Hypericin phototoxicity induces different modes of cell death in melanoma and human skin cells

Hypericin, the major component of St. John's Wort, absorbs light in the UV and visible ranges whereupon it becomes phototoxic through the production of reactive oxygen species. Although photodynamic mechanisms (i.e. through endogenous photosensitizers) play a role in UVA phototherapy for the treatment of skin disorders such as eczema and psoriasis, photodynamic therapy employing exogenous photosensitizers are currently being used only for the treatment of certain forms of non-melanoma skin cancers and actinic keratoses. There are few reports however on its use in treating melanomas. This in vitro study analyses the phototoxic effect of UVA (400-315 nm) - activated hypericin in human pigmented and unpigmented melanomas and immortalised keratinocytes and melanocytes. We show that neither hypericin exposure nor UV irradiation alone reduces cell viability. We show that an exposure to 1 microM UVA-activated hypericin does not bring about cell death, while 3 microM activated hypericin induces a necrotic mode of cell death in pigmented melanoma cells and melanocytes and an apoptotic mode of cell death in non-pigmented melanoma cells and keratinocytes. We hypothesis that the necrotic mode of cell death in the pigmented cells is possibly related to the presence of melanin-containing melanosomes in these cells and that the hypericin-induced increase in reactive oxygen species leads to an increase in permeability of melanosomes. This would result in toxic melanin precursors (of an indolic and phenolic nature) leaking into the cytoplasm which in turn leads to cell death. Hypericin localisation in the endoplasmic reticulum in these cells shown by fluorescent microscopy, further support a disruption in cellular processing and induction of cell death. In contrast, this study shows that cells that do not contain melanosomes (non-pigmented melanoma cells and keratinocytes) die by apoptosis. Further, using a mitochondrial-specific fluorescent dye, we show that intracellular accumulation of hypericin induces a mitochondrial-associated caspase-dependent apoptotic mode of cell death. This work suggests that UVA is effective in activating hypericin and that this phototoxicity may be considered as treatment option in some cases of lentigo maligna or lentigo maligna melanoma that are too large for surgical resection.     

Proliferation, viability, and metabolism of human tumor and normal cells cultured in microcapsule

In this study, we investigated the effect of the microenvironment provided by alginate-poly-l-lysine-alginate (APA) microcapsule with liquefied or gelled core on the proliferation, viability, and metabolism of human cells, including anchorage-dependent MCF-7 breast cancer cells and primary fibroblasts, and anchorage-independent K-562 leukemia cells; cells in conventional culture were used as control. The growth pattern of cells in microcapsule was examined by phase-contrast micrography. The cell viability, proliferation, organization, and gene expression were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, hematoxylin and eosin staining, live/dead staining, 5-bromo-20-deoxyuridine labeling, and immunohistochemistry, respectively. Cell metabolism was determined by measuring glucose and lactate concentrations in medium. The results demonstrate that APA microcapsule with liquefied core provides a microenvironment for both anchorage-dependent and anchorage-independent cells to grow into a large cell aggregate and maintain cell viability at a constant level for a period of time. In conclusion, cells in APA microcapsule are alive and have proliferation potential with lower metabolism rate. APA microcapsule may be a useful tool for in vitro tumor cell modeling and anticancer drug screening as well as for cancer gene therapy. In addition, it lays a solid foundation for the use of microencapsulation in cell culture in vitro and cell implantation in vivo.     

微流控芯片系统在循环肿瘤细胞分离检测中的应用进展

循环肿瘤细胞(CTCs)的分离分析一直是肿瘤相关研究中的热点方向,作为液体活检的重要标志物之一,其在外周血中的含量与癌症病发状况密切相关.然而人体血液中CTCs的含量非常低,通常来说仅有0～10个/mL,因此在开展临床血液样本中CTCs的检测前,往往需要对样本进行前处理,以实现CTCs的分离和富集.微流控芯片技术凭借样...     

