A2E-epoxides damage DNA in retinal pigment epithelial cells. Vitamin E and other antioxidants inhibit A2E-epoxide formation

The autofluorescent pigments that accumulate in retinal pigment epithelial cells with aging and in some retinal disorders have been implicated in the etiology of macular degeneration. The major constituent is the fluorophore A2E, a pyridinium bisretinoid. Light-exposed A2E-laden retinal pigment epithelium exhibits a propensity for apoptosis with light in the blue region of the spectrum being most damaging. Efforts to understand the events precipitating the death of the cells have revealed that during irradiation (430 nm), A2E self-generates singlet oxygen with the singlet oxygen in turn reacting with A2E to generate epoxides at carbon-carbon double bonds. Here we demonstrate that A2E-epoxides, independent of singlet oxygen, exhibit reactivity toward DNA with oxidative base changes being at least one of these lesions. Mass spectrometry revealed that the antioxidants vitamins E and C, butylated hydroxytoluene, resveratrol, a trolox analogue (PNU-83836-E), and bilberry extract reduce A2E-epoxidation, whereas single cell gel electrophoresis and cell viability studies revealed a corresponding reduction in the incidence of DNA damage and cell death. Vitamin E, a lipophilic antioxidant, produced a more pronounced decrease in A2E-epoxidation than vitamin C, and treatment with both vitamins simultaneously did not confer additional benefit. Studies in which singlet oxygen was generated by endoperoxide in the presence of A2E revealed that vitamin E, butylated hydroxytoluene, resveratrol, the trolox analogue, and bilberry reduced A2E-epoxidation by quenching singlet oxygen. Conversely, vitamin C and ginkgolide B were not efficient quenchers of singlet oxygen under these conditions.     

The Cyanobacterial Photoactive Orange Carotenoid Protein Is an Excellent Singlet Oxygen Quencher

Cyanobacteria have developed a photoprotective mechanism that decreases the energy arriving at the photosynthetic reaction centers under high-light conditions. The photoactive orange carotenoid protein (OCP) is essential in this mechanism as a light sensor and energy quencher. When OCP is photoactivated by strong blue-green light, it is able to dissipate excess energy as heat by interacting with phycobilisomes. As a consequence, charge separation and recombination leading to the formation of singlet oxygen diminishes. Here, we demonstrate that OCP has another essential role. We observed that OCP also protects Synechocystis cells from strong orange-red light, a condition in which OCP is not photoactivated. We first showed that this photoprotection is related to a decrease of singlet oxygen concentration due to OCP action. Then, we demonstrated that, in vitro, OCP is a very good singlet oxygen quencher. By contrast, another carotenoid protein having a high similarity with the N-terminal domain of OCP is not more efficient as a singlet oxygen quencher than a protein without carotenoid. Although OCP is a soluble protein, it is able to quench the singlet oxygen generated in the thylakoid membranes. Thus, OCP has dual and complementary photoprotective functions as an energy quencher and a singlet oxygen quencher.     

Photoactivation of the nematicidal compound alpha-terthienyl from roots of marigolds (Tagetes species). A possible singlet oxygen role.

The nematicidal compound alpha-terthienyl from roots of Tagetes species generates upon irradiation with near ultraviolet light reactive oxygen species on which the in vitro nematicidal activity depends. This system was studied by following the inhibition of glucose-6-phosphate dehydrogenase by photoactivated alpha-terthienyl and protection of the enzyme activity in the absence of oxygen and by various additions. Addition of mannitol, benzoate, superoxide dismutase or catalase did not have any effect nor did H2O2. This suggests that OH., O-.2, and H2O2 are not the reactive oxygen species involved. The enzyme was protected against photoactivated alpha-terthienyl in air-saturated solutions by singlet oxygen quenchers such as histidine, methionine, tryptophan, bovine serum albumin, and NaN3. Furthermore, inactivation of the enzyme was about 3.5 times faster in D2O than in H2O. When alpha-terthienyl in CH2Cl2 was irradiated in the presence of the olefin adamantylideneadamantane, a stable dioxetane was formed which decomposed to adamantanone when heated above its melting point. These results indicate a singlet oxygen-mediated process.     

