Search

Background/purpose: Oral submucous fibrosis (OSF) is a precancerous condition of oral cancer with a complex etiology. Our previous work has demonstrated that non-coding RNA miR-1246 contributes to the cancer stemness of oral cancer. In the current study, we sought to investigate the effect of the inhibition of miR-1246 on the oral fibrogenesis. Methods: The expression levels of miR-1246 in OSF tissues and fibrotic buccal mucosal fibroblasts (fBMFs) were examined by qRT-PCR. Collagen gel contraction and migration assays were conducted to evaluate the myofibroblast activities. The relationship between miR-1246 and type I collagen was assessed and the protein expression of type I collagen was determined by Western blot. Results: MiR-1246 expression was upregulated in both OSF specimen and fBMFs compared to the normal counterparts. Inhibition of miR-1246 successfully suppressed the myofibroblast activities, including collagen gel contractility and migration capacity. Moreover, the expression of miR-1246 was positively correlated with type I collagen and the expression of type I collagen was abrogated by repression of miR-1246. Conclusion: MiR-1246 is not only critical to the maintenance of oral stemness but also important to the activation of myofibroblasts. Our results showed that miR-1246 is positively associated with the type I collagen, which may be a downstream effector of miR-1246 and responsible for the fibrosis effect on fBMFs. Keywords: Oral submucous fibrosis, MicroRNA-1246, Type I collagen, Buccal mucosal fibroblasts

To achieve a good understanding of the characteristics of a protein, it is important to study its stability and folding kinetics. Investigations of protein stability have been recently applied to drug-target identification, drug screening, and proteomic studies. The efficiency of the experiments performed to study protein stability and folding kinetics is now a crucial factor that needs to be optimized for these potential applications. However, the standard procedures used to carry out these experiments are usually complicated and time consuming. Large number of measurements is the bottleneck that limits the application of protein folding to large-scale experiments. To overcome this limitation, we developed a method denoted as "one-pot analysis" which is based on taking a single measurement from a mixture of samples rather than from every sample. We combined one-pot analysis with pulse proteolysis to determine the effects of the binding of maltose to maltose-binding protein on the protein folding properties. After carrying out a simple optimization, we demonstrated that protein stability or unfolding kinetics could be measured accurately with just one detection measurement. We then further applied the optimized conditions to cellular thermal shift assay (CETSA). Combining one-pot analysis with CETSA led to a successful determination of the effects of the binding of methotrexate to dihydrofolate reductase in HCT116 cancer cells. Our results demonstrated the applicability of one-pot analysis to energetics-based methods for studying protein folding. We expect the combination of one-pot analysis and energetics-based methods to significantly benefit studies such as drug-target identification, proteomic investigations, and drug screening.

Therapeutic administration of glucocorticoids (GCs) is frequently used as add-on chemotherapy for palliative purposes during breast cancer treatment. Recent studies have shown that GC treatment induces microRNA-708 in ovarian cancer cells, resulting in impaired tumor cell proliferation and metastasis. However, the regulatory functions of GCs on miR-708 and its downstream target genes in human breast cancer cells (BCCs) are poorly understood. In this study, we found that treatment with either the synthetic GC dexamethasone (DEX) or the natural GC mimic, antcin A (ATA) significantly increased miR-708 expression by transactivation of glucocorticoid receptor alpha (GRα) in MCF-7 and MDA-MB-231 human BCCs. Induction of miR-708 by GR agonists resulted in inhibition of cell proliferation, cell-cycle progression, cancer stem cell (CSC)-like phenotype and metastasis of BCCs. In addition, GR agonist treatment or miR-708 mimic transfection remarkably inhibited IKKβ expression and suppressed nuclear factor-kappaB (NF-κB) activity and its downstream target genes, including COX-2, cMYC, cyclin D1, Matrix metalloproteinase (MMP)-2, MMP-9, CD24, CD44 and increased p21CIP1 and p27KIP1 that are known to be involved in proliferation, cell-cycle progression, metastasis and CSC marker protein. BCCs xenograft models indicate that treatment with GR agonists significantly reduced tumor growth, weight and volume. Overall, our data strongly suggest that GR agonists induced miR-708 and downstream suppression of NF-κB signaling, which may be applicable as a novel therapeutic intervention in breast cancer treatment.

Heliomycin (also known as resistomycin) is an antibiotic with a broad spectrum of biological activities. However, low aqueous solubility and poor knowledge of its chemical properties have limited the development of this natural product. Here, we present an original scheme for the introduction of aminoalkylamine residues at positions 3, 5, and 7 of heliomycin and, using this, have prepared a series of novel water-soluble derivatives. The addition of side chains to the heliomycin scaffold significantly improves their interaction with different DNA secondary structures. One derivative, 7-deoxy-7-(2-aminoethyl)amino-10-O-methylheliomycin (8e), demonstrated affinity, stabilization potential, and good selectivity toward i-motif-forming DNA sequences over the duplex and G-quadruplex. Heliomycin derivatives therefore represent promising molecular scaffolds for further development as DNA-i-motif interacting ligands and potential chemotherapeutic agents.

Oral submucous fibrosis (OSF) is a precancerous condition with symptoms of limited mouth opening and areca nut chewing habit has been implicated in its pathogenesis. Hinokitiol, a natural tropolone derived from Chamacyparis taiwanensis, has been reported to improve oral lichen planus and inhibit various cancer cells. Here, we showed that hinokitiol reduced the myofibroblast activities in fBMFs and prevented the arecoline-induced transdifferentiation. Treatment of hinokitiol dose-dependently downregulated the myofibroblast markers as well as various EMT transcriptional factors. In particular, we identified that Snail was able to bind to the E-box in the α-SMA promoter. Our data suggested that exposure of fBMFs to hinokitiol mitigated the hallmarks of myofibroblasts, while overexpression of Snail eliminated the effect of hinokitiol. These findings revealed that the inhibitory effect of hinokitiol on myofibroblasts was mediated by repression of α-SMA via regulation of Snail and showed the anti-fibrotic potential of hinokitiol in the treatment of OSF.

