Photosensitization of the sunscreen octyl p-dimethylaminobenzoate by UVA in human melanocytes but not in keratinocytes.

Sunscreens penetrate human epidermis and modify the biology of proliferating cells. This study addressed the question whether the UV response of cultured human cells is affected by direct treatment with nontoxic levels of sunscreens. Cell survival following exposure to UVC or unfiltered UBV was not altered by preincubation with 25 micrograms/mL of octyl p-dimethylaminobenzoate (o-PABA), 2-ethylhexyl p-methoxycinnamate (EHMC) or oxybenzone. However, UVA or UVB filtered to reproduce the solar UV spectrum penetrating to the basal layer of the epidermis, highly sensitized cells to killing by o-PABA but not by its hydrolysis product, 4-dimethylaminobenzoic acid. Sensitization was found in all cell types tested, except normal keratinocytes, and could be prevented by certain antioxidants particularly pyruvate and the hydroxyl radical scavenger mannitol. o-PABA and EHMC applied without UV reduced the adherence of cells. The results indicate that sunscreens may increase cell mobility and the combination of o-PABA with solar UV may selectively damage melanocytes in the skin.     

RESISTANCE OF PIGMENTED HUMAN CELLS TO KILLING BY SUNLIGHT AND OXYGEN RADICALS

Solar irradiation of a panel of human cell lines revealed three phenomena relevant to understanding the biological role of melanin; a heavily melanised melanoma line (MM418) was considerably more resistant to solar killing compared with HeLa and amelanotic melanoma cells of similar size and DNA content; MM418 cells were also resistant to killing by artificial UVB and by hydrogen peroxide generated in situ with extracellular glucose oxidase; and no difference in survival between the cell lines was found using 254 nm UV or gamma radiation. MM418 cells were resistant to sunlight when irradiated as attached monolayers but not when irradiated in suspension. Further studies showed that resistance to solar radiation in MM418 cells was not due to less DNA damage, as judged by inhibition of semiconservative DNA synthesis, or to enhanced constitutive or induced repair determined by reactivation of irradiated adenovirus. These results indicate that melanisation protects human cells from solar UVB in vitro and that the mechanism is associated with protection from hydrogen peroxide-type damage rather than direct shielding of DNA.     

POTENCY AND SELECTIVE TOXICITY OF TETRA(HYDROXYPHENYL)- AND TETRAKIS(DIHYDROXYPHENYL)PORPHYRINS IN HUMAN MELANOMA CELLS, WITH AND WITHOUT EXPOSURE TO RED LIGHT

Abstract A series of tetra(hydroxyphenyl)-(2-, 3- and 4-hydroxy; THPP) and tetrakis(dihydroxyphenyl)porphyrins (2,3-, 2,4-, 2,5-, 3,4-, and 3,5-dihydroxy; TDHPP) was synthesized and tested for toxicity in HeLa cells and human melanoma cell lines. Irradiation of drug-treated cells with >600 nm light greatly increased the toxicity of all drugs except the 2,5- and 3,5-TDHPP. The THPP were more toxic than TDHPP in all cell lines, with or without irradiation; of the dihydroxy derivatives, the 3,4- and 2,4-isomers were the most toxic and the 2,5-isomer was the least toxic. The MM96E melanoma cell line, shown previously to be sensitive to hydrogen peroxide and superoxide ion, was not hypersensitive to killing by any of the above agents. HeLa cells, which lacked glutathione- S -transferase activity, were sensitive to the 4- and 2,3-isomers after irradiation; similar amounts of all drugs were taken up by HeLa cells. The pigmented melanoma cell line MM418, resistant to UV-B and in situ -generated hydrogen peroxide but sensitive to glutathione (GSH) depletion, was found to be resistant to the 2,3-isomer (no irradiation) and sensitive to the 3,4-isomer. The results indicate that (1) phototoxicity in these phenylporphyrins is not mediated by superoxide ions or hydroxyl radicals, (2) toxicity is dependent on the orientation of the hydroxy groups, (3) GSH transferase and possibly GSH itself offer protection from the 4- and 3,4-derivatives, respectively, and (4) the 3,4-derivative and analogues of similar selectivity should be evaluated further for the treatment of primary melanoma.     