Spectral analysis of laser-induced ultraweak delayed luminescence in cultured normal and tumor human cells: temperature dependence

The emission spectrum of ultraviolet A laser induced ultraweak delayed luminescence in cell cultures of mammalian cells depended on the temperature during irradiation and photonic measurements. A new method using a sophisticated photomultiplier system was developed in order to find differences between normal and tumor cells. The maximal peak of the emitted light for cultures measured at low temperature of 10 degrees C was near 510 nm in the green visible region while following irradiation at 32 degrees C this maximum was shifted to yellow-orange at 570 nm both in normal and melanoma cells. Overall, this ultraweak photonic model of cultured cells provides to be a new and powerful non-invasive tool for developing new strategies in skin cancer detection.     

液相色谱-质谱法比较分析正常人与胃癌患者的胃组织蛋白质

通过蛋白质组学技术筛选胃癌相关标志物是目前胃癌研究的热点,也是早期诊断的关键。针对组织蛋白质提取物非常复杂的特点,并根据疏水性的差异,采用反相液相色谱对正常及癌症组织提取蛋白质进行分离。通过比较正常及癌症组织提取蛋白质的谱图差异,收集并酶解差异最大的保留时间为45～47 min的馏分,采用液相色谱-多级质谱联用(LC-MS/MS)鉴定其酶解产物。鉴定结果显示,正常及癌症组织中的共有蛋白质为9个,正常组织中有6个特异蛋白质,而癌症组织中有17个特异蛋白质。通过进一步分析,筛选出胃癌组织中含有的丰度较高的两个蛋白质。应用生物信息学方法分析这些蛋白质,能为将来的药物靶点、药物作用通路研究提供更多的信息。     

Fluorescence formation during photodynamic therapy in the nucleus of cells incubated with cationic and anionic water-soluble photosensitizers.

The variations of fluorescence during light exposure of the cationic sensitizers methylene blue (MB) and meso-tetra(4N-methylpyridyl)porphyrin (T4MPyP) as well as the anionic meso-tetra(4-sulphonatophenyl)porphyrin (TPPS4) were measured at different intracellular sites using video-intensified microscopy in combination with microspectrofluorometry. Before light exposure the sensitizers were localized in distinct parts of the cytoplasm, especially in fluorescent organelles. During irradiation a drastic fluorescence formation and increase in the cytoplasm and nucleus, which was most pronounced in the nucleoli, could be observed for the cationic sensitizers as well as TPPS4. In the case of MB the increase in fluorescence was concomitant with a spectral shift in the emission spectra. For TPPS4 and T4MPyP the formation of a second species with a Soret band shifted towards longer wavelengths was observed and correlated with the fluorescence increase in the nucleoli. Cell deformations also took place.     

Capillary magnetophoresis of human blood cells and their magnetophoretic trapping in a flow system

The performance of a capillary magnetophoretic device was improved by enhancing the magnetic field gradient using a pair of small iron tips attached to the Nd-Fe-B magnets. The magnetophoretic intensity, B(dB/dx), was determined as a function of distance along the gap between the tips from the magnetophoretic velocity of a 3 microm polystyrene microparticle in 0.6 M manganese(II) chloride solution. The maximum intensity was increased 4.5 times by the attached iron pieces. The magnetophoresis of a single human blood cell in 0.1 M manganese(II) solution was studied by this method and its magnetic susceptibility was estimated. Magnetophoretic trapping of red blood cells was demonstrated under counter-current flow conditions in the capillary.     

Synthesis and cytotoxic activity against human tumor cells of heterocyclic systems derived from 2-thioxo-1,2-dihydro-4H-3,1-benzothazin-4-one

The title compound was prepared and converted to 2-hydrazinyl-1,2-dihydro-4H-benzo[d][1,3]thiazin-4-one which was utilized to synthesize fused heterocyclic systems, namely benzotriazolothiazinone derivatives, as well as, nonfused heterocyclic systems such as pyrazolyl-benzothiazinones, benzothiazinylpyridazine and imidazolylbenzothiazinone derivatives via reaction with formamide, acetic acid, ethyl cyanoacetate, maleic anhydride and benzaldehyde followed by treatment with glycine, respectively. All compounds have been structurally characterized by means of IR, MS, and ¹H-NMR spectra. The synthesized compounds were evaluated in vitro for their antiproliferative activity against HePG-2 and MCF-7 cell lines. 2H-Benzo[d][1,3]thiazine-2,4(1H)-dithione and 2-thioxo-1,2-dihydro-4H-benzo[d][1,3]thiazin-4-one were the most potent against the two cancer cells compared to that of the reference compound doxorubicin. Most of the synthesized compounds also exhibited good cytotoxic activity.     

