Mitoferrin-2-dependent mitochondrial iron uptake sensitizes human head and neck squamous carcinoma cells to photodynamic therapy.

Photodynamic therapy (PDT) is a promising approach to treat head and neck cancer cells. Here, we investigated whether mitochondrial iron uptake through mitoferrin-2 (Mfrn2) enhanced PDT-induced cell killing. Three human head and neck squamous carcinoma cell lines (UMSCC1, UMSCC14A, and UMSCC22A) were exposed to light and Pc 4, a mitochondria-targeted photosensitizer. The three cell lines responded differently: UMSCC1 and UMSCC14A cells were more resistant, whereas UMSCC22A cells were more sensitive to Pc 4-PDT-induced cell death. In non-erythroid cells, Mfrn2 is an iron transporter in the mitochondrial inner membrane. PDT-sensitive cells expressed higher Mfrn2 mRNA and protein levels compared with PDT-resistant cells. High Mfrn2-expressing cells showed higher rates of mitochondrial Fe(2+) uptake compared with low Mfrn2-expressing cells. Bafilomycin, an inhibitor of the vacuolar proton pump of lysosomes and endosomes that causes lysosomal iron release to the cytosol, enhanced PDT-induced cell killing of both resistant and sensitive cells. Iron chelators and the inhibitor of the mitochondrial Ca(2+) (and Fe(2+)) uniporter, Ru360, protected against PDT plus bafilomycin toxicity. Knockdown of Mfrn2 in UMSCC22A cells decreased the rate of mitochondrial Fe(2+) uptake and delayed PDT plus bafilomycin-induced mitochondrial depolarization and cell killing. Taken together, the data suggest that lysosomal iron release and Mfrn2-dependent mitochondrial iron uptake act synergistically to induce PDT-mediated and iron-dependent mitochondrial dysfunction and subsequent cell killing. Furthermore, Mfrn2 represents a possible biomarker of sensitivity of head and neck cancers to cell killing after PDT.

Polysaccharide-rich red algae (Gelidium amansii) hot-water extracts ameliorate the altered plasma cholesterol and hepatic lipid homeostasis in high-fat diet-fed rats.

We have demonstrated that red algae Gelidium amansii (GA) hot-water extract (GHE) is a polysaccharide-rich fraction, containing 68.54% water-soluble indigestible carbohydrate polymers; the molecular weight of major polysaccharide is 892. Here, we investigated the mechanisms of GHE on plasma and hepatic lipid metabolisms in high-fat (HF) diet-fed rats. Rats were divided into: normal diet group, HF-diet group, HF-diet+5% GHE group, and HF-diet+1% cholestyramine group. GHE supplementation for 8 weeks significantly decreased plasma cholesterol, LDL-C, and VLDL-C levels and increased the fecal triglyceride and bile acid excretion in HF diet-fed rats. GHE group has lower lipid contents in the liver and adipose tissues. GHE supplementation decreased the activities of acetyl-CoA carboxylase, fatty acid synthase, and HMG-CoA reductase in the livers. The levels of increased phosphorylated AMP-activated protein kinase (AMPK), peroxisome proliferator activated receptor (PPAR)-α, farnesoid-X receptor (FXR), low density lipoprotein receptor (LDLR), and cytochrome P450-7A1 (CYP7A1) protein expression, and the decreased PPAR-γ protein expression in the livers were observed in GHE group. These results suggest that GHE supplementation is capable of interfering in cholesterol metabolism and increasing hepatic LDLR and CYP7A1 expression to decrease blood cholesterol, and activating FXR and AMPK to inhibit lipogenic enzyme activities and reduce the hepatic lipid accumulation.

PLGA nanoparticle encapsulation reduces toxicity while retaining the therapeutic efficacy of EtNBS-PDT in vitro.

Photodynamic therapy regimens, which use light-activated molecules known as photosensitizers, are highly selective against many malignancies and can bypass certain challenging therapeutic resistance mechanisms. Photosensitizers such as the small cationic molecule EtNBS (5-ethylamino-9-diethyl-aminobenzo[a]phenothiazinium chloride) have proven potent against cancer cells that reside within acidic and hypoxic tumour microenvironments. At higher doses, however, these photosensitizers induce "dark toxicity" through light-independent mechanisms. In this study, we evaluated the use of nanoparticle encapsulation to overcome this limitation. Interestingly, encapsulation of the compound within poly(lactic-co-glycolic acid) (PLGA) nanoparticles (PLGA-EtNBS) was found to significantly reduce EtNBS dark toxicity while completely retaining the molecule's cytotoxicity in both normoxic and hypoxic conditions. This dual effect can be attributed to the mechanism of release: EtNBS remains encapsulated until external light irradiation, which stimulates an oxygen-independent, radical-mediated process that degrades the PLGA nanoparticles and releases the molecule. As these PLGA-encapsulated EtNBS nanoparticles are capable of penetrating deeply into the hypoxic and acidic cores of 3D spheroid cultures, they may enable the safe and efficacious treatment of otherwise unresponsive tumour regions.