In vitro evaluation of antiproliferative effect of ethyl gallate against human oral squamous carcinoma cell line KB

Although some polyphenols are known to possess anticancer activity against different cancer cell lines through induction of apoptosis, the mode of antiproliferative effect of ethyl gallate against human oral squamous carcinoma cell line KB was not studied until now. Therefore, the antiproliferative effect of ethyl gallate was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in comparison with the reference drug paclitaxel. Generation of reactive oxygen species, mitochondrial membrane potential loss, DNA damage and apoptosis were determined using 2,7-diacetyldichlorofluorescein fluorescence, uptake of rhodamine-123 by mitochondria, comet assay and acridine orange/ethidium bromide dual-dye staining method. Both ethyl gallate and paclitaxel exhibited cytotoxicity in a dose-dependent manner. The 50% inhibitory concentration for ethyl gallate was 30 and 20 μg/mL for paclitaxel. A volume of 50 μg/mL of ethyl gallate was found to be significantly effective (P < 0.05) in controlling the cancer cell proliferation leading to acute apoptosis.     

基于标记法的福白菊精油抗真菌机理研究

以福白菊为原料, 通过有机溶剂萃取和水蒸气蒸馏相结合制备福白菊精油. 分别采用健那绿、 细胞膜荧光探针DiI、 碘化丙啶、 罗丹明123以及溴化乙锭等染料标记酵母细胞、 酵母细胞膜、 酵母线粒体和质粒DNA, 研究福白菊精油对酵母细胞、 酵母细胞膜、 线粒体以及质粒DNA的破坏作用, 探讨福白菊精油的抗真菌机理. 研究结果表明, 福白菊精油不仅能破坏真菌的细胞膜, 使其细胞内含物外泄, 还能够穿过细胞膜, 破坏其线粒体膜和DNA, 最终导致真菌细胞死亡.     

Inhibition of Dual Specific Oncolytic Adenovirus on Esophageal Cancer via Activation of Caspases by a Mitochondrial-dependent Pathway

We investigated the anti-tumor effects of dual cancer specific-oncolytic adenovirus Ad-VP on esophageal cancer(EC). The anti-tumor activity of Ad-VP was compared with that of the control recombinant adenoviruses (Ad-GP, Ad-Apoptin, Ad-EGFP) in human esophageal cancer cell EC-109 and human normal liver cell L02 in vitro. In 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assays, the growth of EC-109 cells was slightly inhibited by Ad-GP, Ad-Apoptin and Ad-EGFP. However, Ad-VP induced a significant cytotoxic effect. Infection of EC-109 cells with Ad-VP resulted in a significant induction of apoptosis of them in vitro, detected by 4′,6-diamidino-2-phenylindole(DAPI) or acridine orange and ethidium bromide staining. The results of Western blot and flow cytometric assay indicate the loss of mitochondrial membrane potential(ΔΨ_m), the release of cytochrome c and the activation of caspase-3, 6 and 7 in Ad-VP infected EC-109 cells. In contrast, all these assays show almost no effects of the recombinant adenoviruses on L02 cells. These results demonstrate that the treatment of tumors with Ad-VP selectively inhibits tumor growth and induces apoptosis of esophageal cancer cells. Ad-VP may provide a novel and powerful strategy for cancer gene therapy.     

