A new fluorescent sensor mitoferrofluor indicates the presence of chelatable iron in polarized and depolarized mitochondria.

Mitochondrial chelatable iron contributes to the severity of several injury processes, including ischemia/reperfusion, oxidative stress, and drug toxicity. However, methods to measure this species in living cells are lacking. To measure mitochondrial chelatable iron in living cells, here we synthesized a new fluorescent indicator, mitoferrofluor (MFF). We designed cationic MFF to accumulate electrophoretically in polarized mitochondria, where a reactive group then forms covalent adducts with mitochondrial proteins to retain MFF even after subsequent depolarization. We also show in cell-free medium that Fe2+ (and Cu2+), but not Fe3+, Ca2+, or other biologically relevant divalent cations, strongly quenched MFF fluorescence. Using confocal microscopy, we demonstrate in hepatocytes that red MFF fluorescence colocalized with the green fluorescence of the mitochondrial membrane potential (ΔΨm) indicator, rhodamine 123 (Rh123), indicating selective accumulation into the mitochondria. Unlike Rh123, mitochondria retained MFF after ΔΨm collapse. Furthermore, intracellular delivery of iron with membrane-permeant Fe3+/8-hydroxyquinoline (FeHQ) quenched MFF fluorescence by ∼80% in hepatocytes and other cell lines, which was substantially restored by the membrane-permeant transition metal chelator pyridoxal isonicotinoyl hydrazone. We also show FeHQ quenched the fluorescence of cytosolically coloaded calcein, another Fe2+ indicator, confirming that Fe3+ in FeHQ undergoes intracellular reduction to Fe2+. Finally, MFF fluorescence did not change after addition of the calcium mobilizer thapsigargin, which shows MFF is insensitive to physiologically relevant increases of mitochondrial Ca2+. In conclusion, the new sensor reagent MFF fluorescence is an indicator of mitochondrial chelatable Fe2+ in normal hepatocytes with polarized mitochondria as well as in cells undergoing loss of ΔΨm.

The mitochondrial calcium uniporter mediates mitochondrial Fe2+ uptake and hepatotoxicity after acetaminophen.

Acetaminophen (APAP) overdose disrupts hepatocellular lysosomes, which release ferrous iron (Fe2+) that translocates into mitochondria putatively via the mitochondrial calcium uniporter (MCU) to induce oxidative/nitrative stress, the mitochondrial permeability transition (MPT), and hepatotoxicity. To investigate how MCU deficiency affects mitochondrial Fe2+ uptake and hepatotoxicity after APAP overdose, global MCU knockout (KO), hepatocyte specific (hs) MCU KO, and wildtype (WT) mice were treated with an overdose of APAP both in vivo and in vitro. Compared to strain-specific WT mice, serum ALT decreased by 88 and 56%, respectively, in global and hsMCU KO mice at 24 h after APAP (300 mg/kg). Hepatic necrosis also decreased by 84 and 56%. By contrast, when MCU was knocked out in Kupffer cells, ALT release and necrosis were unchanged after overdose APAP. Intravital multiphoton microscopy confirmed loss of viability and mitochondrial depolarization in pericentral hepatocytes of WT mice, which was decreased in MCU KO mice. CYP2E1 expression, hepatic APAP-protein adduct formation, and JNK activation revealed that APAP metabolism was equivalent between WT and MCU KO mice. In cultured hepatocytes after APAP, loss of cell viability decreased in hsMCU KO compared to WT hepatocytes. Using fructose plus glycine to prevent cell killing, mitochondrial Fe2+ increased progressively after APAP, as revealed with mitoferrofluor (MFF), a mitochondrial Fe2+ indicator. By contrast in hsMCU KO hepatocytes, mitochondrial Fe2+ uptake after APAP was suppressed. Rhod-2 measurements showed that Ca2+ did not increase in mitochondria after APAP in either WT or KO hepatocytes. In conclusion, MCU mediates uptake of Fe2+ into mitochondria after APAP and plays a central role in mitochondrial depolarization and cell death during APAP-induced hepatotoxicity.

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.

Mitochondrial calcium and the permeability transition in cell death.