On the mechanism of Candida spp. photoinactivation by hypericin

The photoprocesses involved in hypericin photoinactivation of three different Candida species (C. albicans, C. parapsilosis and C. krusei) have been examined. Production of singlet oxygen from the triplet state and of superoxide from both the triplet state and the semiquinone radical anion are demonstrated. Hydrogen peroxide is formed downstream of these early events. The outcome of the photodynamic treatments is dictated by the intracellular distribution of hypericin, which is different in the three species and affects the ability of hypericin to produce the different reactive oxygen species and trigger cell-death pathways. The results are in line with the previously-observed different susceptibilities of the three Candida species to hypericin photodynamic treatments.     

Targeted cross-linking of a molten globule form of acetylcholinesterase by the virucidal agent hypericin.

The natural product hypericin is a photosensitive polycyclic aromatic dione compound, which has been widely investigated because of its virucidal and antitumor properties. Although it has been suggested that singlet oxygen or a radical species might be responsible for its biological action, its mechanism of action remains unknown. Due to its amphiphilic characteristics, we considered the possibility that it might interact preferentially with partially unfolded proteins which exhibit exposed hydrophobic surfaces. We here demonstrate that hypericin binds to a molten globule species generated from Torpedo acetylcholinesterase, but not to the corresponding native enzyme. Irradiation with visible light, under aerobic conditions, causes chemical cross-linking of the catalytic subunits, to dimers and heavier species, under conditions where no cross-linking is observed for the native enzyme. Both anaerobiosis and sodium azide greatly reduce the extent of cross-linking, suggesting that singlet oxygen is responsible for the phenomenon. This agrees with our observation, using spin traps, that mainly singlet oxygen is produced by the complex of hypericin with the molten globule of acetylcholinesterase. Cross-linking is enhanced in the presence of liposomes to which the molten globule of acetylcholinesterase is quantitatively adsorbed. This may be due to high local concentrations of both hypericin and the protein resulting in close proximity, and hence in a high yield of cross-linking. Molten globule species are believed to be intermediates in both protein folding and translocation through biological membranes. Thus, hypericin may serve as a valuable tool for trapping such intermediates. This might also explain its therapeutic effectiveness toward virus-infected or tumor cells.     

Photoactivation of hypericin decreases the viability of RINm5F insulinoma cells through reduction in JNK/ERK phosphorylation and elevation of caspase-9/caspase-3 cleavage and Bax-to-Bcl-2 ratio

Insulinomas cause neuroglycopenic symptoms, permanent neurological damage and even death. Current available therapies cannot satisfactorily treat malignant insulinomas and some benign insulinomas. The promising phototherapeutic results and harmless side effects of hypericin in some cancer treatments prompted us to explore possible anti-growth activity of photoactivated hypericin against RINm5F insulinoma cells and underlying mechanisms. We now show that detectable and maximal internalization of hypericin in RINm5F insulinoma cells occurred in 20 and 60 min respectively. Hypericin was considerably associated with the plasma membrane, appreciably localized in the sub-plasma membrane region and substantially accumulated in the cytoplasm. Photoactivated hypericin decreased the viability of RINm5F insulinoma cells due to its anti-proliferative and apoptotic actions. Photoactivation of hypericin inhibited cell proliferation reflected by decreased expression of the proliferation marker Ki-67 and cell-cycle arrest in the G₀/G₁-phase. The anti-proliferative effect resulted from down-regulation of phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular-signal-regulated kinase (ERK). Photoactivated hypericin triggered apoptosis through activation of caspase-3 and caspase-9 and elevation of the Bax-to B-cell lymphoma 2 (Bcl-2) ratio. The findings lay a solid foundation for implementation of hypericin-mediated photodynamic therapy in treatment of insulinomas.     

Expression of the EGF Family in Gastric Cancer: Downregulation of HER4 and Its Activating Ligand NRG4

Gastric cancer is a major cause of cancer-related deaths in both men and women. The epidermal growth factor receptors are EGFR, HER2, HER3 and HER4. Of the four epidermal growth factor receptors, EGFR and HER2 are well-known oncogenes involved in gastric cancer. Little, however, is known about the role played by HER3 and HER4 in this disease. We obtained paired samples from the tumor and the adjacent normal tissue from the same patient undergoing surgery for gastric cancer. Using RT-qPCR, we quantified the mRNA expression of the four receptors including the HER4 splicing isoforms and all the ligands activating these receptors. Using immunohistochemistry, the protein expression of HER4 was also quantified. We found that HER2 mRNA expression was upregulated in the tumor tissue compared to the matched normal tissue (p = 0.0520). All ligands with affinity for EGFR were upregulated, whereas the expression of EGFR was unchanged. Interestingly, we found the mRNA expression of HER4 (p = 0.0002) and its ligand NRG4 (p = 0.0009) to be downregulated in the tumor tissue compared to the matched normal tissue. HER4 downregulation was demonstrated for all the alternatively spliced isoforms of this receptor. These results support the involvement of EGFR and HER2 in gastric cancer and suggest an interesting association of reduced HER4 expression with development of gastric cancer.     