The in vitro Antitumour Activity of Substituted Dibutyl-1,3,2-dioxastannolanes

Six dibutyl-1,3,2-dioxastannolanes, including two enantiomeric pairs, exhibited greater in vitro antitumour activity towards a variety of human tumour cell lines than cisplatin but with little discrimination, suggesting hydrolysis to a common cytotoxic intermediate. A cell line hypersensitive to mitochondrial inhibitors (CI80–13S) was not sensitive to any of the test compounds, suggesting that cell mechanisms other than, or in addition to, mitochondrial function are targeted by tin antitumour agents. A pigmented melanoma cell line (MM418c5) was resistant to the test compounds, which were found to be sequestered by melanin. This resistance was not observed with triphenyltin hydroxide. © 1997 John Wiley & Sons, Ltd.     

Synthesis and spectroscopic characterisation of a combinatorial library based on the fungal natural product 3-chloro-4-hydroxyphenylacetamide

Parallel solution-phase chemistry has yielded a series of secondary amide analogues of the fungal natural product 3-chloro-4-hydroxyphenylacetamide. 3-Chloro-4-hydroxyphenylacetic acid was coupled to a variety of primary amines using 1-ethyl-3-(3'-dimethylamino- propyl)-carbodiimide hydrochloride. The desired products were obtained in good yield and high purity following rapid silica purification. All analogues were spectroscopically characterised using NMR, UV, IR and MS data. One compound displayed moderate cytotoxicity against the human melanoma and prostate cell lines, MM96L and DU145.     

Synthesis and Antitumor Activity of a New 7-Azaindole Derivative

We designed and synthesized a 7-azaindole derivative（TH1082）, which was characterized by 1^H NMR and 13^C-NMR. We investigated its antitumor effects on human melanoma A375 cells, human liver cancer SMMC cells and human breast cancer MCF-7 cells in vitro via 3-（4,5-dimethyldiazol-2-yl）-2,5-diphenyl-tetrazolium bromide （MTT） assay and also explored the mechanism of antiproliferation of them. The results show that TH1082 significantly inhibited the proliferation of these cells to different extent. The IC50 values for A375 cells, SMMC cells and MCF-7 cells were 25.38, 48.70 and 76.94 μg/mL at 24 h, respectively. To observe cell morphological changes, acridine orange/ethidium bromide（AO/EB） staining and Hoechest33342/Pl staining were carried out. These results indicate that TH1082 could induced the apoptosis of A375 cells. The apoptotic rates were （9.5±2.09）%, （18.9±2.25）% and （39.5±2.02）%（5, 10 and 20 μg/mL） for A375, SMMC and MCF-7 cell lines, respectively. Further, we determined the activities of caspase-3 and caspase-9 in A375 cells treated with TH1082 at different concentrations（0, 5, 10 and 20 μg/mL） or Z-VAD-FMK（20 μmol/L）, a pan-caspase inhibitor for 24 h. The results show that TH1082 activated caspase-3 and caspase-9, and the activation could be blocked by Z-VAD-FMK. Taken together, these findings indicate that TH 1082 could inhibit the proliferation ofA375 cells via activating caspase-3 and caspase-9.     

Functional assays in high-resolution clear native gels to quantify mitochondrial complexes in human biopsies and cell lines

We introduce high resolution clear native electrophoresis (CNE) as a powerful technique to resolve enzymatically active mitochondrial complexes from cultured human cell lines and skeletal muscle biopsy samples. Quantitative enzymatic assays can be performed using small amounts of cultured cells with low mitochondria content, for example, around 10 mg of sedimented osteosarcoma cells (wet weight) which is equivalent to around 10 million cells. High resolution CNE offers general advantages for in-gel catalytic activity assays compared to blue native electrophoresis. It seems especially suited for assaying mitochondrial ATP synthase and respiratory chain complexes I and II in cell models of human mitochondrial disorders and for detailed analyses of patient cells and tissues with defects in oxidative phosphorylation.     