Dysregulation of Ca(2+) has long been implicated to be important in cell injury. A Ca(2+)-linked process important in necrosis and apoptosis (or necrapoptosis) is the mitochondrial permeability transition (MPT). In the MPT, large conductance permeability transition (PT) pores open that make the mitochondrial inner membrane abruptly permeable to solutes up to 1500 Da. The importance of Ca(2+) in MPT induction varies with circumstance. Ca(2+) overload is sufficient to induce the MPT. By contrast after ischemia-reperfusion to cardiac myocytes, Ca(2+) overload is the consequence of bioenergetic failure after the MPT rather than its cause. In other models, such as cytotoxicity from Reye-related agents and storage-reperfusion injury to liver grafts, Ca(2+) appears to be permissive to MPT onset. Lastly in oxidative stress, increased mitochondrial Ca(2+) and ROS generation act synergistically to produce the MPT and cell death. Thus, the exact role of Ca(2+) for inducing the MPT and cell death depends on the particular biologic setting.

Structural factors and mechanisms underlying the improved photodynamic cell killing with silicon phthalocyanine photosensitizers directed to lysosomes versus mitochondria.

The phthalocyanine photosensitizer Pc 4 has been shown to bind preferentially to mitochondrial and endoplasmic reticulum membranes. Upon photoirradiation of Pc 4-loaded cells, membrane components, especially Bcl-2, are photodamaged and apoptosis, as indicated by activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase, is triggered. A series of analogs of Pc 4 were synthesized, and the results demonstrate that Pcs with the aminopropylsiloxy ligand of Pc 4 or a similar one on one side of the Pc ring and a second large axial ligand on the other side of the ring have unexpected properties, including enhanced cell uptake, greater monomerization resulting in greater intracellular fluorescence and three-fold higher affinity constants for liposomes. The hydroxyl-bearing axial ligands tend to reduce aggregation of the Pc and direct it to lysosomes, resulting in four to six times more killing of cells, as defined by loss of clonogenicity, than with Pc 4. Whereas Pc 4-PDT photodamages Bcl-2 and Bcl-xL, Pc 181-PDT causes much less photodamage to Bcl-2 over the same dose-response range relative to cell killing, with earlier cleavage of Bid and slower caspase-3-dependent apoptosis. Therefore, within this series of photosensitizers, these hydroxyl-bearing axial ligands are less aggregated than is Pc 4, tend to localize to lysosomes and are more effective in overall cell killing than is Pc 4, but induce apoptosis more slowly and by a modified pathway.

A bioactivatable self-quenched nanogel for targeted photodynamic therapy.

Photodynamic therapy has attracted significant attention due to its localized treatment advantage. However, the non-specific distribution of photosensitizers and the subsequent potential toxicity caused by sunshine exposure hinder its wide adoption in cancer treatment. To minimize these unwanted effects and improve its efficacy, we developed a bioactivatable self-quenched nanogel, which remains in its inactive state in healthy tissues. Anti-EGFR Affibody decorated nanogels can effectively target head and neck cancer and release activated pheophorbide A in a reducing environment, such as in the tumor stroma and cytoplasm. Consequently, the EGFR targeted nanogel coupled with NIR irradiation alleviates tumor burden by 94.5% while not inducing systemic toxicity.

Click-Engineered, Bioresponsive, and Versatile Particle-Protein-Dye System.

We present a multifunctional polymer based nanoparticle platform for personalized nanotheranostic applications, which include photodynamic therapy and active targeting. In this system, poly(propargyl acrylate) (PA) particles were surface-modified with organic ligands and fluorophores (the payload) through an environmentally-sensitive linker. An azide modified bovine serum albumin (azBSA) was employed as the linker. This system prevents opsonization and, upon digestion, releases the payload. Attachment of the emitting payload to the particle through azide-modified bovine serum albumin (BSA) quenches emission, which can be again activated with digestion of the azBSA. The emission "turn-on" at a specific location will increase the signal-to-noise ratio. By utilizing human head and neck squamous carcinoma cells (UMSCC22A), photodynamic therapy studies with these particles gave promising reductions in cell growth. Additionally, the particle-protein-dye system is versatile as different fluorophores (such as silicon phthalocyanine or cyanine 3) can be attached to the protein and the same activation/deactivation behavior is observed. Active targeting can be employed to enhance the concentration of the payload in the designated tumor. Human lung carcinoma cells (A549) were utilized in toxicity studies where PA-azBSA particles were modified with a Survivin targeting ligand and indicated an enhanced cell death with the modified particles relative to the "free" Survivin targeting ligand.

Renal Glomerular Mitochondria Function in Salt-Sensitive Hypertension.