PMA-induced up-regulation of MMP-9 is regulated by a PKCalpha-NF-kappaB cascade in human lung epithelial cells

Expression of matrix metalloproteinase-9 (MMP-9) is associated with airway remodeling and tissue injury in asthma. However, little is known about how MMP-9 is up-regulated in airway epithelial cells. In this study, we show that phorbol myristate acetate (PMA) induces MMP-9 expression via a protein kinase Calpha (PKCalpha)-dependent signaling cascade in BEAS-2B human lung epithelial cells. Pretreatment with either GF109203X, a general PKC inhibitor, or Go6976, a PKCalpha/beta isozyme inhibitor, inhibited PMA-induced activation of the MMP-9 promoter, as did transient transfection with PKCalpha antisense oligonuclotides. PMA activated NF-kappaB by phosphorylating IkappaB in these cells and this was also inhibited by GF109203X and Go6976, suggesting that PKCa acts as an upstream regulator of NF-kappaB in PMA-induced MMP-9 induction. Our results indicate that a "PKCalpha-NF- kappaB"-dependent cascade is involved in the signaling leading to PMA-induced MMP-9 expression in the lung epithelium.     

Metabolism of selenite in human lung cancer cells: X-ray absorption and fluorescence studies

Selenite is an inorganic form of selenium that has a cytotoxic effect against several human cancer cell lines: one or more selenite metabolites are considered to be responsible for its toxicity. X-ray absorption spectroscopy was used to monitor Se speciation in A549 human lung cancer cells incubated with selenite over 72 h. As anticipated, selenodiglutathione and elemental Se both comprised a large proportion of Se in the cells between 4 and 72 h after treatment, which is in accordance with the reductive metabolism of selenite in the presence of glutathione and glutathione reductase/NADPH system. Selenocystine was also present in the cells but was only detected as a significant component between 24 and 48 h concomitant with a decrease in the proportion of selenocysteine and the viability of the cells. The change in speciation from the selenol, selenocysteine, to the diselenide, selenocystine, is indicative of a change in the redox status of the cells to a more oxidizing environment, likely brought about by metabolites of selenite. X-ray fluorescence microscopy of single cells treated with selenite for 24 h revealed a punctate distribution of Se in the cytoplasm. The accumulation of Se was associated with a greater than 2-fold increase in Cu, which was colocalized with Se. Selenium K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy revealed Se-Se and Se-S bonding, but not Se-Cu bonding, despite the spatial association of Se and Cu. Microprobe X-ray absorption near-edge structure spectroscopy (μ-XANES) showed that the highly localized Se species was mostly elemental Se.     

A comparative study of the cellular uptake, localization and phototoxicity of meta-tetra(hydroxyphenyl) chlorin encapsulated in surface-modified submicronic oil/water carriers in HT29 tumor cells

The poor selectivity of photosensitizers for tumor tissue remains a drawback in photodynamic therapy (PDT) and could be improved by adapted formulations. The cellular uptake, localization and phototoxicity of meta-tetra(hydroxyphenyl)chlorin (mTHPC) encapsulated in submicronic colloidal carriers have been studied in macrophage-like J774 cells and HT 29 human adenocarcinoma cells. Nanocapsules with an external layer made of poly(D,L lactic acid) (PLA NCs), PLA grafted with polyethylene glycol (PLA-PEG NCs), PLA coated with poloxamer 188 (polox PLA NCs) and oil/water nanoemulsion (NE) have been examined. The cellular uptake by J774, as determined by microspectroflorimetry, is reduced with mTHPC encapsulated into surface-modified NCs--PLA-PEG and polox PLA--compared with naked PLA, indicating a possible limitation of the clearance of such carriers by the reticuloendothelial system. Encapsulation also modifies the interaction between mTHPC and HT29 cells. Compared with the manufacturer's solution (PEG, ethanol, water), the cellular uptake is strongly reduced. However, the HT29 phototoxicity is much less affected and a protecting effect against plasma proteins is observed. Fluorescence microscopy reveals a specific punctate fluorescence pattern with PLA-PEG and polox PLA NCs in contrast to a more diffuse distribution with NE and solution, indicating that photodamage targeting could be different. These findings suggest that photosensitizers encapsulated into surface-modified nanocapsules could be a promising approach for improving PDT efficacy and this has to be confirmed in vivo.     