HER2、VEGF和MVD与胃癌患者临床病理特征的关系

目的：探讨HER2、VEGF的表达和MVD水平及这三者与胃癌的临床病理和生物学特性的关系。方法：胃癌患者26例,其中14例患者接受了全胃切除术,12例患者行胃癌活检,在癌组织中心获得有效标本设为胃癌组,以距离癌组织中心5cm的正常癌组织为正常组。RT-PCR法检测两组胃组织中HER2mRNA的表达;免疫组织化学检测胃组织中VEGF的表达;计算各组微血管密度。结果：与正常组相比,胃癌组HER2mRNA表达明显增加;正常组胃组织VEGF平均灰度值为21．51±4．64,而胃癌组胃组织VEGF平均灰度值为167．23±18．85,两组相比有明显差异;正常组胃组织MVD为13．44±5．34个,与正常组相比,胃癌组胃组织MVD水平（65．74±9．87个）明显增高;HER2与患者TNM分期成正相关,VEGF与患者性别及TNM分期均成正相关,而MVD与患者临床病理特征无相关性。结论：血管内皮生长因子的表达及与胃癌患者临床病理特征的相关性表明,HER2、VEGF两个分子生物标志物在肿瘤的发生发展中具有重要作用,其能够被用来作为预测的侵袭性胃癌预后的重要参数。     

The cytotoxic activity of hematoheme: evidence for two different mechanisms

Hematoheme displays a potent cytolytic activity toward erythrocytes either in the presence of hydrogen peroxide and a halide ion (system I) or in the presence of oxygen and a reducing agent (system II). In system I it resembles the cytotoxic activity of various peroxidases, whereas in system II it resembles the destructive activity of bleomycin and a variety of metal complexes. Both types of reactions presumably involve the generation of active oxygen species, which are responsible for the damaging effects. In a first attempt to compare the chemical mechanisms of the two types of reactions we used various traps and scavengers of active oxygen species. Tryptophan as well as tyrosine and uric acid were found to be potent inhibitors of the hematoheme-H2O2-halide reaction but do not significantly inhibit the hematoheme-O2-ascorbate reaction. Pyridine, on the other hand, inhibits the oxygen-mediated reaction, but does not affect the peroxide-halide-mediated activity. The cytolytic activity of photoactivated hematoporphyrin, which involves the generation of singlet oxygen, is activated by pyridine and is strongly inhibited by diphenylisobenzofuran. The latter compound is a weak activator of both hematoheme reactions. We conclude that the two hematoheme reactions proceed by two different mechanisms and probably generate different toxic intermediates. The results further suggest that the toxic intermediate generated by photoactivated hematoporphyrin (singlet oxygen) does not play a dominant role in either of the two hematoheme reactions.     

Targeted generation of DNA strand breaks using pyrene-conjugated triplex-forming oligonucleotides

Gene targeting by triplex-forming oligonucleotides (TFOs) has proven useful for gene modulation in vivo. Photoreactive molecules have been conjugated to TFOs to direct sequence-specific damage in double-stranded DNA. However, the photoproducts are often repaired efficiently in cells. This limitation has led to the search for sequence-specific photoreactive reagents that can produce more genotoxic lesions. Here we demonstrate that photoactivated pyrene-conjugated TFOs (pyr-TFOs) induce DNA strand breaks near the pyrene moiety with remarkably high efficiency and also produce covalent pyrene-DNA adducts. Free radical scavenging experiments demonstrated a role for singlet oxygen activated by the singlet excited state of pyrene in the mechanism of pyr-TFO-induced DNA damage. In cultured mammalian cells, the effect of photoactivated pyr-TFO-directed DNA damage was to induce mutations, in the form of deletions, approximately 7-fold over background levels, at the targeted site. Thus, pyr-TFOs represent a potentially powerful new tool for directing DNA strand breaks to specific chromosomal locations for biotechnological and potential clinical applications.     

