Extracellular reactive oxygen species are generated by a plasma membrane oxidative phosphorylation system.

Although the oxidative phosphorylation (OXPHOS) system has been found in mitochondria and the plasma membrane of various mammalian cell lines, understanding the physiological functions of the plasma membrane OXPHOS system is challenging. Here, we demonstrated that OXPHOS I, II, III, IV and V subunits were expressed in the plasma membrane of HepG2 cells and primary mouse hepatocytes, as determined by non-permeabilized immunofluorescence, total internal reflection fluorescence (TIRF) microscopy, cell surface-biotin labeling and plasma membrane and lipid raft isolation. Next, we demonstrated that NADH administration generated extracellular superoxide and improved insulin signaling in HepG2 cells and primary mouse hepatocytes. The NADH-dependent generation of extracellular superoxide was prevented by knockdown of NDUFV-1, the first subunit of OXPHOS I receiving electrons from NADH and the NADH-improved insulin signaling was abolished by extracellular catalase. Thus, we conclude that the OXPHOS system in the plasma membrane may be required for the generation of extracellular ROS and the regulation of insulin signaling.

Selective Cannabinoid Receptor-1 Agonists Regulate Mast Cell Activation in an Oxazolone-Induced Atopic Dermatitis Model.

BACKGROUND: Many inflammatory mediators, including various cytokines (e.g. interleukins and tumor necrosis factor [TNF]), inflammatory proteases, and histamine are released following mast cell activation. However, the endogenous modulators for mast cell activation and the underlying mechanism have yet to be elucidated. Endogenous cannabinoids such as palmitoylethanolamide (PEA) and N-arachidonoylethanolamine (anandamide or AEA), were found in peripheral tissues and have been proposed to possess autacoid activity, implying that cannabinoids may downregulate mast cell activation and local inflammation. OBJECTIVE: In order to investigate the effect of cannabinoid receptor-1 (CB1R) agonists on mast cell activation, AEA-derived compounds were newly synthesized and evaluated for their effect on mast cell activation. METHODS: The effects of selected compounds on FcεRI-induced histamine and ß-hexosaminidase release were evaluated in a rat basophilic leukemia cell line (RBL-2H3). To further investigate the inhibitory effects of CB1R agonist in vivo, an oxazolone-induced atopic dermatitis mouse model was exploited. RESULTS: We found that CB1R inhibited the release of inflammatory mediators without causing cytotoxicity in RBL-2H3 cells and that CB1R agonists markedly and dose-dependently suppressed mast cell proliferation indicating that CB1R plays an important role in modulating antigen-dependent immunoglobulin E (IgE)-mediated mast cell activation. We also found that topical application of CB1R agonists suppressed the recruitment of mast cells into the skin and reduced the level of blood histamine. CONCLUSION: Our results indicate that CB1R agonists down-regulate mast cell activation and may be used for relieving inflammatory symptoms mediated by mast cell activation, such as atopic dermatitis, psoriasis, and contact dermatitis.

Withaferin-A Inhibits Colon Cancer Cell Growth by Blocking STAT3 Transcriptional Activity.

BACKGROUND: Withania somnifera (known as Ashwagandha) is a medicinal plant used in the ayurvedic medicines in India. Withaferin-A, a withanolide derived from the leaf extract of W. somnifera, has been reported to exhibit anti-tumor activity against various cancer cells, such as leukemia, breast cancer and colon cancer cells. METHODS: We investigated the anti-cancer effects of withaferin-A on the proliferation and migration of human colorectal cancer (HCT116) cells. And we evaluated the effects of withaferin-A on the transcriptional activity of STAT3 and the growth of HCT116 cells in xenograft mouse tumor model. RESULTS: In the present study, we found that withaferin-A inhibited the proliferation and migration of HCT116 cells in a concentration-dependent manner. Treatment of HCT116 cells with withaferin-A attenuated interleukin-6-induced activation of STAT3, which has been implicated in the development and progression of colon cancer. To examine the effect of withaferin-A on HCT116 cells proliferation in vivo, we generated HCT116 cells xenograft tumors in Balb/c nude mice and treated the tumor bearing mice with or without withaferin-A intraperitoneally. Treatment with withaferin-A exhibited significant decrease in the volume and weight of tumors as compared to untreated controls. CONCLUSIONS: The present study suggests that withaferin-A holds the potential to be developed as a small molecule inhibitor of STAT3 for the treatment of HCT116.