Ways to enhance chemosensitizing of tumor cells with NO donors in tumor therapy with cytostatics and hydroxamic acids

Russian Chemical Bulletin - The influence of an organic NO donor belonging to non-steroidal anti-inflammatory drugs (NSAIDs) — NO-indomethacin (NO-Ind) on the antitumor effect of...     

Establishment of a microplate-formatted cell-based immunoassay for rapid analysis of nucleotide excision repair ability in human primary cells

DNA photolesions induced by UV, cyclobutane pyrimidine dimer (CPD) and (6-4) photoproduct (6-4PP), are repaired by nucleotide excision repair (NER) in human cells. Various immunoassays using monoclonal antibodies specific for the photolesions have been developed and widely used for the analysis of cellular NER activity. In this study, we have newly developed a microplate-formatted cell-based immunoassay, based on indirect immunofluorescence staining with lesion-specific antibodies combined with an infrared imaging system. Using this assay, we show the repair kinetics of CPD and 6-4PP in various fibroblasts from newborn and adult donors with no age-related difference. Furthermore, epidermal keratinocytes and melanocytes exhibit comparable NER activity, and calcium ion-induced differentiation of keratinocytes has no significant impacts on their NER activity. We also evaluated the effects of a proteasome inhibitor, MG132, and a histone deacetylase inhibitor, sodium butyrate, on NER efficiency using this assay. All these results suggest that the new assay is highly useful for the rapid and quantitative analysis of NER activity in various primary cells with limited growth activity and is applicable to a screening system for drugs affecting NER efficiency.     

211At labeling of a monoclonal antibody and its Fab fragment: Cytotoxicity on human gastric cancer cells and biodistribution in nude mice with tumor xenografts

An antigastric cancer monoclonal antibody, 3H11 and its Fab fragment, were labeled with #-emitter ²¹¹At using p-[²¹¹At] astatobenzoic acid (PAtBA) intermediate. The astatinated antibodies had conspicuous cytotoxic effect on human gastric cancer cell M85 in vitro. Tissue distribution of the astatinated antibodies were investigated in nude mice with subcutaneous tumor xenografts by i.v. injection. The astatinated Fab fragment was better suitable for 7.2-hour half life of ²¹¹At, since its tumor uptake remained higher (9.48–8.42 I.D%/g) than the astatinated intact antibody (~4.0 I.D%/g) from 3 to 14-hour post injection. However, the undesired high ²¹¹At uptake of the astatinated antibodies in some normal tissues, such as stomach, kidney and lung, suggested that the ²¹¹At labeled antibodies should be further explored.     

Gas chromatographic system equipped with a mass detector and a selective nitrogen-phosphorous detector operating simulataneously in the analysis of pesticide residues

The possibility to perform gas chromatographic analysis using both a mass detector and a selective nitrogen-phosphorous detector will enable highly sensitive analytical results and an optimal identification reliability. In this work we describe an easily workable technical solution, which has been accomplished in our laboratory and the application of the above system to analytical procedures for the detection and determination of nitrogenous herbicides in drinking water and phosphorylated pesticides in agricultural products is discussed.     

UVB irradiation of normal human skin favors the development of type-2 T-cells in vivo and in primary dermal cell cultures