// Huei-Yu Chen 1 , Hsiao-Ling Cheng 1 , Yi-Hui Lee 1 , Tien-Ming Yuan 1, 2 , Shi-Wen Chen 2 , You-Yu Lin 1 , Pin Ju Chueh 1, 3, 4, 5 1 Institute of Biomedical Sciences, National Chung Hsing University, Taichung, 40227, Taiwan 2 Department of Surgery, Feng-Yuan Hospital, Ministry of Health and Welfare, Taichung 42055, Taiwan 3 Graduate Institute of Basic Medicine, China Medical University, Taichung, 40402, Taiwan 4 Department of Medical Research, China Medical University Hospital, Taichung, 40402, Taiwan 5 Department of Biotechnology, Asia University, Taichung, 41354, Taiwan Correspondence to: Pin Ju Chueh, email: pjchueh@dragon.nchu.edu.tw Keywords: apoptosis, deacetylase, oxaliplatin, tumor-associated NADH oxidase (tNOX or ENOX2), sirtuin 1 (SIRT1) Received: October 20, 2016 Accepted: January 09, 2017 Published: January 21, 2017 ABSTRACT Oxaliplatin belongs to the platinum-based drug family and has shown promise in cancer treatment. The major mechanism of action of platinum compounds is to form platinum–DNA adducts, leading to DNA damage and apoptosis. Accumulating evidence suggests that they might also target non-DNA molecules for their apoptotic activity. We explored the effects of oxaliplatin on a tumor-associated NADH oxidase (tNOX) in gastric cancer lines. In AGS cells, we found that the oxaliplatin-inhibited tNOX effectively attenuated the NAD + /NADH ratio and reduced the deacetylase activity of an NAD + -dependent sirtuin 1, thereby enhancing p53 acetylation and apoptosis. Similar results were also observed in tNOX-knockdown AGS cells. In the more aggressive MKN45 and TMK-1 lines, oxaliplatin did not inhibit tNOX, and induced only minimal apoptosis and cytotoxicity. However, the downregulation of either sirtuin 1 or tNOX sensitized TMK-1 cells to oxaliplatin-induced apoptosis. Moreover, tNOX-depletion in these resistant cells enhanced spontaneous apoptosis, reduced cyclin D expression and prolonged the cell cycle, resulting in diminished cancer cell growth. Together, our results demonstrate that oxaliplatin targets tNOX and SIRT1, and that the tNOX-NAD + -sirtuin 1 axis is essential for oxaliplatin-induced apoptosis.

Background Oxaliplatin belongs to the platinum-based drug family and has shown promise in treating cancer by binding to DNA to induce cytotoxicity. However, individual patients show diverse therapeutic responses toward oxaliplatin due to yet-unknown underlying mechanisms. We recently established that oxaliplatin also exert its anti-cancer activity in gastric cancer cell lines by targeting tumor-associated NADH oxidase (tNOX), attenuate NAD+ generation and reduce NAD+-dependent sirtuin 1 (SIRT1) deacetylase activity, which in turn enhances p53 acetylation and apoptosis. Methods In this study, differential cellular outcomes in response to oxaliplatin exposure of p53-wild-type versus p53-null HCT116 human colon cancer cells were examined. Cell growth profile was determined by cell impedance measurements and apoptosis was analyzed by flow cytometry. The engagement between oxaliplatin and tNOX protein was studied by cellular thermal shift assay. Furthermore, western blot analysis revealed that p53 was important in regulating tNOX expression in these cell lines. Results In p53-wild-type cells, we found that oxaliplatin inhibited cell growth by inducing apoptosis and concurrently down-regulating tNOX at both the transcriptional and translational levels. In p53-null cells, in contrast, oxaliplatin moderately up-regulated tNOX expression and yielded no apoptosis and much less cytotoxicity. Further experiments revealed that in p53-wild-type cells, oxaliplatin enhanced ROS generation and p53 transcriptional activation, leading to down-regulation of the transcriptional factor, POU3F2, which enhances the expression of tNOX. Moreover, the addition of a ROS scavenger reversed the p53 activation, POU3F2 down-regulation, and apoptosis induced by oxaliplatin in p53-wild-type cells. In the p53-null line, on the other hand, oxaliplatin treatment triggered less ROS generation and no p53 protein, such that POU3F2 and tNOX were not down-regulated and oxaliplatin-mediated cytotoxicity was attenuated. Conclusion Our results show that oxaliplatin mediates differential cellular responses in colon cancer cells depending on their p53 status, and demonstrate that the ROS-p53 axis is important for regulating POU3F2 and its downstream target, tNOX. Notably, the depletion of tNOX sensitizes p53-null cells to both spontaneous and oxaliplatin-induced apoptosis. Our work thus clearly shows a scenario in which targeting of tNOX may be a potential strategy for cancer therapy in a p53-inactivated system.

A series of new 4,11-diaminoanthra[2,3-b]furan-5,10-dione derivatives with different side chains were synthesized. Selected 2-unsubstituted derivatives 11-14 showed high antiproliferative potency on a panel of mammalian tumor cell lines including multidrug resistance variants. Compounds 11-14 utilized multiple mechanisms of cytotoxicity including inhibition of Top1/Top2-mediated DNA relaxation, reduced NAD(+)/NADH ratio through tNOX inhibition, suppression of a NAD(+)-dependent sirtuin 1 (SIRT1) deacetylase activity, and activation of caspase-mediated apoptosis. Here, for the first time, we report that tumor-associated NADH oxidase (tNOX) and SIRT1 are important cellular targets of antitumor anthracene-9,10-diones.