Simultaneous quantitative detection of relevant biomarkers in breast cancer by quantitative real-time PCR

The assessment of ERa, PgR and HER2 status is routinely performed today to determine the endocrine responsiveness of breast cancer samples. Such determination is usually accomplished by means of immunohistochemistry and in case of HER2 amplification by means of fluorescent in situ hybridization (FISH). The analysis of these markers can be improved by simultaneous measurements using quantitative real-time PCR (Qrt-PCR). In this study we compared Qrt-PCR results for the assessment of mRNA levels of ERa, PgR, and the members of the human epidermal growth factor receptor family, HER1, HER2, HER3 and HER4. The results were obtained in two independent laboratories using two different methods, SYBR Green I and TaqMan probes, and different primers. By linear regression we demonstrated a good concordance for all six markers. The quantitative mRNA expression levels of ERa, PgR and HER2 also strongly correlated with the respective quantitative protein expression levels prospectively detected by EIA in both laboratories. In addition, HER2 mRNA expression levels correlated well with gene amplification detected by FISH in the same biopsies. Our results indicate that both Qrt-PCR methods were robust and sensitive tools for routine diagnostics and consistent with standard methodologies. The developed simultaneous assessment of several biomarkers is fast and labor effective and allows optimization of the clinical decision-making process in breast cancer tissue and/or core biopsies.     

Cellular Defense against Singlet Oxygen-induced Oxidative Damage by Cytosolic NADP + -dependent Isocitrate Dehydrogenase

Singlet oxygen ( 1 O 2 ) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. Recently, we have shown that NADP + -dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study, we investigated the role of cytosolic form of NADP + -dependent isocitrate dehydrogenase (IDPc) against singlet oxygen-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to singlet oxygen generated from photoactivated dye, the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against singlet oxygen, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against singlet oxygen-induced oxidative injury.     

The photoactivated toxin cercosporin as a tool in fungal photobiology

Cercospora species are a highly successful group of fungi which pathogenize diverse species of economically important plants. Many Cercospora species produce a unique photoactivated and photoinduced polyketide toxin, cercosporin, which has been implicated as a pathogenicity factor. Illuminated cercosporin interacts with molecular oxygen to produce highly toxic singlet oxygen. Although nearly all organisms tested, including plants, mice and most fungi, are susceptible to cercosporin-mediated cell damage, Cercospora species are resistant. In general, little is known about how organisms protect themselves against singlet oxygen. Studies on how Cercospora species avoid autotoxicity are proving to be a valuable model in understanding this process in other systems. Furthermore, advances are being made in the understanding of how light regulates gene expression and cercosporin synthesis in Cercospora species. These studies are helping to elucidate mechanisms of gene regulation and light signal transduction for an environmental signal important in numerous fungal developmental processes, including secondary metabolite production.     

Cellular defense against singlet oxygen-induced oxidative damage by cytosolic NADP+-dependent isocitrate dehydrogenase

Singlet oxygen ( 1 O 2 ) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. Recently, we have shown that NADP + -dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study, we investigated the role of cytosolic form of NADP + -dependent isocitrate dehydrogenase (IDPc) against singlet oxygen-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to singlet oxygen generated from photoactivated dye, the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against singlet oxygen, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against singlet oxygen-induced oxidative injury.     

Green fluorescent protein photobleaching: a model for protein damage by endogenous and exogenous singlet oxygen

Characterization of protein damage during photosensitization of chlorin e6-treated cells was performed using the green fluorescent protein (GFP). The GFP-chromophore damage caused by singlet oxygen was studied in COS 7 kidney cells and E. coli bacteria following light irradiation. Electron spin resonance (ESR) revealed the generation of endogenous singlet oxygen (1O2) by photoactivated GFP, an effect similar to that produced by the exogenous photosensitizer chlorin e6. A light dose-dependent photobleaching effect of GFP was pronounced at low pH or upon photosensitization with chlorin e6. However, the 1O2 quenchers beta-carotene and sodium azide minimized GFP photo-bleaching. Gel electrophoresis of photosensitized GFP followed by fluorescence multi-pixel spectral imaging revealed the binding of chlorin e6 to GFP, affecting the photobleaching efficacy. Fluorescence multi-pixel spectral imaging of GFP-transfected COS 7 cells demonstrated the presence of GFP in the cytoplasm and nucleus, while chlorin e6 was found to be concentrated in the perinuclear vesicles. Exposure of the cells to light induced GFP photobleaching in the close vicinity of chlorin e6 vesicles. We conclude that photoactivated GFP generates endogenous 1O2, inducing chromophore damage, which can be enhanced by the cooperation of exogenous chlorin e6.     