Protein triggered fluorescence switching of near-infrared emitting nanoparticles for contrast-enhanced imaging.

Sub-100 nm colloidal particles which are surface-functionalized with multiple environmentally-sensitive moieties have the potential to combine imaging, early detection, and the treatment of cancer with a single type of long-circulating "nanodevice". Deep tissue imaging is achievable through the development of particles which are surface-modified with fluorophores that operate in the near-infrared (NIR) spectrum and where the fluorophore's signal can be maximized by "turning-on" the fluorescence only in the targeted tissue. We present a general approach for the synthesis of NIR emitting nanoparticles that exhibit a protein triggered activation/deactivation of the emission. Dispersing the particles into an aqueous solution, such as phosphate buffered saline (PBS), resulted in an aggregation of the hydrophobic fluorophores and a cessation of emission. The emission can be reinstated, or activated, by the conversion of the surface-attached fluorophores from an aggregate to a monomeric species with the addition of an albumin. This activated probe can be deactivated and returned to a quenched state by a simple tryptic digestion of the albumin. The methodology for emission switching offers a path to maximize the signal from the typically weak quantum yield inherent in NIR fluorophores.

Lysosomal signaling enhances mitochondria-mediated photodynamic therapy in A431 cancer cells: role of iron.

In photodynamic therapy (PDT), light activates a photosensitizer added to a tissue, resulting in singlet oxygen formation and cell death. The photosensitizer phthalocyanine 4 (Pc 4) localizes primarily to mitochondrial membranes in cancer cells, resulting in mitochondria-mediated cell death. The aim of this study was to determine how lysosomes contribute to PDT-induced cell killing by mitochondria-targeted photosensitizers such as Pc 4. We monitored cell killing of A431 cells after Pc 4-PDT in the presence and absence of bafilomycin, an inhibitor of the vacuolar proton pump of lysosomes and endosomes. Bafilomycin was not toxic by itself, but greatly enhanced Pc 4-PDT-induced cell killing. To investigate whether iron loading of lysosomes affects bafilomycin-induced killing, cells were incubated with ammonium ferric citrate (30 µM) for 30 h prior to PDT. Ammonium ferric citrate enhanced Pc 4 plus bafilomycin-induced cell killing without having toxicity by itself. Iron chelators (desferrioxamine and starch-desferrioxamine) and the inhibitor of the mitochondrial calcium (and ferrous iron) uniporter, Ru360, protected against Pc 4 plus bafilomycin toxicity. These results support the conclusion that chelatable iron stored in the lysosomes enhances the efficacy of bafilomycin-mediated PDT and that lysosomal disruption augments PDT with Pc 4.

Signaling From Lysosomes Enhances Mitochondria-Mediated Photodynamic Therapy In Cancer Cells.

In photodynamic therapy (PDT), visible light activates a photosensitizing drug added to a tissue, resulting in singlet oxygen formation and cell death. Assessed by confocal microscopy, the photosensitizer phthalocyanine 4 (Pc 4) localizes primarily to mitochondrial membranes in cancer cells, resulting in mitochondria-mediated cell death. A Pc 4 derivative, Pc 181, accumulates into lysosomes. In comparison to Pc 4, Pc 181 was a more effective photosensitizer promoting killing cancer cells after PDT. The mode of cell death after Pc 181-PDT is predominantly apoptosis, and pancaspase and caspase-3 inhibitors prevent onset of the cell death. To assess further how lysosomes contribute to PDT, we monitored cell killing of A431cells after PDT in the presence and absence of bafilomycin, an inhibitor of the acidic vacuolar proton pump that collapses the pH gradient of the lysosomal/endosomal compartment. Bafilomycin by itself did not induce toxicity but greatly enhanced Pc 4-PDT-induced cell killing. In comparison to Pc 4, less enhancement of cell killing by bafilomycin occurred after Pc 181-PDT at photosensitizer doses producing equivalent cell killing in the absence of bafilomycin. These results indicate that lysosomal disruption can augment PDT with Pc 4, which targets predominantly mitochondria, but less so after PDT with Pc 181, since Pc 181 already targets lysosomes.