC) proliferation and adipogenesis need additional study. Here, we investigated the changes in obesity at early stage and how Wnt5a regulates rASC regeneration, proliferation, and adipogenesis. Our data revealed that obesity at early stage measured by Lee index presented a state with impaired adipogenesis and more infiltrated inflammatory cells but without significant changes in adipocyte sizes and inflammatory factors. The process might be associated with anti‐canonical Wnt pathway and a reciprocal Wnt5a/JNK pathway. Besides the gene expression of Wnt5a decreased from cell passage 1 to passage 3. The cell proliferation was regulated by increasing dose of Wnt5a with the maximal effect at 50 ng/mL and 50 ng/mL Wnt5a suppressed adipogenic differentiation at middle‐late stage of adipogenesis via anti‐β‐catenin and a mitogen‐activated protein kinase (MAPK) signaling‐independent manner. Accordingly, the research helps to gain further insights into the early stage of obesity and its associated changes on a cellular and molecular level.     

Definition and Evaluation of the Spatio‐Temporal Variations in Chlorophyll Fluorescence during the Phases of CAM and during Endogenous Rhythms in Continuous Light,in Thick Leaves of Kalanchoë daigremontiana5

Abstract: We used chlorophyll fluorescence imaging to examine the homogeneity of photosynthetic metabolism during CAM in the thick leaves of Kalanchoë daigremontiana Hamet et Perrier de la Bǎthie. Intense, persistent fluorescence from a DCMU treated thin leaf of Clematis sp placed beneath a K. daigremontiana leaf was readily detected through the thick leaf. Evidently reabsorption of fluorescence was qualitatively unimportant in the system used. Chlorophyll fluorescence images from 7 mm tissue discs excised from Kalanchoë leaves were collected at 60 s intervals during 20 min transients elicited by red excitation light. Information about patchiness and subsurface processes was gained by statistical factor analysis and Fourier transform. Although small, highly resolved rings of bright chlorophyll fluorescence surrounding discs of low fluorescence were observed from cells near the surface, no independent regional temporal variation in fluorescence was evident in the surface‐biased images. Temporally independent chlorophyll fluorescence was present in images biased towards sub‐epidermal sources, in most phases of CAM, and during endogenous rhythm. These asynchronous changes were several millimetres apart. This patchy fluorescence was confirmed when attached leaves were excited with blue light in a leaf chamber while CO₂ and H₂O exchange was monitored. Large spatio‐temporal variations in the efficiency of photosystem II were always observed during phases II and IV of CAM, when both CO₂ fixation cycles are active, and during the maximum rate of CO₂ fixation during the endogenous rhythm in continuous light. These data are discussed in terms of metabolic isolation in the thick but uniform tissues in which gas diffusion may be largely confined to wet cell walls, thereby rendering the tissue functionally heterobaric. Prolonged, but in some instances, reversible alterations in PSII efficiency could be produced by injection of metabolic inhibitors, confirming that patchy fluorescence may reflect the differing energy costs of photosynthesis in different CAM phases.