EPR Investigations to Study the Impact of Mito-Metformin on the Mitochondrial Function of Prostate Cancer Cells

Background: Mito-metformin10 (MM10), synthesized by attaching a triphenylphosphonium cationic moiety via a 10-carbon aliphatic side chain to metformin, is a mitochondria-targeted analog of metformin that was recently demonstrated to alter mitochondrial function and proliferation in pancreatic ductal adenocarcinoma. Here, we hypothesized that this compound may decrease the oxygen consumption rate (OCR) in prostate cancer cells, increase the level of mitochondrial ROS, alleviate tumor hypoxia, and radiosensitize tumors. Methods: OCR and mitochondrial superoxide production were assessed by EPR (9 GHz) in vitro in PC-3 and DU-145 prostate cancer cells. Reduced and oxidized glutathione were assessed before and after MM10 exposure. Tumor oxygenation was measured in vivo using 1 GHz EPR oximetry in PC-3 tumor model. Tumors were irradiated at the time of maximal reoxygenation. Results: 24-hours exposure to MM10 significantly decreased the OCR of PC-3 and DU-145 cancer cells. An increase in mitochondrial superoxide levels was observed in PC-3 but not in DU-145 cancer cells, an observation consistent with the differences observed in glutathione levels in both cancer cell lines. In vivo, the tumor oxygenation significantly increased in the PC-3 model (daily injection of 2 mg/kg MM10) 48 and 72 h after initiation of the treatment. Despite the significant effect on tumor hypoxia, MM10 combined to irradiation did not increase the tumor growth delay compared to the irradiation alone. Conclusions: MM10 altered the OCR in prostate cancer cells. The effect of MM10 on the superoxide level was dependent on the antioxidant capacity of cell line. In vivo, MM10 alleviated tumor hypoxia, yet without consequence in terms of response to irradiation.     

Lipoproteins modulate growth and differentiation of cultured human epidermal keratinocytes

The effects of various lipoproteins on the growth and the differentiation of cultured normal human keratinocytes were investigated. Primary cultures of human epidermal keratinocytes were obtained from neonatal foreskin, and then added with lipoproteins, very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL). Cell growth potential was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. VLDL and LDL enhanced keratinocytes growth and LDL receptor expression at the plasma membrane level. These effects were more remarkably observed in cells cultured with VLDL than in cells cultured with LDL. Apolipoprotein E (ApoE) was highly increased in VLDL treated cells. These results suggest that VLDL binds with high affinity to cell surface receptors and induces cell proliferation.     

Selenium nanoparticles fabricated in Undaria pinnatifida polysaccharide solutions induce mitochondria-mediated apoptosis in A375 human melanoma cells

Selenium nanoparticle (Nano-Se) is a novel Se species with novel biological activities and low toxicity. In the present study, we demonstrated a simple method for synthesis of size-controlled Nano-Se by adding Undaria pinnatifida polysaccharides to the redox system of selenite and ascorbic acid. A panel of four human cancer cell lines was shown to be susceptible to Nano-Se, with IC(50) values ranging from 3.0 to 14.1 microM. Treatment of A375 human melanoma cells with the Nano-Se resulted in dose-dependent cell apoptosis as indicated by DNA fragmentation and phosphatidylserine translocation. Further investigation on intracellular mechanisms found that Nano-Se treatment triggered apoptotic cell death in A375 cells with the involvement of oxidative stress and mitochondrial dysfunction. Our results suggest that Nano-Se may be a candidate for further evaluation as a chemopreventive and chemotherapeutic agent for human cancers, especially melanoma cancer.     

High-performance liquid chromatographic method for the comparison of the photostability of five sunscreen agents

Sunscreen agents are commonly used in cosmetic products to filter out noxious radiation in sunlight. A convenient high-performance liquid chromatographic (HPLC) method for the quantification of five sunscreens after irradiation has been selected. We used this analytical method to compare the photostability of benzophenone-3, PEG-25 PABA, octyl dimethyl PABA, 4-methylbenzylidene camphor and butyl methoxydibenzoylmethane, at levels in the range of 25-60 microM. The assay was carried out, using a C8 column with a methanol--water mobile phase. The detector was set at a wavelength of 300 nm. The assay was linear with the following limits: 0.2 microgram ml-1 for benzophenone-3, 1 microgram ml-1 for PEG-25 PABA, 0.15 microgram ml-1 for octyl dimethyl PABA, 0.1 microgram ml-1 for methylbenzylidene camphor and 0.05 microgram ml-1 for butyl methoxydibenzoylmethane. The half-lives calculated indicate a very good photostability of the sunscreens studied and permit to classify amongst themselves.     

