Loss of AP-2alpha results in deregulation of E-cadherin and MMP-9 and an increase in tumorigenicity of colon cancer cells in vivo.

Activator protein-2 (AP-2) is a transcription factor that regulates proliferation and differentiation in mammalian cells and has been implicated in the acquisition of the metastatic phenotype in several types of cancer. Herein, we examine the role of AP-2alpha in colon cancer progression. We provide evidence for the lack of AP-2alpha expression in the late stages of colon cancer cells. Re-expression of the AP-2alpha gene in the AP-2alpha-negative SW480 colon cancer cells suppressed their tumorigenicity following orthotopic injection into the cecal wall of nude mice. The inhibition of tumor growth could be attributed to the increased expression of E-cadherin and decreased expression and activity of matrix-metalloproteinase-9 (MMP-9) in the transfected cells, as well as a substantial loss of their in vitro invasive properties. Conversely, targeting constitutive expression of AP-2alpha in AP-2-positive KM12C colon cancer cells with small interfering RNA resulted in an increase in their invasive potential, downregulation of E-cadherin and increased expression of MMP-9. In SW480 cells, re-expression of AP-2alpha resulted in a fourfold increase in the activity of E-cadherin promoter, and a 5-14-fold decrease in the activity of MMP-9 promoter, indicating transcriptional regulation of these genes by AP-2alpha. Chromatin immunoprecipitation assay showed that re-expressed AP-2alpha directly binds to the promoter of E-cadherin, where it has been previously reported to act as a transcriptional activator. Furthermore, chromatin immunoprecipitation assay revealed AP-2alpha binding to the MMP-9 promoter, which ensued by decreased binding of transcription factor Sp-1 and changes in the recruitment of transcription factors to a distal AP-1 element, thus, contributing to the overall downregulation of MMP-9 promoter activity. Collectively, our data provide evidence that AP-2alpha acts as a tumor suppressor gene in colon cancer..

Photodynamic properties of meta-tetra(hydroxyphenyl)chlorin in human tumor cells.

The photodynamic properties of a second-generation photodynamic sensitizer, meta-tetra(hydroxyphenyl)chlorin (mTHPC) were studied by dye-sensitized photoinactivation (650 nm) of HT29 human adenocarcinoma cells in culture. The photocytotoxicity of mTHPC in vitro depended on the presence of molecular oxygen. A strong inhibition of the photocytotoxicity of mTHPC was observed upon addition of sodium azide, a known singlet oxygen quencher. Photocytotoxicity was not inhibited by scavengers of superoxide anion radical, hydrogen peroxide and hydroxyl radicals. We suggest that mTHPC photosensitizes cell killing predominantly by type II, singlet oxygen-mediated photodynamic reactions. Illumination of cells preloaded with mTHPC induced peroxidation of membrane lipids. Inhibition of photoperoxidation by alpha-tocopherol (0.1 mM) present during illumination did not result in any decrease in toxicity, suggesting that reactions of lipid peroxidation play only a minor role in the overall photocytotoxic effect of mTHPC.

Effects of glutathione peroxidase and catalase on hemolysis and methemoglobin modifications induced by photooxidized psoralen.

Psoralen photooxidation products (POP products) were obtained by UVA irradiation (365 nm, 180-640 W/m2) of an aqueous psoralen solution with fluences of 0-800 kJ/m2. Preincubation of POP products with glutathione peroxidase (GSHPer) or catalase, as well as presence of catalase during UVA irradiation of the aqueous psoralen solution did not influence their hemolytic activity. However, both GSHPer and catalase inhibited POP-induced conversion of methemoglobin. This indicates that hydrogen peroxide and psoralen peroxides destructible by GSHPer, which are being produced during psoralen photooxidation, do not possess hemolytic activity. Furthermore, hydrogen peroxide does not appear to serve as an intermediate in the process of hemolysin formation. Hydrogen peroxide generated during psoralen photooxidation is apparently the main POP product responsible for MetHb conversion.

Subcellular localization of meta-tetra (hydroxyphenyl) chlorin in human tumor cells subjected to photodynamic treatment.

Subcellular localization of meta-tetra (hydroxyphenyl) chlorin (mTHPC) in HT29 human colon adenocarcinoma cells has been studied by means of fluorescence microscopy. The observed diffuse intracellular distribution of mTHPC fluorescence outside the nucleus indicates general staining of cellular organelles. No changes in dye fluorescence pattern are evident during and immediately after cell illumination. Alternatively, the changes in mTHPC fluorescence pattern are observed upon subsequent cell incubation, and are characterized by the appearance of distinct bright fluorescence zones. Reaching a maximum 1 h after illumination, modifications of the fluorescence pattern then gradually disappear in parallel with the formation of plasma membrane blebs, suggesting that cell necrotic lysis is taking place. Photosensitized damage to mitochondria and the Golgi apparatus has been studied using fluorescent probes 1 h after irradiation, the stage of extensive cytoplasm vacuolization, and reveal alterations of these organelles. Changes in the mTHPC fluorescence pattern and mTHPC photocytotoxicity, as measured by the MTT test 24 h after illumination, are inhibited by sodium azide, a singlet oxygen quencher.

Enhancement of meta-tetrahydroxyphenylchlorin-sensitized photodynamic treatment on human tumor xenografts using a water-soluble vitamin E analogue, Trolox.

Photodynamic therapy (PDT) using meta-tetrahydroxyphenylchlorin (mTHPC) performed on HT29 human colon adenocarcinoma xenografts in nude mice was shown to be enhanced by Trolox, a water-soluble vitamin E analogue. Trolox, injected i.p. at 250 mg/kg body weight 90 min before PDT, delayed tumor doubling time from 13 (PDT only) to 19 days. Enhancement of the tumoricidal effect of PDT by Trolox required the presence of the drug at the photochemical stage since its injection after irradiation is ineffective. HPLC measurements indicated that 1 hr after injection the Trolox concentration in plasma was as high as 0.8 mM. In vivo measurements of mTHPC fluorescence in mice treated by PDT with or without Trolox injection showed that Trolox did not protect mTHPC from photodegradation. Laser flash photolysis studies performed in solution demonstrated that Trolox reduces triplet mTHPC efficiently (reaction rate constant 2 x 10(7) M(-1) * sec(-1)) leading to the formation of radical products. Kinetic considerations suggest that the Trolox-mediated radical pathway can work in relay with singlet oxygen in hypoxic conditions, providing a possible explanation for the observed enhancement of mTHPC-sensitized PDT by Trolox.