Chemical modifications of the anthraquinone scaffold are aimed at optimization of this exceptionally productive class of antitumor drugs. In particular, our previously reported anthra[2,3-b]furan-3-carboxamides demonstrated a high cytotoxic potency in cell culture and in vivo. In this study, we expanded our series of anthra[2,3-b]furan-3-carboxamides by modifying the key functional groups and dissected the structure-activity relationship within this chemotype. The majority of new compounds inhibited the growth of mammalian tumor cell lines at submicromolar to low micromolar concentrations. We found that 4,11-hydroxy groups as well as the carbonyl moiety in the carboxamide fragment were critical for cytotoxicity whereas the substituent at the 2-position of anthra[2,3-b]furan was not. Importantly, the new derivatives were similarly potent against wild type cells and their variants resistant to doxorubicin due to P-glycoprotein (Pgp) expression or p53 inactivation. The most cytotoxic derivatives 6 and 9 attenuated plasmid DNA relaxation by topoisomerase 1. Finally, we demonstrated that 6 and 9 at 1 μM induced intracellular oxidative stress, accumulation in G2/M phase of the cell cycle, and apoptosis in gastric carcinoma cell lines regardless of their p53 status. These results further substantiate the potential of anthra[2,3-b]furan-3-carboxamides as antitumor drug candidates.

Oral submucous fibrosis (OSF) has been recognized as one of the oral potentially malignant disorders. Areca nut chewing is implicated in this pathological fibrosis, and it causes chronic inflammation and persistent activation of myofibroblasts. As yet, existing treatments only provide temporary symptomatic relief and there is a lack of an effective intervention to cure OSF. Therefore, development of approaches to ameliorate myofibroblast activities becomes a crucial objective to prevent the malignant progression of OSF. In this study, we examined the inhibitory effect of glabridin, an isoflavane extracted from licorice root, on the myofibroblast characteristics in human fibrotic buccal mucosal fibroblasts (fBMFs). Our results showed that myofibroblast activities, including collagen gel contractility, migration, invasion and wound healing abilities were reduced after exposure of glabridin in a dose-dependent manner. Most importantly, we demonstrated that the arecoline-induced myofiroblast activities were abolished by glabridin treatment. Additionally, the expression of the myofibroblast marker α-smooth muscle actin and other fibrogenic marker, type I collagen, in fBMFs were dose-dependently downregulated. Moreover, we showed that the production of TGF-β was suppressed by glabridin in fBMFs and the protein expression of phospho-Smad2 was decreased as well. In summary, our data suggested that glabridin repressed the myofibroblast features in fBMFs via TGF-β/Smad2 signaling pathway. Glabridin also prevented the arecoline-increased myofibroblast activities, and could serve as a natural anti-fibrosis compound for OSF.

A series of 4-aminomethyl derivatives of heliomycin 1 was prepared using the Mannich reaction. The modification significantly improved aqueous solubility of the initially poorly soluble antibiotic. Testing the antiproliferative efficacy revealed a potent activity of heliomycin as well as its new derivatives on a panel of mammalian tumor cells including drug resistant variants. In contrast to 1 the new derivatives 7a, 7l, 7p generated a high level of ROS associated with induction of apoptosis in T24 bladder cancer cells. Introduction of 4-aminomethyl moiety increased the affinity to DNA and the ability to inhibit topoisomerase 1 making 7p the most promising candidate for further preclinical evaluation. Thus, aminomethylation is the first-in-class successful transformation of the antibiotic 1 resulting in an improved water solubility of derivatives and promising properties in search of novel anticancer drug candidates.

Capsaicin (8-methyl-N-vanillyl-6-noneamide), which is an active component in red chili peppers, is used as a chemopreventive agent that shows favorable cytotoxicity against cancer cells. Accumulating evidence indicates that capsaicin preferentially inhibits a tumor-associated NADH oxidase (tNOX, ENOX2) that is ubiquitously expressed in cancer but not in non-transformed cells. This attenuates cancer cell growth by inducing apoptosis. The capsaicin-mediated inhibition of tNOX was recently shown to prolong the cell cycle. However, the molecular events underlying this regulation have not yet been investigated. In the present study, we used a cellular thermal shift assay (CETSA) to detect &ldquo, target engagement&rdquo, of capsaicin and its consequent impact on cell cycle progression. Our results indicated that capsaicin engaged with tNOX and triggered the proteasomal degradation of tNOX, which leads to the inhibition of NAD+-dependent SIRT1 deacetylase. Ultimately, the acetylation levels of c-Myc and p53 were enhanced, which suppressed the activation of G1 cyclin/Cyclin-dependent kinase complexes and triggered cell cycle arrest in cancer cells. The results obtained when tNOX was overexpressed in non-cancer cells validated its importance in cell cycle progression. These findings provide the first molecular insights into the regulatory role of tNOX and the anti-proliferative property of capsaicin in regulating the cell cycle of bladder cancer cells.

Background Many in vitro studies have revealed that the interference of dye molecules in traditional nanoparticle cytotoxicity assays results in controversial conclusions. The aim of this study is to establish an extensive and systematic method for evaluating biological effects of gold nanoparticles in mammalian cell lines. Methods We establish the cell-impedance measurement system, a label-free, real-time cell monitoring platform that measures electrical impedance, displaying results as cell index values, in a variety of mammalian cell lines. Cytotoxic effects of gold nanoparticles are also evaluated with traditional in vitro assays. Results Among the six cell lines, gold nanoparticles induce a dose-dependent suppression of cell growth with different levels of severity and the suppressive effect of gold nanoparticles was indirectly associated with their sizes and cellular uptake. Mechanistic studies revealed that the action of gold nanoparticles is mediated by apoptosis induction or cell cycle delay, depending on cell type and cellular context. Although redox signaling is often linked to the toxicity of nanoparticles, in this study, we found that gold nanoparticle-mediated reactive oxygen species generation was not sustained to notably modulate proteins involved in antioxidative defense system. Conclusion The cell-impedance measurement system, a dye-free, real-time screening platform, provides a reliable analysis for monitoring gold nanoparticle cytotoxicity in a variety of mammalian cell lines. Furthermore, gold nanoparticles induce cellular signaling and several sets of gene expression to modulate cellular physical processes. General significance The systematic approach, such as cell-impedance measurement, analyzing the toxicology of nanomaterials offers convincing evidence of the cytotoxicity of gold nanomaterials.