Photodynamic Effects of Hypericin on Lipid Peroxidation and Antioxidant Status in Melanoma Cells

Photodynamic-induced cytotoxicity by hypericin (HYP) was studied on three human melanoma cell lines: one pigmented cell line (G361) and two amelanotic cell lines (M18 and M6). No significant variation in the rate of uptake and in the maximum level of HYP incorporation for the different cells was observed. In the dark, no cytotoxicity was observed in the range0–10^?6M HYP for the three cell lines. Amelanotic cells were found to be more sensitive than pigmented cells to irradiation of HYP with visible light (Λ > 590 nm). In addition, for the three cell lines HYP-induced photocytotoxicity was found to be drug-dose and light-dose dependent. Under the conditions used, thiobarbituric acid-reacting substances (TBARs) were significantly increased in amelanotic cells after irradiation (P < 0.0001). By contrast, the amount of TBARS remained unchanged in pigmented cells. Antioxidant defenses including enzymes and glutathione (GSH) were assayed before and after HYP photosensitization. Significantly increased total SOD activity was observed after photosensitization for amelanotic cells (P < 0.05), while glutathione peroxidase (GSHPx) and catalase (Cat) activities but also GSH levels were significantly decreased (P < 0.01). In pigmented cells a significantly increased Cat activity was found (P < 0.05), whereas GSHPx was unaffected after irradiation. It can be inferred that (a) HYP may be an effective PDT agent for melanoma and (b) there is a relationship between melanin content and sensitivity to HYP phototoxicity in human melanoma cells.     

Proliferation and Invasion of Melanoma Are Suppressed by a Plant Protease Inhibitor,Leading to Downregulation of Survival/Death-Related Proteins

Cell adhesion and migration are crucial for cancer progression and malignancy. Drugs available for the treatment of metastatic melanoma are expensive and unfit for certain patients. Therefore, there is still a need to identify new drugs that block tumor cell development. We investigated the effects of Enterolobium contortisiliquum trypsin inhibitor (EcTI), a protease inhibitor, on cell viability, cell migration, invasion, cell adhesion, and cell death (hallmarks of cancer) in vitro using human melanoma cells (SK-MEL-28 and CHL-1). Although EcTI did not affect non-tumor cells, it significantly inhibited the proliferation, migration, invasion, and adhesion of melanoma cells. Investigation of the underlying mechanisms revealed that EcTI triggered apoptosis and nuclear shrinkage, increased PI uptake, activated effector caspases-3/7, and produced reactive oxygen species (ROS). Furthermore, EcTI disrupted the mitochondrial membrane potential, altered calcium homeostasis, and modified proteins associated with survival and apoptosis/autophagy regulation. Acridine orange staining indicated acidic vesicular organelle formation upon EcTI treatment, demonstrating a cell death display. Electronic microscopy corroborated the apoptotic pattern by allowing the visualization of apoptotic bodies, mitochondrial cristae disorganization, and autophagic vesicles. Taken together, these results provide new insights into the anti-cancer properties of the natural EcTI protein, establishing it as a promising new therapeutic drug for use in melanoma treatment.     