Lipid raft proteome reveals that oxidative phosphorylation system is associated with the plasma membrane.

Although accumulating proteomic analyses have supported the fact that mitochondrial oxidative phosphorylation (OXPHOS) complexes are localized in lipid rafts, which mediate cell signaling, immune response and host-pathogen interactions, there has been no in-depth study of the physiological functions of lipid-raft OXPHOS complexes. Here, we show that many subunits of OXPHOS complexes were identified from the lipid rafts of human adipocytes, C2C12 myotubes, Jurkat cells and surface biotin-labeled Jurkat cells via shotgun proteomic analysis. We discuss the findings of OXPHOS complexes in lipid rafts, the role of the surface ATP synthase complex as a receptor for various ligands and extracellular superoxide generation by plasma membrane oxidative phosphorylation complexes.

Differential regulation of the antiapoptotic action of B-cell lymphoma 2 (Bcl-2) and B-cell lymphoma extra long (Bcl-xL) by c-Jun N-terminal protein kinase (JNK) 1-involved pathway in neuroglioma cells.

Here, we confirmed that stable expression of B-cell lymphoma-xL (Bcl-xL) in N18TG neuroglioma cells could suppress c-Jun N-terminal protein kinase (JNK) activation, nuclear fragmentation, and cell death caused by etoposide treatment. Moreover, additional overexpression of JNK1 led to partially antagonize the antiapoptotic environment attained by Bcl-xL, implying that JNK1-involved pathway may play a role in down-regulation of the antiapoptotic effect of Bcl-xL. However, the antagonistic effect of JNK1 on the antiapoptotic action of Bcl-xL was significantly weaker than that on the action of Bcl-2. Interestingly, we found that overexpression of JNK1 led to increase of Bcl-xL expression. Thus, these results suggest that Bcl-xL and Bcl-2 may induce its antiapoptotic effect in a different mechanism, provoking the possibility of involvement of JNK1-involved pathway in Bcl-xL expression.

Secretion of adenylate kinase 1 is required for extracellular ATP synthesis in C2C12 myotubes.

Extracellular ATP (exATP) has been known to be a critical ligand regulating skeletal muscle differentiation and contractibility. ExATP synthesis was greatly increased with the high level of adenylate kinase 1 (AK1) and ATP synthase beta during C2C12 myogenesis. The exATP synthesis was abolished by the knock-down of AK1 but not by that of ATP synthase beta in C2C12 myotubes, suggesting that AK1 is required for exATP synthesis in myotubes. However, membrane-bound AK1beta was not involved in exATP synthesis because its expression level was decreased during myogenesis in spite of its localization in the lipid rafts that contain various kinds of receptors and mediate cell signal transduction, cell migration, and differentiation. Interestingly, cytoplasmic AK1 was secreted from C2C12 myotubes but not from C2C12 myoblasts. Taken together all these data, we can conclude that AK1 secretion is required for the exATP generation in myotubes.

Sustained ERK activation is involved in the kaempferol-induced apoptosis of breast cancer cells and is more evident under 3-D culture condition.

