Apoptosis and expression of cytokines triggered by pyropheophorbide-a methyl ester-mediated photodynamic therapy in nasopharyngeal carcinoma cells.

The photodynamic properties of pyropheophorbide-a methyl ester (MPPa), a semi-synthetic photosensitizer derived from chlorophyll a, were evaluated in a human nasopharyngeal carcinoma HONE-1 cell line. MPPa was non-toxic to the HONE-1. At the concentrations of 0.5-2µM, MPPa-mediated a drug dose-dependent photocytotoxicity in the HONE-1 cells. Confocal microscopy revealed a subcellular localization of MPPa in mitochondria and the Golgi apparatus. MPPa PDT-induced apoptosis was associated with the collapse of mitochondrial membrane potential, release of cytochrome c, the up-regulation of endoplasmic reticulum (ER) stress proteins (calnexin, Grp 94 and Grp78), and the activation of caspases-3 and -9. The photocytotoxicity was reduced by the corresponding specific caspase inhibitors. MPPa PDT-treated HONE-1 cells also up-regulated the gene expression of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α) and beta-chemokines (MIP-1ß, MPIF-1, and MPIF-2). These results suggest that the MPPa may be developed as a chlorophyll-based photosensitizer for the treatment of nasopharyngeal carcinoma.

Denervation exposes cryptic concanavalin A binding sites in skeletal muscle membranes.

Denervation produces significant changes in glycoproteins on the surfaces of skeletal muscles which can be detected as an increase in membrane-bound carbohydrate and an increase in specific binding of labelled lectins. The increased binding of concanavalin A to denervated membranes is detectable only in intact membranes and is abolished by treatments which disrupt or perturb membrane structure whereas the increased binding of Ricinus communis agglutinin is detectable under both conditions. We suggest that, in addition to chemical modification of existing carbohydrate chains in glycoproteins and the synthesis of new glycoproteins, denervation results in an altered geometric arrangement of existing membrane glycoproteins.

Glycosyltransferase activities in chicken brain synaptic junctions.

Isolated synaptic junction fractions from adult chicken brain were found to contain a number of glycoprotein glycosyltransferase with different donor and acceptor specificities. The activities of sialyl- and galactosyltransferases towards endogenous acceptors in the synaptic junction fraction were low but increased greatly after the addition of deglycosylated fetuin or mucin as exogenous acceptors. The activity of the fucosyltransferases towards endogenous acceptors in the fraction was much higher than that of the other transferases. The addition of deglycosylated fetuin caused a smaller increase in the activity than with the other transferases and deglycosylated mucin had no effect. We find no evidence for the enrichment of any glycosyltransferase in the synaptic junction. If anything, our results suggest that the synaptic junction may be specifically depleted in one type of fucosyltransferase.

Photodynamic therapy with pyropheophorbide-a methyl ester in human lung carcinoma cancer cell: efficacy, localization and apoptosis.

Pyropheophorbide-a methyl ester (MPPa) is a semisynthetic photosensitizer derived from chlorophyll a. The absorption peak of MPPa in organic solvent and in cells was at 667 and 674 nm, respectively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay showed that MPPa had no dark cytotoxicity. In vitro photodynamic activity was extensively evaluated using a human lung carcinoma cancer cell line (NCI-h446). MPPa exhibited no genotoxicity, as assayed by single-cell gel electrophoresis. Using confocal laser scanning microscopy and organelle-specific fluorescent probes, MPPa was found to localize in the intracellular membrane system, namely the endoplasmic reticulum, Golgi apparatus, lysosomes and mitochondria, in the NCI-h446 cells. Furthermore, nuclear staining and DNA gel electrophoresis revealed that DNA condensation and fragmentation occurred post-photodynamic therapy, indicating the cell death was in the apoptotic mode.

Photodynamic effects of mTHPC on human colon adenocarcinoma cells: photocytotoxicity, subcellular localization and apoptosis.