To determine the effect of UVB exposure on the balance of type-1 or type-2 T-cells in skin, we examined the expression of key markers interferon (IFN)-gamma and interleukin (IL)-4 in cryostat sections. IFN-gamma mRNA was clearly detectable in nonirradiated control skin, and IFN-gamma protein was found in 2% of the dermal CD3pos T-cells, whereas IL-4 mRNA was hardly detectable, and no IL-4 protein was found. In contrast, IL-4 mRNA expression increased upon irradiation, and IL-4 was found in 2% of the T-cells at day 2 after UVB-exposure. Concomitantly, IFN-gamma mRNA expression decreased, and IFN-gamma protein became absent. We also analyzed T-cells present in primary dermal cell cultures, which were used as an in vitro equivalent of the in vivo situation. As compared with T-cells from control skin, T-cells in dermal cell cultures from UVB-exposed skin displayed an increased IL-4 and decreased IFN-gamma expression. No such skewing occurred when the T-cells from irradiated skin were cloned in the absence of a dermal microenvironment. Except for an occasional positive T-cell, type-1-associated cell-surface markers (CCR5, CXCR3) or type-2 markers (CCR3, CD30, CRTH2) were undetectable in situ. But these markers were expressed on cultured dermal T-cells from UVB-exposed and control skin at a comparable level, but did not correlate with the IFN-gamma and IL-4 production. Altogether, UVB-induced changes of the dermal microenvironment favor the development of type-2 T-cells.     

Epithelial‐to‐mesenchymal transition of human lung alveolar epithelial cells in a microfluidic gradient device

Epithelial‐to‐mesenchymal transition (EMT), a process in which epithelial cells undergo phenotypic transitions to fibrotic cells, is induced by stimulants including transforming growth factor‐beta1 (TGF‐β1). In the present study, we developed a microfluidic gradient device to reproduce EMT in A549 human lung alveolar epithelial cells in response to TGF‐β1 gradients. The device was directly mounted on the cells that had grown in cell culture plates and produced a stable concentration gradient of TGF‐β1 with negligible shear stress, thereby providing a favorable environment for the anchorage‐dependent cells. A549 cells elongated with the characteristic spindle‐shaped morphological changes with upregulation of alpha‐smooth muscle actin, a mesenchyme marker, in a gradient‐dependent manner, suggestive of EMT progression. We observed that at higher TGF‐β1 concentrations ranging from 5 to 10 ng/mL, the cultures in the microfluidic device allowed to quantitatively pick up subtle differences in the EMT cellular response as compared with plate cultures. These results suggest that the microfluidic gradient device would accurately determine the optimal concentrations of TGF‐β1, given that epithelial cells of different tissue origins greatly vary their responses to TGF‐β1. Therefore, this microfluidic device could be a powerful tool to monitor EMT induced by a variety of environmental stresses including cigarette smoke with high sensitivity.     

Label-free electrical discrimination of cells at normal, apoptotic and necrotic status with a microfluidic device

As a label-free alternative of conventional flow cytometry, chip-based impedance measurement for single cell analysis has attracted increasing attentions in recent years. In this paper, we designed a T-shape microchannel and fabricated a pair of gold electrodes located horizontally on each side of the microchannel using a transfer printing method. Instant electric signals of flowing-through single cells were then detected by connecting the electrodes to a Keithley resistance and capacitance measurement system. Experimental results based on the simultaneous measurement of resistance and capacitance demonstrated that HL-60 and SMMC-7721 cells could be differentiated effectively. Moreover, SMMC-7721 cells at normal, apoptotic and necrotic status can also be discriminated in the flow. We discussed the possible mechanism for the discrimination of cell size and cell status by electrical analysis, and it is believed that the improvement of detection with our design results from more uniform distribution of the electric field. This microfluidic design may potentially become a promising approach for the label-free cell sorting and screening.     

Continuous on-line derivatization and selective separation of D-aspartic acid by a capillary electrophoresis system with a continuous sample introduction interface

A rapid and selective method is described for the separation of D-aspartic acid (D-Asp) using a continuous on-line derivatization system coupled to capillary electrophoresis (CE). D-Asp was derivatized using o-phthaldialdehyde/N-acetyl-L-cysteine (OPA/NAC). By on-line derivatization, amino acid enantiomers were automatically and reproducibly converted to the UV-absorbing diastereomer derivatives which were separated by capillary zone electrophoresis (CZE) in the presence of 10 mmol/L beta-cyclodextrin (beta-CD). Under the investigated separation conditions, D-Asp is resolved from L-aspartic acid (L-Asp) and other amino acids in a standard mixture of amino acids. The separation could be achieved within 4 min and the sample throughput rate can reach up to 16 h(-1). The repeatability (defined as relative standard deviation, RSD) was 3.21%, 3.58% with peak area evaluation and 3.72%, 4.03% with peak height evaluation for L-Asp and D-Asp.