Salt-sensitive (SS) hypertension is accompanied with an early onset of proteinuria, which results from the loss of glomerular podocytes. Here, we hypothesized that glomerular damage in the SS hypertension occurs in part due to mitochondria dysfunction, and we used a unique model of freshly isolated glomeruli to test this hypothesis. In order to mimic SS hypertension, we used Dahl SS rats, an established animal model. Animals were fed a 0.4% NaCl (normal salt, NS) diet or challenged with a high salt (HS) 4% NaCl diet for 21 days to induce an increase in blood pressure (BP). Similar to previous studies, we found that HS diet caused renal hypertrophy, increased BP, glomerulosclerosis, and renal lesions such as fibrosis and protein casts. We did not observe changes in mitochondrial biogenesis in the renal cortex or isolated glomeruli fractions. However, Seahorse assay performed on freshly isolated glomeruli revealed that basal mitochondrial respiration, maximal respiration, and spare respiratory capacity were lower in the HS compared to the NS group. Using confocal imaging and staining for mitochondrial H2O2 using mitoPY1, we detected an intensified response to an acute H2O2 application in the podocytes of the glomeruli isolated from the HS diet fed group. TEM analysis showed that glomerular mitochondria from the HS diet fed group have structural abnormalities (swelling, enlargement, less defined cristae). Therefore, we report that glomerular mitochondria in SS hypertension are functionally and structurally defective, and this impairment could eventually lead to loss of podocytes and proteinuria. Thus, the glomerular-mitochondria axis can be targeted in novel treatment strategies for hypertensive glomerulosclerosis.

Fluorescence resonance energy transfer reveals a binding site of a photosensitizer for photodynamic therapy.

Phthalocyanine (Pc) 4, like many photosensitizers for photodynamic therapy (PDT), localizes to intracellular membranes, especially mitochondria. Pc 4-PDT photodamages Bcl-2 and Bcl-xL, antiapoptotic proteins interacting with the permeability transition pore complex that forms at contact sites between the inner and outer mitochondrial membranes. These complexes and the inner membrane are unique in containing the phospholipid cardiolipin. Nonyl-acridine orange (NAO) is a specific probe of cardiolipin. Here we show evidence for fluorescence resonance energy transfer from NAO to Pc 4, defining a binding site for the photosensitizer. This observation establishes an innovative tool for exploring the localization of other photosensitizers and additional fluorescent, mitochondrion-localizing drugs having appropriate spectral properties.

Apoptosis repressor with caspase recruitment domain (ARC) is expressed in cancer cells and localizes to nuclei.

Apoptosis repressor with caspase recruitment domain is expressed at high levels in brain and myogenic tissues, consistent with a role to inhibit apoptosis in the terminally differentiated cells. Expression of ARC in cancers is not known. In this study, we reported that ARC was highly expressed in various non-myogenic and non-neurogenic human and rat cancer cell lines. Unexpectedly, ARC was localized almost exclusively to the nuclei of cancer cells, which was unlike the cytoplasmic localization of ARC in non-cancer cells. Furthermore, nuclear ARC in cancer cells did not co-localize with nucleolus protein of 30 kDa, an alternatively spliced ARC isoform. These findings indicate that ARC is distributed differently in cancer cells than non-cancer cells and thus might play a role in neoplastic transformation.

Apoptosis and necrosis in health and disease: role of mitochondria.

Mitochondria play an important role in both the life and death of cells. Mitochondria are the powerhouse of the cell, providing over 90% of adenosine triphosphate (ATP) consumed by the cell. Mitochondrial energy production, however, is disrupted in various pathological situations leading to cellular Injury. The mechanisms causing the injury are turning out to be more complex than originally expected. For instance, calcium, oxidant chemicals, ischemia/ reperfusion, and a range of other agents promote onset of the mitochondrial permeability transition in mitochondria from liver, heart, and other tissues. Often the consequence of this event is ATP depletion, ion deregulation, mitochondrial and cellular swelling, activation of degradative enzymes, plasma membrane failure, and cell lysis. This is referred to as necrotic cell death. The mitochondrial permeability transition is also involved in apoptotic cell death. In this mode of death, the role of the permeability transition is to release proapoptotic proteins from mitochondria into the cytosol where with the aid of cellular ATP they complete the apoptotic cascade. Therefore, mitochondria contribute to both apoptotic and necrotic death.

Promotion of photodynamic therapy-induced apoptosis by the mitochondrial protein Smac/DIABLO: dependence on Bax.