The photodynamic properties of meta-tetra(hydroxyphenyl)chlorin (mTHPC), a promising second-generation photosensitizer, were investigated using a human colon adenocarcinoma cell line (Colo 201 cells). The study on photocytotoxicity using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay showed that mTHPC was an effective photosensitizer on Colo 201 cells. The photocytotoxicity of mTHPC showed both drug and light dose-dependent characteristics. To reach LD50, namely, the dose at which 50% of the cells were killed, only 0.45+/-0.15 microg/mL of mTHPC and 3 J/cm2 of light dose were required. The presence of 10% fetal calf serum in culture medium significantly decreased the incorporation of mTHPC into cells and resulted in the reduction of photodynamic efficacy. Using confocal laser scanning microscopy, mTHPC was first shown to localize in lysosomes rather than in mitochondria. Furthermore, nuclear stainings demonstrated that photodynamic therapy with mTHPC induced apoptosis in Colo 201 cells.

Subcellular localization of merocyanine 540 (MC540) and induction of apoptosis in murine myeloid leukemia cells.

Subcellular localization of photosensitizers is thought to play a critical role in determining the mode of cell death after photodynamic treatment (PDT) of leukemia cells. Using confocal laser scanning microscopy and fluorescent organelle probes, we examined the subcellular localization of merocyanine 540 (MC540) in the murine myeloid leukemia M1 and WEHI 3B (JCS) cells. Two patterns of localization were observed: in JCS cells, MC540 was found to localize on the plasma membrane and mitochondria; and in M1 leukemia cells, MC540 was found to localize on lysosomes. The relationship between subcellular localization of MC540 and PDT-induced apoptosis was investigated. Apoptotic cell death, as judged by the formation of apoptotic nuclei, was observed 4 h after irradiation in both leukemia cell lines. Typical ladders of apoptotic DNA fragments were also detected by DNA gel electrophoresis in PDT-treated JCS and M1 cells. At the irradiation dose of 46 kJ/m2 (LD90 for JCS and LD86 for M1 cells), the percentage of apoptotic JCS and M1 cells was 78 and 38%, respectively. This study provided substantial evidence that MC540 localized differentially in the mitochondria, and the subsequent photodamage of the organelle played an important role in PDT-mediated apoptosis in myeloid leukemia cells.

The binding characteristics and intracellular localization of temoporfin (mTHPC) in myeloid leukemia cells: phototoxicity and mitochondrial damage .

The state of aggregation of the photosensitizer meso-tetrahydroxyphenylchlorin (mTHPC) in both cell free and intracellular environment was elucidated by comparing its absorption and excitation spectra. In methanol, mTHPC existed as monomers and strongly fluoresced. In aqueous solutions such as phosphate-buffered saline (PBS), mTHPC formed nonfluorescent aggregates. Some portion of mTHPC monomerized in the presence of 10% fetal calf serum PBS. In murine myeloid leukemia M1 and WEHI-3B (JCS) cells, cytoplasmic mTHPC were monomeric. By using organelle-specific fluorescent probes, it was found that mTHPC localized preferentially at the mitochondria and the perinuclear region. Photodynamic treatment of mTHPC-sensitized leukemia cells caused rapid appearance of the apoptogenic protein cytochrome c in the cytosol. Results from flow cytometric analysis showed that the release of cytochrome c was especially pronounced in JCS cells, and well correlated with the extent of apoptotic cell death as reported earlier. Electron microscopy revealed the loss of integrity of the mitochondrial membrane and the appearance of chromatin condensation as early as 1 h after light irradiation. We conclude that rapid release of cytochrome c from photodamaged mitochondria is responsible for the mTHPC-induced apoptosis in the myeloid leukemia JCS and M1 cells.

A comparison of the photodynamic effects of temoporfin (mTHPC) and MC540 on leukemia cells: efficacy and apoptosis.