In order to determine the effects of a variety of flavonoids, we applied differing amounts of several flavonoids to human breast cancer cells. Kaempferol treatment resulted in significant reduction of cell viability in the MCF-7 cells, although it exerted only minor effect on the cell viability of MDA-MB-231 or mammary epithelial HC-11 cells. Kaempferol was demonstrated to induce sustained ERK activation concomitantly with MEK1 and ELK1 activation, and this kaempferol-induced apoptosis was suppressed by treatment with PD98059, the overexpression of a kinase-inactive ERK mutant, or ERK siRNA. Kaempferol treatment was shown to profoundly induce the generation of fluorescent DCF in the MCF-7 cells, and treatment with N-acetyl cysteine suppressed kaempferol-induced PARP cleavage. Moreover, because breast cancer is associated with increased collagen synthesis and accumulation, we utilized a collagen-based 3D culture method. Under the 3-dimensional culture condition employed herein, kaempferol treatment was shown to result in a significant reduction in cell viability, an effect which occurred in a dose-dependent manner. Compared with what was observed under conventional 2D culture condition, we observed more evident apoptotic cell death and ERK activation as the result of kaempferol treatment in a collagen-based 3D culture environment. Similar to the case of conventional 2D cultured cells, the addition of PD98059 significantly suppressed intracellular ROS production. Collectively, these results show that the sustained activation of the ERK signaling pathway is markedly involved in kaempferol-induced apoptosis of breast cancer MCF-7 cells, and that this effect is more evident under 3D culture condition.

Regulation of apoptosis by modified naringenin derivatives in human colorectal carcinoma RKO cells.

Flavonoids are micronutrients that are widely detected in foods of plant origin and have been ascribed pharmacological properties. Several biological functions of flavonoids have been thus far identified, whereas there currently exists a lack of evidence to support the relationship between the structure-activity relationship and apoptosis-inducing activity. In an attempt to determine the importance of the OH group or substitution of the 5- or 7-carbon in the diphenylpropane skeleton of flavonoids, we selected 14 different flavonoids with different structures, particularly with regard to the 5- or 7-carbon, and found that naringenin treatment caused a slight decrease in the cell viability of the human colorectal carcinoma RKO cells. Next, in order to characterize the effects of specific substitutions of the 7-carbon of naringenin on apoptosis-regulatory activities, and in an attempt to develop anti-proliferative flavonoid derivatives that would be more effective against colon cancer, we originally synthesized several modified naringenin derivatives (MNDs) including 7-O-benzyl naringenin (KUF-1) and 7-O-(m-metoxybenzyl) naringenin (KUF-2). Treatment with KUF-1 or KUF-2 resulted in significant apoptosis-inducing effects concomitant with losses in mitochondrial membrane potential, caspase activation, intracellular ROS production, and sustained ERK activation. Our data show that KUF-1 or KUF-2 regulate the apoptosis of RKO cells via intracellular ROS production coupled with the concomitant activation of the ERK signaling pathway, thereby implying that hydroxylation or substitution at C7 is critical for the apoptosis-inducing activity of flavonoids.

Induction of apoptotic cell death by synthetic naringenin derivatives in human lung epithelial carcinoma A549 cells.

Although flavonoids, which are both qualitatively and quantitatively one of the largest groups of natural products, exhibit a variety of beneficial health effects, the exact molecular mechanism of the cellular activities is still not fully explained and there currently exists a lack of evidence for any relationship between the structure-activity relationship and apoptosis-inducing activity. In order to determine the importance of the OH group or substitution of the 5 or carbon-7 in the diphenylpropane skeleton of flavonoids, we originally synthesized several modified naringenin derivatives, including 7-O-benzyl naringenin (KUF-1) and 7-O-(MeO-L-Leu-D-Pro-carbonylmethyl) naringenin (KUF-7). Treatment with KUF-1 or KUF-7 resulted in significant apoptosis-inducing effects concomitant with chromatin condensation, caspase activation, and intracellular ROS production. Our data indicate that originally synthesized naringenin derivatives, KUF-1 and KUF-7 differentially regulate the apoptosis of A549 cells via intracellular ROS production coupled with the concomitant activation of the caspase cascade signaling pathway, thereby implying that hydroxylation or substitution at Carbon-7 is critical for the apoptosis-inducing activity of flavonoids.