Ursolic acid (UA), a naturally occurring pentacyclic triterpene acid found in many medicinal herbs and edible plants, triggers apoptosis in several tumor cell lines but not in human bone cancer cells. Most recently, we have demonstrated that UA exposure reduces the viability of human osteosarcoma MG-63 cells through enhanced oxidative stress and apoptosis. Interestingly, an inhibitor of osteoclast-mediated bone resorption, zoledronic acid (ZOL), also a third-generation nitrogen-containing bisphosphonate, is effective in the treatment of bone metastases in patients with various solid tumors. In this present study, we found that UA combined with ZOL to significantly suppress cell viability, colony formation, and induce apoptosis in two lines of human osteosarcoma cells. The pre-treatment of the antioxidant had reversed the oxidative stress and cell viability inhibition in the combined treatment, indicating that oxidative stress is important in the combined anti-tumor effects. Moreover, we demonstrated that ZOL combined with UA significantly induced autophagy and co-administration of autophagy inhibitor reduces the growth inhibitory effect of combined treatment. Collectively, these data shed light on the pathways involved in the combined effects of ZOL and UA that might serve as a potential therapy against osteosarcoma.

Ursolic acid (UA), a naturally occurring pentacyclic triterpene acid found in many medicinal herbs and edible plants, has been shown to trigger apoptosis in several lines of tumor cells in vitro. We found that treatment with UA suppressed the viability of human osteosarcoma MG-63 cells and induced cell cycle arrest at sub-G1 and G2/M phases. Furthermore, exposure to UA induced intracellular oxidative stress and collapse of mitochondrial membrane permeability, resulting in the subsequent activation of apoptotic caspases 8, 9, and 3 as well as PARP cleavage, and ultimately apoptosis in MG-63 cells. Moreover, protein analysis of mitogen-activated protein kinase (MAPK)-related protein expression showed an increase in activated ERK1/2, JNK, and p38 MAPK in UA-treated MG-63 cells. In addition, UA-induced apoptosis was significantly abolished in MG-63 cells that had been pretreated with inhibitors of caspase 3, 8, and 9 and ERK1/2. Furthermore, UA-treated MG-63 cells also exhibited an enhancement in Bax/Bcl-2 ratio, whereas anti-apoptotic XIAP and survivin were down-regulated. Taken together, we provide evidence demonstrating that UA mediates caspase-dependent and ERK1/2 MAPK-associated apoptosis in osteosarcoma MG-63 cells.

Bladder cancer is one of the most frequent cancers among males, and its poor survival rate reflects problems with aggressiveness and chemo-resistance. Recent interest has focused on the use of chemopreventatives (nontoxic natural agents that may suppress cancer progression) to induce targeted apoptosis for cancer therapy. Capsaicin, which has anti-cancer properties, is one such agent. It is known to preferentially inhibit a tumor-associated NADH oxidase (tNOX) that is preferentially expressed in cancer/transformed cells. Here, we set out to elucidate the correlation between tNOX expression and the inhibitory effects of capsaicin in human bladder cancer cells. We showed that capsaicin downregulates tNOX expression and decreases bladder cancer cell growth by enhancing apoptosis. Moreover, capsaicin was found to reduce the expression levels of several proteins involved in cell cycle progression, in association with increases in the cell doubling time and enhanced cell cycle arrest. Capsaicin was also shown to inhibit the activation of ERK, thereby reducing the phosphorylation of paxillin and FAK, which leads to decreased cell migration. Finally, our results indicate that RNA interference-mediated tNOX depletion enhances spontaneous apoptosis, prolongs cell cycle progression, and reduces cell migration and the epithelial-mesenchymal transition. We also observed a downregulation of sirtuin 1 (SIRT1) in these tNOX-knockdown cells, a deacetylase that is important in multiple cellular functions. Taken together, our results indicate that capsaicin inhibits the growth of bladder cancer cells by inhibiting tNOX and SIRT1 and thereby reducing proliferation, attenuating migration, and prolonging cell cycle progression.

Gastric cancer is one of the most common human malignancies, and its prevalence has been shown to be well-correlated with cancer-related deaths worldwide. Regrettably, the poor prognosis of this disease is mainly due to its late diagnosis at advanced stages after the cancer has already metastasized. Recent research has emphasized the identification of cancer biomarkers in the hope of diagnosing cancer early and designing targeted therapies to reverse cancer progression. One member of a family of growth-related nicotinamide adenine dinucleotide (NADH or hydroquinone) oxidases is tumor-associated NADH oxidase (tNOX; ENOX2). Unlike its counterpart CNOX (ENOX1), identified in normal rat liver plasma membranes and shown to be stimulated by growth factors and hormones, tNOX activity purified from rat hepatoma cells is constitutively active. Its activity is detectable in the sera of cancer patients but not in those of healthy volunteers, suggesting its clinical relevance. Interestingly, tNOX expression was shown to be present in an array of cancer cell lines. More importantly, inhibition of tNOX was well correlated with reduced cancer cell growth and induction of apoptosis. RNA interference targeting tNOX expression in cancer cells effectively restored non-cancerous phenotypes, further supporting the vital role of tNOX in cancer cells. Here, we review the regulatory role of tNOX in gastric cancer cell growth.

In view of the extensive use of nanoparticles in countless applications, a fast and effective method for assessing their potential adverse effects on the environment and human health is extremely important. At present, in vitro cell-based assays are the standard approach for screening chemicals for cytotoxicity because of their relative simplicity, sensitivity, and cost-effectiveness compared with animal studies. Regrettably, such cell-based viability assays encounter limitations when applied to determining the biological toxicity of nanomaterials, which often interact with assay components and produce unreliable outcomes. We have established a cell-impedance-based, label-free, real-time cell-monitoring platform suitable for use in a variety of mammalian cell lines that displays results as cell index values. In addition to this real-time screening platform, other traditional cytotoxicity assays were employed to validate cytotoxicity assessments. We suggest that the cell impedance measurement approach is effective and better suited to determining the cytotoxicity of nanomaterials for environmental safety screening. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1170-1182, 2017.