The photodynamic effects of temoporfin (meso-tetrahydroxyphenylchlorin, mTHPC) and merocyanine 540 (MC540) in murine myeloid leukemia M1 and WEHI 3B (JCS) cells were compared. The mTHPC was found to be more potent and selective. At a lethal dosage of 90% killing (LD90), only 1.3 microM of mTHPC and 4.2 kJ/m2 of light irradiation was required, which was a 20-fold lower drug concentration and 11-fold smaller light dose than that required when using MC540. Meanwhile, three times less, or 15%, of the coincubated erythrocytes were destroyed by mTHPC than by MC540. Confocal micrographs showed that both drugs accumulated diffusely inside the cytoplasm in a very similar fashion, but mTHPC induced a more extensive apoptosis in photosensitized JCS cells. For example, at LD90, mTHPC practically killed all JCS cells via apoptosis and cleaved the DNA to extremely small 150 base-pair fragments. In contrast, among the JCS cells killed by MC540, about 88% died via apoptosis and large DNA fragments were abundant. Relative to MC540, the ability of mTHPC to trigger large-scale and thorough apoptosis in leukemia cells may help explain its potency and selectivity.

Modulatory effects of peroxovanadates on insulin receptor binding.

The insulin-mimetic effects exhibited by vanadate, hydrogen peroxide, and some peroxovanadates have recently been shown to occur, at least in part, through an activation of the insulin receptor tyrosine kinase activity. In this study, we examine the effects of these compounds on insulin receptor binding using receptor preparations from human placental membranes. Among the 16 vanadium(V)-peroxo complexes studied, the [VO(O2)2(bipy)]- ion, where bipy = 2,2'-bipyridine, was found to increase insulin receptor binding by 24%, whereas the [VO(O2)2(en)]- ion, where en = ethylenediamine, was found to reduce insulin receptor binding by about the same amount under steady-state conditions. Scatchard analysis of the binding data indicates that the observed effect of the [VO(O2)2(bipy)]- ion on insulin receptor binding is exerted mainly at the high-capacity low-affinity sites. Furthermore, this modulatory effect is reversible and requires a continuous presence of the compound. By perturbing the membrane environment of the insulin receptor, we have shown that an intact membrane structure is essential for an observable effect. The observed modulation of insulin receptor binding by peroxovanadates is interpreted in terms of a ternary complex model in which the peroxovanadate acts as an allosteric effector modulating the binding equilibrium between insulin and its receptor.

A study of the binding of merocyanine 540 to myeloid leukemia M1 cells using an intensified charge-coupled device for fluorescence imaging microscopy.

The binding of merocyanine 540 (MC540) to murine myeloid leukemia (M1) cells and normal erythrocytes was measured by fluorescence digital imaging microscopy using an intensified charge-coupled device. It was found that, on average, about three times more MC540 were bound to a unit membrane area of M1 cells than erythrocytes, a result consistent with previous studies. However, it was shown for the first time that MC540 binding varied significantly from one M1 cell to the next, and about 15% of the sensitized M1 cells were as MC540-negative as normal erythrocytes. Using the leukemic inhibitory factor as a differentiation inducer, M1 cells were induced to differentiate into mature macrophage-like cells in vitro. Such treatment lowered the average MC540 binding by about one-third but did not affect the cell-to-cell variation significantly.

The structural specificity of carbohydrate in the initiation of rat sperm motility.

Mammalian spermatozoa are stored in the cauda epididymis (CE) in a quiescent state and become motile when diluted with seminal plasma upon ejaculation. The structural specificity of a variety of sugars and sugar derivatives as diluents that are capable of initiating a transition of CE spermatozoa from the quiescent to the actively motile state was examined. It was found that monosaccharides, except those containing less than five-carbon skeletons, were good motility initiators; a trisaccharide tested showed reduced activity. The initiation activity was also independent of the structural stereospecificity and the nutritional value of the sugar. Based on these observations, a mechanism involving a receptor which handles sugar transport or sugar recognition in a transport process is proposed to be responsible for generating a signal that triggers CE spermatozoal motility.

Effect of denervation on sarcolemmal proteins and glycoproteins of fast and slow mammalian skeletal muscle.