Photodynamic therapy (PDT) using the second-generation photosensitizer phthalocyanine (Pc) 4 causes mitochondrial damage and induces apoptosis through the release of cytochrome c to the cytosol. Another protein of the mitochondrial intermembrane space, Smac/DIABLO (second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI), is also released to the cytosol in response to apoptotic stimuli and promotes caspase activation by binding IAP. To investigate the possible role of Smac/DIABLO in apoptosis induced by Pc 4-PDT, we transfected Smac/DIABLO (tagged at its C-terminus with green fluorescent protein [GFP]) into MCF-7c3 cells (human breast cancer MCF-7 cells stably transfected with procaspase-3) and DU-145 cells (human prostate cancer cells that express no Bax because of a frameshift insertion mutation). Confocal microscopy showed that recombinant Smac/DIABLO, like cytochrome c, localized to mitochondria and colocalized with MitoTracker Red. Three hours after exposure of MCF-7c3 cells to PDT (200 nM Pc 4 and 150 mJ/cm2 red light), Smac/DIABLO-GFP, as well as cytochrome c, was found largely in the cytosol. In contrast, for DU-145 cells, both Smac/DIABLO-GFP and cytochrome c remained in the mitochondria after PDT. By staining with Hoechst 33,342, typical apoptotic nuclei were observed in MCF-7c3 cells, but not in DU-145 cells, after Pc 4-PDT. These results suggest that the release of Smac/DIABLO from mitochondria may be regulated by a Bax-mediated mechanism and that Smac/DIABLO may cooperate with the cytochrome c-dependent apoptosis pathway. In addition, in MCF-7c3 cells transfected by Smac/DIABLO-GFP, apoptosis induced by Pc 4-PDT was greater than in cells transfected with the GFP vector alone or in untransfected cells, as determined by flow cytometry. Thus, Smac/DIABLO promotes apoptosis after Pc 4-PDT in a Bax-dependent manner and may facilitate the passage of PDT-treated cells through the late steps of apoptosis.

Triple-responsive expansile nanogel for tumor and mitochondria targeted photosensitizer delivery.

A pH, thermal, and redox potential triple-responsive expansile nanogel system (TRN), which swells at acidic pH, temperature higher than its transition temperature, and reducing environment, has been developed. TRN quickly expands from 108 nm to over 1200 nm (in diameter), achieving more than 1000-fold size enlargement (in volume), within 2 h in a reducing environment at body temperature. Sigma-2 receptor targeting-ligand functionalized TRN can effectively target head and neck tumor, and help Pc 4 targeting mitochondria inside cancer cells to achieve enhanced photodynamic therapy efficacy.

N-acetyl-L-cysteine sensitizes pancreatic cancers to gemcitabine by targeting the NFκB pathway.

First-line therapy for pancreatic cancer is gemcitabine. Although tumors may initially respond to the gemcitabine treatment, soon tumor resistance develops leading to treatment failure. Previously, we demonstrated in human MIA PaCa-2 pancreatic cancer cells that N-acetyl-l-cysteine (NAC), a glutathione (GSH) precursor, prevents NFκB activation via S-glutathionylation of p65-NFκB, thereby blunting expression of survival genes. In this study, we documented the molecular sites of S-glutathionylation of p65, and we investigated whether NAC can suppress NFκB signaling and augment a therapeutic response to gemcitabine in vivo. Mass spectrometric analysis of S-glutathionylated p65-NFκB protein in vitro showed post-translational modifications of cysteines 38, 105, 120, 160 and 216 following oxidative and nitrosative stress. Circular dichroism revealed that S-glutathionylation of p65-NFκB did not change secondary structure of the protein, but increased tryptophan fluorescence revealed altered tertiary structure. Gemcitabine and NAC individually were not effective in decreasing MIA PaCa-2 tumor growth in vivo. However, combination treatment with NAC and gemcitabine decreased tumor growth by approximately 50%. NAC treatment also markedly enhanced tumor apoptosis in gemcitabine-treated mice. Compared to untreated tumors, gemcitabine treatment alone increased p65-NFκB nuclear translocation (3.7-fold) and DNA binding (2.5-fold), and these effects were blunted by NAC. In addition, NAC plus gemcitabine treatment decreased anti-apoptotic XIAP protein expression compared to gemcitabine alone. None of the treatments, however, affected extent of tumor hypoxia, as assessed by EF5 staining. Together, these results indicate that adjunct therapy with NAC prevents NFκB activation and improves gemcitabine chemotherapeutic efficacy.