Cancer chemoprevention is employed to block or reverse the progression of malignancies. To date, several thousands of agents have been found to possess chemopreventative activity, one of which is capsaicin, a component of chili peppers that exhibits antigrowth activity against various cancer cell lines. However, the role of capsaicin in tumorigenesis remains controversial because both cancer prevention and promotion have been proposed. Here, we made the unexpected discovery that treatment with low concentrations of capsaicin up-regulates tNOX (tumor-associated NADH oxidase) expression in HCT116 human colon carcinoma cells in association with enhanced cell proliferation and migration, as evidenced by down-regulation of epithelial markers and up-regulation of mesenchymal markers. Importantly, tNOX-knockdown in HCT116 cells by RNA interference reversed capsaicin-induced cell proliferation and migration in vitro and decreased tumor growth in vivo. Collectively, these findings provide a basis for explaining the tumor-promoting effect of capsaicin and might imply that caution should be taken when using capsaicin as a chemopreventive agent.

DNA-damaging agents are commonly used as anticancer therapeutics. Unfortunately, such drugs induced DNA damages as well as DNA repair are important in mediating drug resistance to cancer treatments. To evaluate changes in DNA repair proteins that occur in DNA damage agent treatment, we challenged human A549 lung adenocarcinoma cells with cisplatin. hHR23/RAD23, an accessory protein involved in nucleotide-excision repair (NER) at an early lesion-recognition step, was upregulated by cisplatin in a dose- and time-dependent manner. Upregulation of hHR23 expression by low-dose cisplatin was accompanied by an increase in p53, p21, and XPC protein levels. Importantly, knockdown of hHR23B by RNA interference decreased DNA repair activity, cell survival, and induction of p53 and XPC following treatment with cisplatin. Conversely, overexpression of hHR23B enhanced repair activity towards cisplatin-damaged DNA. Inhibition of MEK/ERK and phosphoinositide 3-kinase (PI3K)/AKT signaling pathways attenuated cisplatin-induced hHR23 expression, indicating that these pathways are involved in the process. The increase in hHR23 protein expression mediated by MEK/ERK signaling was due to increased translational efficiency resulting from phosphorylation/activation of the translation-initiating factor eIF-4B. Taken together, these results suggest that cisplatin-induced increases in hHR23 levels are regulated by proliferative signaling pathways and important for DNA repair.

Gastric cancer is a common human malignancy and a major contributor to cancer-related deaths worldwide. Unfortunately, the prognosis of most gastric cancer patients is poor because they are generally diagnosed at a late stage after the cancer has already metastasized. Most current research, therefore, emphasizes selective targeting of cancer cells by apoptosis-inducing agents. One such therapeutic agent is capsaicin, a component of chili peppers that has been shown to possess anti-growth activity against various cancer cell lines. Here, we examined the effect of capsaicin on SNU-1 and TMC-1 gastric cancer cells and found differing outcomes between the two cell lines. Our results show that capsaicin induced significant cytotoxicity with increases in oxidative stress, PARP cleavage, and apoptosis in sensitive SNU-1 cells. In contrast, TMC-1 cells were much less sensitive to capsaicin, exhibiting low cytotoxicity and very little apoptosis in response to capsaicin treatment. Capsaicin-induced apoptosis in SNU-1 cells was associated with down-regulation of tumor-associated NADH oxidase (tNOX) mRNA and protein. On the contrary, tNOX expression was scarcely affected by capsaicin in TMC-1 cells. We further showed that tNOX-knockdown sensitized TMC-1 cells to capsaicin-induced apoptosis and G1 phase accumulation, and led to decreased cell growth, demonstrating that tNOX is essential for cancer cell growth. Collectively, these results indicate that capsaicin induces divergent effects of the growth of gastric cancer cells that parallel its effects on tNOX expression, and demonstrate that forced tNOX down-regulation restored capsaicin-induced growth inhibition in TMC-1 cells.

Tumor-associated NADH oxidase (tNOX) is a growth-related protein expressed in transformed cells. tNOX knockdown using RNA interference leads to a significant reduction in HeLa cell proliferation and migration, indicating an important role for tNOX in growth regulation and the cancer phenotype. Here, we show that tNOX is down-regulated during apoptosis in HCT116 cells. Treatment with diverse stresses induced a dose- and time-dependent decrease in tNOX expression that was concurrent with apoptosis. Moreover, shRNA-mediated tNOX knockdown rendered cells susceptible to apoptosis, whereas re-expression of tNOX partially recovered cell proliferation. Our results indicate that tNOX is suppressed during apoptosis and demonstrate that tNOX down-regulation sensitizes cells to stress-induced growth reduction, suggesting that tNOX is required for transformed cell growth.

Capsaicin is considered a chemopreventive agent by virtue of its selective antigrowth activity, commonly associated with apoptosis, against cancer cells. However, noncancerous cells possess relatively higher tolerance to capsaicin, although the underlying mechanism for this difference remains unclear. Hence, this study aimed to elucidate the differential effects of capsaicin on cell lines from lung tissues by addressing the signal pathway leading to two types of cell death. In MRC-5 human fetal lung cells, capsaicin augmented silent mating type information regulation 1 (SIRT1) deacetylase activity and the intracellular NAD(+)/NADH ratio, decreasing acetylation of p53 and inducing autophagy. In contrast, capsaicin decreased the intracellular NAD(+)/NADH ratio, possibly through inhibition of tumor-associated NADH oxidase (tNOX), and diminished SIRT1 expression leading to enhanced p53 acetylation and apoptosis. Moreover, SIRT1 depletion by RNA interference attenuated capsaicin-induced apoptosis in A549 cancer cells and autophagy in MRC-5 cells, suggesting a vital role for SIRT1 in capsaicin-mediated cell death. Collectively, these data not only explain the differential cytotoxicity of capsaicin but shed light on the distinct cellular responses to capsaicin in cancerous and noncancerous cell lines.