Negative regulation of SEK1 signaling by serum- and glucocorticoid-inducible protein kinase 1.

Serum- and glucocorticoid-inducible protein kinase 1 (SGK1) has been implicated in diverse cellular activities including the promotion of cell survival. The molecular mechanism of the role of SGK1 in protection against cellular stress has remained unclear, however. We have now shown that SGK1 inhibits the activation of SEK1 and thereby negatively regulates the JNK signaling pathway. SGK1 was found to physically associate with SEK1 in intact cells. Furthermore, activated SGK1 mediated the phosphorylation of SEK1 on serine 78, resulting in inhibition of the binding of SEK1 to JNK1, as well as to MEKK1. Replacement of serine 78 of SEK1 with alanine abolished SGK1-mediated SEK1 inhibition. Oxidative stress upregulated SGK1 expression, and depletion of SGK1 by RNA interference potentiated the activation of SEK1 induced by oxidative stress in Rat2 fibroblasts. Moreover, such SGK1 depletion prevented the dexamethasone-induced increase in SGK1 expression, as well as the inhibitory effects of dexamethasone on paclitaxel-induced SEK1-JNK signaling and apoptosis in MDA-MB-231 breast cancer cells. Together, our results suggest that SGK1 negatively regulates stress-activated signaling through inhibition of SEK1 function.

Interplay between PI3K/Akt and MAPK signaling pathways in DNA-damaging drug-induced apoptosis.

In order to elucidate the role of the mitogen-activated protein kinases, including JNK, p38 MAPK and ERK, as well as the survival-associated PI3K/Akt signaling pathway, in the response to chemotherapy, we have conducted a comparative study regarding the effects of doxorubicin on these pathways. Doxorubicin was determined to elicit the apoptosis of NIH3T3 cells in a dose-dependent manner. Prior to cell death, both Akt and p38 MAPK were transiently activated, and subsequently inactivated almost wholly, whereas ERK and JNK evidenced sustained activations in response to the drug treatment. The inhibition of PI3K/Akt and p38 MAPK both accelerated and enhanced doxorubicin-induced apoptosis and ERK inhibition apparently exerted negative effect on apoptosis. The modulation of PI3K/Akt activation by treatment of LY294002 or expression of Akt mutants such as Akt-DN or Myr-Akt exerted a significant effect on the activation of ERK1/2. We also observed that PI3K/Akt and sustained ERK activation were associated intimately with the etoposide-induced apoptosis. Taken together, our results clearly suggest that the differential regulation of the PI3K/Akt, ERK1/2, and p38 MAPK signaling pathways are crucial in the context of DNA-damaging drug-induced apoptosis, and this has compelled us to propose that the sustained activation of ERK1/2 pathway may be generally involved in the apoptosis induced by anticancer DNA-damaging drugs, including doxorubicin and etoposide.

Extracellular ATP is generated by ATP synthase complex in adipocyte lipid rafts.

Mitochondrial biogenesis is known to accompany adipogenesis to complement ATP and acetyl-CoA required for lipogenesis. Here, we demonstrated that mitochondrial proteins such as ATP synthase alpha and beta, and cytochrome c were highly expressed during the 3T3-L1 differentiation into adipocytes. Fully-differentiated adipocytes showed a significant increase of mitochondria under electron microscopy. Analysis by immunofluorescence, cellular fractionation, and surface biotinylation demonstrated the elevated levels of ATP synthase complex found not only in the mitochondria but also on the cell surface (particularly lipid rafts) of adipocytes. High rate of ATP (more than 30 microM) synthesis from the added ADP and P(i) in the adipocyte media suggests the involvement of the surface ATP synthase complex for the extracellular ATP synthesis. In addition, this ATP synthesis was significantly inhibited in the presence of oligomycin, an ATP synthase inhibitor, and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an ATP synthase uncoupler. Decrease of extracellular ATP synthesis in acidic but not in basic media further indicates that the surface ATP synthase may also be regulated by proton gradient through the plasma membrane.