We compared the protein and glycoprotein composition of a sarcolemmal membrane fraction isolated from normal and denervated rat extensor digitorum longus (EDL) and soleus muscles. Membranes from EDL and soleus muscles showed significantly different protein compositions. A relatively small number of glycoproteins, which were all minor proteins, accounted for the majority of concanavalin A (ConA) and Ricinus communis agglutinin (RCA120) binding. These glycoproteins appear to be common to EDL and soleus but bound different relative amounts of lectin in the two muscles. A large proportion of the ConA binding sites in EDL, but not soleus, were cryptic (not accessible by ConA unless the membrane structure was disrupted). Denervation had a differential effect on sarcolemma from the two muscles with EDL exhibiting large changes and soleus changing little if at all. Several major proteins changed their relative concentrations after denervation and the relative amount of RCA120 bound to the major glycoproteins also changed. The major ConA-binding glycoproteins did not change in either membrane but denervation resulted in the exposure of most of the cryptic ConA-binding sites in EDL membranes. Endogenous sialyl- and galactosyl-transferase activities in the membrane fractions significantly increased in EDL, but did not change in soleus, suggesting that the turnover of the glycoproteins is increased in EDL after denervation.

The effect of denervation on mammalian sarcolemmal proteins and glycoproteins.

The effects of surgical denervation on proteins and glycoproteins of sarcolemmal membranes have been investigated using sarcolemmal fractions prepared from mixed rat muscles. Denervation did not cause any gross change in the protein composition but some consistent quantitative changes were detected. Denervation caused a significant increase in the binding of 125I-labeled concanavalin A (ConA), Ricinus communis agglutinin (RCA120), and wheat germ agglutinin (WGA) to intact membranes. This increased binding appears to be brought about by two mechanisms: the synthesis of more binding-sites with the same apparent KD and the unmasking of previously cryptic binding sites. The activity of sialyl-, galactosyl-, and N-acetylglucosaminyltransferases in the sarcolemmal fractions increased, whereas fucosyl-glycoprotein-transferase activity decreased following denervation. Kinetic analysis of the sialyl- and galactosyltransferase activities showed that the change was due to an increase in Vmax with no change in Km. These results are consistent with an increase in the turnover of sarcolemmal glycoproteins following denervation.

The effect of tetrodotoxin on mammalian sarcolemmal proteins and glycoproteins.

This study evaluates the relative contributions of neural impulse activity and neurotrophic (non-impulse) factors by comparing the effects of denervation and the blockage of neural impulse activity on various biochemical parameters of mixed muscle sarcolemma. Muscle paralysis was induced by repeated injection of Tetrodotoxin (TTx) to the sciatic nerve. After seven days of inactivity the isolated sarcolemma was analyzed for protein and glycoprotein composition, Na+/K+ ATPase activity, Concanavalin A binding to intact sarcolemma and to carbohydrate components separated in SDS-polyacrylamide gels and sialyl and galactosyl transferase activity. The results have shown that following muscle inactivity all of the parameters except glycosyl transferases changed in a similar manner but to a lesser degree than denervation. It is concluded that trophic factors in addition to neural impulse activity play a role in the regulation of a number of surface membrane properties.

Three new saponins from Tribulus terrestris.

Three new steroidal saponins 1-3, together with five known steroidal saponins, L-mannitol and an inorganic salt were isolated from Tribulus terrestris L. (Zygophyllaceae). The structures of the new steroidal saponins were elucidated as hecogenin 3-O-beta-xylopyranosyl(1-->3)-beta-glucopyranosyl(1-->4)-beta-galactopyr anoside (1), hecogenin 3-O-beta-glucopyranosyl(1-->2)-beta-glucopyranosyl(1-->4)- beta-galactopyranoside (2) and 3-O-[beta-xylopyranosyl(1-->2)-[beta-xylopyranosyl(1-->3)]-beta- glucopyranosyl(1-->4)-[alpha-rhamnopyranosyl(1-->2)]-beta-galactopyranos yl]- 26-O-beta-glucopyranosyl-22-methoxy-(3 beta,5 alpha,25R)-furostan-3,26-diol (3). Structure elucidation was accomplished by 1D and 2D NMR spectra (13C-1H COSY, HMQC, HMBC, 1H-1H COSY, TOCSY, and NOESY), mass spectrometry (FABMS, ESIMS) and chemical methods.