Epithelial-to-mesenchymal transition (EMT) is a crucial event involved metastasis of certain tumors. Thus, identifying chemical agents that can block EMT is highly warranted for the development of anti-cancer chemoprevention/chemotherapies. In this study, we found that Antrodin C (ADC), a maleimide derivative isolated from Antrodia cinnamomea health food product inhibits TGF-β1-induced EMT and breast cancer cell metastasis in vitro. Pretreatment of MCF-7 cells with ADC significantly blocked TGF-β1-induced phenotypic changes and actin cytoskeleton remodeling. In addition, ADC was able to up-regulate epithelial markers such as E-cadherin and occludin, whereas mesenchymal markers including N-cadherin and vimentin were significantly inhibited, possibly through the modulation of transcriptional regulators Smad/Smad3. ADC blocked TGF-β1-induced migration and invasion of MCF-7 cells through the down-regulation of matrix-metalloproteinases (MMP-2, -9) and urokinase plasminogen activator (uPA). The inhibition of MMPs and uPA activity by ADC was reasoned by suppression of its corresponding transcription factor β-catenin. Taken together, our data suggested that ADC attenuates the TGF-β1-induced EMT, migration and invasion of human breast carcinoma through the suppression of Smad2/3 and β-catenin signaling pathways.

Phenoxodiol, a synthetic isoflavene with clinical efficacy in the management of ovarian and other forms of human cancer, blocked the activity of a cancer-specific and growth-related cell surface ECTO-NOX protein with both oxidative (hydroquinone) and protein disulfide-thiol interchange activity designated tNOX. Purified recombinant tNOX bound phenoxodiol with high affinity (Kd of 50 nM). The tNOX protein appeared to be both necessary and sufficient for the cancer-specific cytotoxicity of phenoxodiol. Growth inhibition of fibroblasts from embryos of mice expressing a tNOX transgene, but not from wild-type mice, was inhibited by phenoxodiol followed by apoptosis. Both the oxidative and protein disulfide-thiol interchange activities that alternate to generate the complex set of oscillations with a period length of 22 min (24 min for the constitutive counterpart CNOX) that characterize ECTO-NOX proteins respond to phenoxodiol. Oxidation of NADH or reduced coenzyme Q10 was rapidly blocked by phenoxodiol. In contrast, the protein disulfidethiol interchange activity measured either by the restoration of activity to scrambled and inactive RNase or from the cleavage of dithiodipyridine (EC50 of 50 nM) was inhibited progressively over an interval of 60 min that spanned three cycles of activity. Inhibition of the latter paralleled the inhibition of cell enlargement and the consequent inability of inhibited cells to initiate traverse of the cell cycle. Activities of constitutive ECTO-NOX (CNOX) forms of either cancer or noncancer cells were unaffected by phenoxodiol to help explain how the cytotoxic effects of phenoxodiol may be restricted to cancer cells.

Transforming growth factor-beta (TGF-beta) has been associated with the onset of cardiac cell hypertrophy, but the mechanisms underlying this dissociation are not completely understood. By a previous study, we investigated the involvement of a MAP3K, ZAK, which in cultured H9c2 cardiac cells is a positive mediator of cell hypertrophy. Our results showed that expression of a dominant-negative form of ZAK inhibited the characteristic TGF-beta-induced features of cardiac hypertrophy, including increased cell size, elevated expression of atrial natriuretic factor (ANF), and increased organization of actin fibers. Furthermore, dominant-negative MKK7 effectively blocked both TGF-beta-and ZAK-induced ANF expression. In contrast, a JNK/SAPK specific inhibitor, sp600125, had little effect on TGF-beta- or ZAK-induced ANF expression. Our findings suggest that a ZAK mediates TGF-beta-induced cardiac hypertrophic growth via a novel TGF-beta signaling pathway that can be summarized as TGF-beta>ZAK>MKK7>ANF.

Monoclonal antibodies were generated in mice to a 34-kDa circulating form of a drug-responsive hydroquinone (NADH) oxidase with a protein disulfide-thiol interchange activity specific to the surface of cancer cells and the sera of cancer patients. Screening used Western blots with purified 34-kDa tNOX from HeLa cells and the sera of cancer patients. Epitopes were sought that inhibited the drug-responsive oxidation of NADH with the sera of cancer patients, but which had no effect on NADH oxidation with the sera of healthy volunteers. Two such antisera were generated. One, designated monoclonal antibody (mAb) 12.1, was characterized extensively. The NADH oxidase activity inhibited by mAb 12.1 also was inhibited by the quinone site inhibitor capsaicin (8-methyl- N-vanillyl-6-noneamide). The inhibition was competitive for the drug-responsive protein disulfide-thiol interchange activity assayed either by restoration of activity to scrambled RNase or by cleavage of a dithiodipyridine substrate, and was uncompetitive for NADH oxidation. Both the mAb 12.1 and the postimmune antisera immunoprecipitated drug-responsive NOX activity and identified the same 34-kDa tNOX protein in the sera of cancer patients that was absent from sera of healthy volunteers, and was utilized as immunogen. Preimmune sera from the same mouse as the postimmune antisera was without effect. Both mouse ascites containing mAb 12.1 and postimmune sera (but not preimmune sera) slowed the growth of human cancer cell lines in culture, but did not affect the growth of non-cancerous cell lines. Immunocytochemical and histochemical findings showed that mAb 12.1 reacted with the surface membranes of human carcinoma cells and tissues.

NOX proteins are growth-related cell surface proteins that catalyze both hydroquinone or NADH oxidation and protein disulfide interchange and exhibit prion-like properties. The two enzymatic activities alternate to generate a regular period length of about 24 min. Here we report the expression, cloning, and characterization of a tumor-associated NADH oxidase (tNOX). The cDNA sequence of 1830 bp is located on gene Xq25-26 with an open reading frame encoding 610 amino acids. The activities of the bacterially expressed tNOX oscillate with a period length of 22 min as is characteristic of tNOX activities in situ. The activities are inhibited completely by capsaicin, which represents a defining characteristic of tNOX activity. Functional motifs identified by site-directed mutagenesis within the C-terminal portion of the tNOX protein corresponding to the processed plasma membrane-associated form include quinone (capsaicin), copper and adenine nucleotide binding domains, and two cysteines essential for catalytic activity. Four of the six cysteine to alanine replacements retained enzymatic activity, but the period lengths of the oscillations were increased. A single protein with two alternating enzymatic activities indicative of a time-keeping function is unprecedented in the biochemical literature.