Isolation and activity of the photodynamic pigment hypericin

Abstract. Hypericin, a photodynamic pigment, occurring in members of the Hypericaceae, can induce photosensitivity in grazing animals. The pigment has been isolated from the glandular trichomes located on the calyx of Hypericum hirsulum. Hypericin is shown to be capable of sensitizing the photo-oxidation of methyl linolenate. This activity is reduced in the presence of crocin, a carotenoid. Evidence for the generation of singlet molecular oxygen by hypericin is provided by the monitoring of oxygen consumption during the photosensitized oxidation of imidazole. Rates of oxygen consumption were modified by deuterium oxide and sodium azide. The photodynamic action of hypericin on pea leaf discs results in the promotion of photo-oxidative damage, measured by pigment loss and ethane production. These results are discussed in relation to the possible function of hypericin within the plant and the role of photo-dynamic reactions in nature.     

FOXO3a反馈上调人表皮生长因子受体3表达介导HER2+乳腺癌细胞酪氨酸激酶抑制剂治疗耐受

近年来，酪氨酸激酶抑制剂（tyrosine kinase inhibitors，TKI）类药物治疗HER2+乳腺癌进展迅速，但出现治疗耐受仍是迫切需要解决的问题。本研究采用TKI（AEE788、Lapatinib）处理HER2+乳腺癌细胞BT474和SKBR3，发现HER3在mRNA和蛋白质水平上的表达均上调。MTS及克隆形成实验结果显示，siRNA干扰HER3的表达能够显著抑制BT474、SKBR3细胞的增殖，表明干扰HER3可增强细胞对TKI的敏感性。为进一步考察TKI促进HER3表达的可能机制，Western 印迹及免疫荧光检测发现，AEE788、Lapatinib能够上调FOXO3a的表达且促进其入核。干扰FOXO3a可逆转TKI对HER3的诱导作用，说明TKI通过激活FOXO3a上调HER3的表达。综上所述，FOXO3a反馈上调HER3表达介导HER2+乳腺癌细胞TKI治疗耐受。这一研究发现，为临床解决TKI治疗耐受提供一定的理论基础。     

Up-regulation of acetyl-CoA carboxylase alpha and fatty acid synthase by human epidermal growth factor receptor 2 at the translational level in breast cancer cells

Expression of the HER2 oncogene is increased in approximately 30% of human breast carcinomas and is closely correlated with the expression of fatty acid synthase (FASN). In the present study, we determined the mechanism by which FASN and acetyl-CoA carboxylase alpha (ACCalpha) could be induced by HER2 overexpression. SK-BR-3 and BT-474 cells, breast cancer cells that overexpress HER2, expressed higher levels of FASN and ACCalpha compared with MCF-7 and MDA-MB-231 breast cancer cells in which HER2 expression is low. The induction of FASN and ACCalpha in BT474 cells were not mediated by the activation of SREBP-1. Exogenous HER2 expression in MDA-MB-231 cells induced the expression of FASN and ACCalpha, and the HER2-mediated increase in ACCalpha and FASN was inhibited by both LY294002, a phosphatidylinositol 3-kinase inhibitor, and rapamycin, a mammalian target of rapamycin (mTOR) inhibitor. In addition, the activation of mTOR by the overexpression of RHEB in MDA-MB-231 cells increased the synthetic rates of both FASN and ACCalpha. On the other hand, FASN and ACCalpha were reduced in BT-474 cells by a blockade of the mTOR signaling pathway. These changes observed in their protein levels were not accompanied by changes in their mRNA levels. The 5'- and 3'-untranslated regions of both FASN and ACCalpha mRNAs were involved in selective translational induction that was mediated by mTOR signal transduction. These results strongly suggest that the major mechanism of HER2-mediated induction of FASN and ACCalpha in the breast cancer cells used in this study is translational regulation primarily through the mTOR signaling pathway.