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.

A requirement for bid for induction of apoptosis by photodynamic therapy with a lysosome- but not a mitochondrion-targeted photosensitizer.

Photodynamic therapy (PDT) with lysosome-targeted photosensitizers induces the intrinsic pathway of apoptosis via the cleavage and activation of the BH3-only protein Bid by proteolytic enzymes released from photodisrupted lysosomes. To investigate the role of Bid in apoptosis induction and the role of damaged lysosomes on cell killing by lysosome-targeted PDT, we compared the responses of wild type and Bid-knock-out murine embryonic fibroblasts toward a mitochondrion/endoplasmic reticulum-binding photosensitizer, Pc 4, and a lysosome-targeted sensitizer, Pc 181. Whereas apoptosis and overall cell killing were induced equally well by Pc 4-PDT in both cell lines, Bid(-/-) cells were relatively resistant to induction of apoptosis and to overall killing following PDT with Pc 181, particularly at low PDT doses. Thus, Bid is critical for the induction of apoptosis caused by PDT with the lysosome-specific sensitizers, but dispensable for PDT targeted to other membranes.

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.

Glutathione supplementation potentiates hypoxic apoptosis by S-glutathionylation of p65-NFkappaB.

In murine embryonic fibroblasts, N-acetyl-L-cysteine (NAC), a GSH generating agent, enhances hypoxic apoptosis by blocking the NFkappaB survival pathway (Qanungo, S., Wang, M., and Nieminen, A. L. (2004) J. Biol. Chem. 279, 50455-50464). Here, we examined sulfhydryl modifications of the p65 subunit of NFkappaB that are responsible for NFkappaB inactivation. In MIA PaCa-2 pancreatic cancer cells, hypoxia increased p65-NFkappaB DNA binding and NFkappaB transactivation by 2.6- and 2.8-fold, respectively. NAC blocked these events without having an effect on p65-NFkappaB protein levels and p65-NFkappaB nuclear translocation during hypoxia. Pharmacological inhibition of the NFkappaB pathway also induced hypoxic apoptosis, indicating that the NFkappaB signaling pathway is a major protective mechanism against hypoxic apoptosis. In cell lysates after hypoxia and treatment with N-ethylmaleimide (thiol alkylating agent), dithiothreitol (disulfide reducing agent) was not able to increase binding of p65-NFkappaB to DNA, suggesting that most sulfhydryls in p65-NFkappaB protein were in reduced and activated forms after hypoxia, thereby being blocked by N-ethylmaleimide. In contrast, with hypoxic cells that were also treated with NAC, dithiothreitol increased p65-NFkappaB DNA binding. Glutaredoxin (GRx), which specifically catalyzes reduction of protein-SSG mixed disulfides, reversed inhibition of p65-NFkappaB DNA binding in extracts from cells treated with hypoxia plus NAC and restored NFkappaB activity. This finding indicated that p65-NFkappaB-SSG was formed in situ under hypoxia plus NAC conditions. In cells, knock-down of endogenous GRx1, which also promotes protein glutathionylation under hypoxic radical generating conditions, prevented NAC-induced NFkappaB inactivation and hypoxic apoptosis. The results indicate that GRx-dependent S-glutathionylation of p65-NFkappaB is most likely responsible for NAC-mediated NFkappaB inactivation and enhanced hypoxic apoptosis.

Mdm2 and HIF-1alpha interaction in tumor cells during hypoxia.

The interaction between HIF-1alpha, Mdm2, and p53 proteins during hypoxia has received recent attention. Here, we investigated the consequences of interaction between HIF-1alpha and Mdm2 under hypoxic conditions. Endogenous HIF-1alpha and Mdm2 proteins were co-immunoprecipitated from lysates of hypoxic HCT116 p53WT and p53(-/-) cells, suggesting that association of these two proteins is a p53-independent event. The cellular Mdm2 protein content was not significantly altered in hypoxic tumor cells. Overexpression of Mdm2 resulted in an increase in HIF-1alpha protein content in hypoxic cells and increased hypoxia-induced vascular endothelial growth factor (VEGF) transcriptional activation. These results point toward a novel and p53-independent function of Mdm2 to promote tumor cell adaptations to hypoxia by interacting with and promoting HIF-1 activation.