NADH oxidases of low specific activities from urine of cancer patients were found to be inhibited or stimulated by the vanilloid capsaicin (8-methyl-N-vanillyl-6-noneamide). Similar activities, inhibited or stimulated by capsaicin, were reported previously for sera of cancer patients but not for sera of normal volunteers or for patients with disorders other than cancer. Like those from sera, the activities from urine were resistant to heat and to digestion with proteinase K. Two different fractions with capsaicin-responsive NADH oxidase activities were obtained by FPLC. One fraction in which the 33-kDa band was the major component exhibited NADH oxidase activity stimulated by capsaicin. Another fraction in which 66-kDa and 45-kDa bands were major components exhibited NADH oxidase activities inhibited by capsaicin. A monoclonal antibody generated to a ca 34-kDa form of the NADH oxidase from sera reacted with a urine protein of a ca 33-kDa band in the capsaicin-stimulated fraction. The 33-kDa protein was of low abundance and was estimated to be present in amounts between 5 and 100 microgram/L, depending on the particular patient.

Our laboratory described a ca. 34-kDa protein of the HeLa S cell surface that bound an antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl) urea (LY181984) with high affinity and that exhibited NADH oxidase and protein disulfide-thiol interchange activities also inhibited by LY181984. The quinone site inhibitor 8-methyl-N-vanillyl-6-noneamide (capsaicin) also blocked these same enzymatic activities. Using capsaicin inhibition as the criterion, the drug-responsive oxidase was released from the surface of HeLa S cells and purified. The activity of the released capsaicin-inhibited oxidase was resistant to heating at 50 degrees C and to protease digestion. After heating and proteinase K digestion, the activity was isolated in >90% yield by FPLC as an apparent 50- to 60-kDa multimer. Final purification by preparative SDS-PAGE yielded a capsaicin-inhibited NADH oxidase activity of a specific activity indicative of >500-fold purification relative to the plasma membrane. The final activity correlated with a ca. 34-kDa band on SDS-PAGE. Matrix-assisted laser desorption mass spectroscopy as well as reelectrophoresis of the 34-kDa band indicated that the ca. 34-kDa material was a stable mixture of 22-, 17-, and 9.5-kDa components which occasionally migrated as a ca. 52-kDa complex. The purified complex tended to multimerize and formed insoluble 10- to 20-nm-diameter amyloid rods. The components of the purified 34-kDa complex were blocked to N-terminal amino acid sequencing and were resistant to further protease digestion. After multimerization into amyloid rods, the protein remained resistant to proteases even under denaturing conditions and to cyanogen bromide either with or without prior alkylation.

Gold nanoparticles (AuNPs) possess unique properties that have been exploited in several medical applications. However, a more comprehensive understanding of the environmental safety of AuNPs is imperative for use of these nanomaterials. Here, we describe the impacts of AuNPs in various mammalian cell models using an automatic and dye-free method for continuous monitoring of cell growth based on the measurement of cell impedance. Several well-established cytotoxicity assays were also used for comparison. AuNPs induced a concentration-dependent decrease in cell growth. This inhibitory effect was associated with apoptosis induction in Vero cells but not in MRC-5 or NIH3T3 cells. Interestingly, cDNA microarray analyses in MRC-5 cells supported the involvement of DNA damage and repair responses, cell-cycle regulation, and oxidative stress in AuNP-induced cytotoxicity and genotoxicity. Moreover, autophagy appeared to play a role in AuNPs-induced attenuation of cell growth in NIH3T3 cells. In this study, we present a comprehensive overview of AuNP-induced cytotoxicity in a variety of mammalian cell lines, comparing several cytotoxicity assays. Collectively, these assays offer convincing evidence of the cytotoxicity of AuNPs and support the value of a systematic approach for analyzing the toxicology of nanoparticles.

A ca. 33.5-kDa protein has been identified as a soluble NADH oxidase activity of culture media conditioned by growth of HeLa cells. The protein appears to be derived from a 34-kDa protein of the HeLa plasma membrane. Both proteins are characterized by an ability to oxidize NADH in the absence of exogenous electron acceptors. The activity is inhibited by 8-methyl-N-vanillyl-6-noneamide (capsaicin). The soluble and the plasma membrane forms of the activity exhibit a similar EC50 of about 5 nM for inhibition of the activity by capsaicin. The activity was purified from culture media conditioned by growth of HeLa cells using DEAE ion exchange chromatography, G-200 size exclusion chromatography, and preparative SDS-PAGE. Purification was monitored on the basis of the capsaicin-inhibited oxidation of NADH, including the final electrophoretic purification. Activity was restored following SDS-PAGE by reduction with dithiothreitol or reduced glutathione in the presence of NADH followed by the addition of 0.03% hydrogen peroxide and preincubation in the presence of NADH for 5-15 min. For affinity purification, the vanillylamine portion of capsaicin was linked to agarose. The agarose-linked vanillylamine bound a ca. 33.5-kDa protein band with capsaicin-inhibited NADH activity from total defined culture media conditioned by growth of HeLa cells. The NADH oxidase activity of both the soluble and the plasma membrane-associated form of the activity was inhibited by antisera corresponding to the 33.5-kDa protein. The antisera also immunoprecipitated and reacted on Western blots with both the soluble (33.5 kDa) and plasma membrane (34 kDa)-associated forms of the capsaicin-inhibited activity. The results identify the capsaicin-inhibited NADH oxidase of the conditioned media of HeLa cells as being a ca. 33.5-kDa shed form of the previously reported capsaicin-inhibited NADH oxidase of the HeLa cell plasma membrane.