Biochemical Composition and Antioxidant Properties of Lavandula angustifolia Miller Essential Oil are Shielded by Propolis Against UV Radiations

UV radiations are principal causes of skin cancer and aging. Suntan creams were developed to protect epidermis and derma layers against photodegradation and photooxidation. The addition of antioxidant plant extracts (i.e. essential oil) to sunscreens is habitually performed, to increase their UV protective effects and to contrast pro‐radical and cytotoxic compounds present in these solutions. According to these observations, in the present work, the alteration of chemical composition and bioactive properties of Lavandula angustifolia Miller essential oil, exposed to UV light, was investigated. UV induced a significant deterioration of lavender oil biochemical profile. Moreover, the antioxidant activity of this solution, in in vitro tests and directly on B16‐F10 melanoma cells, greatly decreased after UV treatment. Our results also showed that essential oil was shielded from UV stress by propolis addition. Even after UV treatment, bee glue highly protected lavender oil secondary metabolites from degradation and also preserved their antiradical properties, both in in vitro antioxidant assays and in cell oxidative damage evaluations. This research proposed propolis as highly efficient UV protective and antiradical additive for sunscreens, cosmetics and alimentary or pharmaceutical products containing plant extracts.     

Cellular consequences of copper complexes used to catalyze bioorthogonal click reactions

Copper toxicity is a critical issue in the development of copper-based catalysts for copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reactions for applications in living systems. The effects and related toxicity of copper on mammalian cells are dependent on the ligand environment. Copper complexes can be highly toxic, can induce changes in cellular metabolism, and can be rapidly taken up by cells, all of which can affect their ability to function as catalysts for CuAAC in living systems. Herein, we have evaluated the effects of a number of copper complexes that are typically used to catalyze CuAAC reactions on four human cell lines by measuring mitochondrial activity based on the metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to study toxicity, inductively coupled plasma mass spectrometry to study cellular uptake, and coherent anti-Stokes Raman scattering (CARS) microscopy to study effects on lipid metabolism. We find that ligand environment around copper influences all three parameters. Interestingly, for the Cu(II)-bis-L-histidine complex (Cu(his)(2)), cellular uptake and metabolic changes are observed with no toxicity after 72 h at micromolar concentrations. Furthermore, we show that under conditions where other copper complexes kill human hepatoma cells, Cu(I)-L-histidine is an effective catalyst for CuAAC labeling of live cells following metabolic incorporation of an alkyne-labeled sugar (Ac(4)ManNAl) into glycosylated proteins expressed on the cell surface. This result suggests that Cu(his)(2) or derivatives thereof have potential for in vivo applications where toxicity as well as catalytic activity are critical factors for successful bioconjugation reactions.     

Synthesis and Anti-tumor Activity of Novel Glycyrrhetinic Acid Derivatives

Twenty-five derivatives of glycyrrhetinic acid(GA)modified on the A-ring,at C30 and C11 positions were synthesized.Their in vitro cytotoxicity against various cancer cell lines[henrietta lacks strain o...     

Soapwort (Saponaria officinalis L.) Extract vs. Synthetic Surfactants—Effect on Skin-Mimetic Models

Our skin is continuously exposed to different amphiphilic substances capable of interaction with its lipids and proteins. We describe the effect of a saponin-rich soapwort extract and of four commonly employed synthetic surfactants: sodium lauryl sulfate (SLS), sodium laureth sulfate (SLES), ammonium lauryl sulfate (ALS), cocamidopropyl betaine (CAPB) on different human skin models. Two human skin cell lines were employed: normal keratinocytes (HaCaT) and human melanoma cells (A375). The liposomes consisting of a dipalmitoylphosphatidylcholine/cholesterol mixture in a molar ratio of 7:3, mimicking the cell membrane of keratinocytes and melanoma cells were employed as the second model. Using dynamic light scattering (DLS), the particle size distribution of liposomes was analyzed before and after contact with the tested (bio)surfactants. The results, supplemented by the protein solubilization tests (albumin denaturation test, zein test) and oil emulsification capacity (using olive oil and engine oil), showed that the soapwort extract affects the skin models to a clearly different extent than any of the tested synthetic surfactants. Its protein and lipid solubilizing potential are much smaller than for the three anionic surfactants (SLS, ALS, SLES). In terms of protein solubilization potential, the soapwort extract is comparable to CAPB, which, however, is much harsher to lipids.