Tumor-associated NADH oxidase (tNOX; ENOX2) is a growth-related protein expressed in transformed cells. Consistent with this function, tNOX knockdown by RNA interference leads to a significant reduction in cell proliferation and migration in HeLa cells, whereas tNOX overexpression confers an aggressive phenotype. Here, for the first time, we report that tNOX is phosphorylated by protein kinase Cδ (PKCδ) both in vitro and in vivo. Replacement of serine-504 with alanine significantly reduces phosphorylation by PKCδ. Co-immunoprecipitation experiments reveal an interaction between tNOX and PKCδ. Moreover, whereas overexpression of wild-type tNOX in NIH3T3 cells increases cell proliferation and migration, overexpression of the S504A tNOX mutant leads to diminished cell proliferation and migration, reflecting reduced stability of the unphosphorylatable tNOX mutant protein. Collectively, these results suggest that phosphorylation of serine-504 by PKCδ modulates the biological function of tNOX.

Background Tumor-associated NADH oxidase (tNOX; ENOX2) is a growth-related protein expressed in transformed cells. High concentrations of numerous chemotherapeutic agents have shown to inhibit tNOX activity and protein levels leading to a reduction in cell growth while little is known for the effects of low concentrations of chemotherapeutic agents on tNOX expression. Methods Effects of chemotherapeutic agents on cell function were evaluated with traditional in vitro assays and the xCELLigence System. Western blot analyses were used to study protein expression profiles of the epithelial-to-mesenchymal transition. Results We showed that doxorubicin treatment transiently up-regulates tNOX expression in human lung carcinoma A549 cells in association with enhanced cell migration. Similar results were observed in tamoxifen-exposed A549 cells. Furthermore, protein marker analyses revealed that the enhanced migration induced by tamoxifen was correlated with epithelial-to-mesenchymal transition, as evidenced by down-regulation of epithelial markers and up-regulation of mesenchymal markers. Importantly, tNOX overexpression enhanced cell migration, confirming the essential role of tNOX in cell migration. Conclusions Based on these findings, we conclude that doxorubicin and tamoxifen induce a transient up-regulation of tNOX expression, leading to enhanced cell migration and EMT. General significance These findings establish an essential role for tNOX in cell migration and survival and may provide a rational framework for the further development of tNOX inhibitors as a novel class of antitumor agents.

A new polyacetylenic compound, araliadiol, was isolated from the leaves of Aralia cordata Thunb. (Araliaceae). The structure of araliadiol was determined to be 3( S),8( R)-pentadeca-1,9( Z)-diene-4,6-diyne-3,8-diol by MS, NMR, IR, and UV spectroscopic analysis as well as Mosher ester reaction. Araliadiol displayed a significant inhibitory effect on the growth of a human breast adenocarcinoma cell line (MCF-7), with an IC (50) value for cytotoxicity of 6.41 µg/mL. Cell cycle analysis revealed that the proportion of cells in the G (1) phase of the cell cycle increased in a dose-dependent manner (from 54.7 % to 72.0 %) after 48 h exposure to araliadiol at dosages ranging from 0 to 80 µM. The results suggest that araliadiol inhibits cell cycle progression of MCF-7 at the G (1)-S transition. After treatment with araliadiol, phosphorylation of retinoblastoma protein (Rb) in MCF-7 cells was inhibited, accompanied by a decrease in the levels of cyclin D (3) and cyclin-dependent kinase 4 (cdk4) and an increase in the expression of p21 (WAF-1/Cip1). However, the expression of phosphorylated p53 (Ser15) and Chk2 was not altered in MCF-7 cells. These findings indicate that araliadiol exhibits its growth-inhibitory effects on MCF-7 cells through downregulation of cdk4 and cyclin D (3), and upregulation of p21 (WAF-1/Cip1) by a p53-independent mechanism.

ECTO-NOX proteins are growth-related cell surface proteins that catalyze both hydroquinone or NADH oxidation and protein disulfide interchange and exhibit time-keeping and prion-like properties. A bacterially expressed truncated recombinant 46 kDa ENOX2 with full ENOX2 activity bound ca 2 moles copper and 2 moles of zinc per mole of protein. Unfolding of the protein in trifluoroacetic acid in the presence of the copper chelator bathocuproine resulted in reversible loss of both enzymatic activities and of a characteristic pattern in the Amide I to Amide II ratios determined by FTIR with restoration by added copper. The H546-V-H together with His 562 form one copper binding site and H582 represents a second copper site as determined from site-directed mutagenesis. Bound copper emerges as having an essential role in ENOX2 both for enzymatic activity and for the structural changes that underly the periodic alternations in activity that define the time-keeping cycle of the protein.

Tumor-associated NADH oxidase (tNOX, also known as ENOX2) is a growth-related protein expressed in transformed cells. Previous reports have revealed that the inhibition of tNOX activity by the anti-cancer drug, capsaicin, correlates with a reduction in growth of cancer cells, indicating a close relationship between tNOX activity and cell growth. Moreover, the study of depleted tNOX expression by RNA interference in HeLa cells suggests that it may be associated with the ability of tumor cells to acquire an aggressive phenotype, particularly in relation to cell proliferation. A key role for tNOX in regulating cell growth is further supported by the observation that the growth rate of MEF cells from tNOX-overexpressing transgenic mice is approximately two-fold greater than that of wild-type cells. The purpose of this study was to investigate the anti-proliferative effect of capsaicin on tNOX expression level in stomach cancer cells. We showed that capsaicin induced cytotoxicity in SCM cells concomitantly with apoptosis, PARP cleavage, and down-regulation of tNOX protein.

Nanomaterials and nanoparticles have received considerable attention recently because of their unique properties and diverse biotechnology and life sciences applications. Nanosilver products, which have well-known antimicrobial properties, have been used extensively in a range of medical settings. Despite the widespread use of nanosilver products, relatively few studies have been undertaken to determine the biological effects of nanosilver exposure. The purpose of this study was to evaluate the toxicity of nanosilver and to elucidate possible molecular mechanisms underlying the biological effects of nanosilver. Here, we show that nanosilver is cytotoxic, inducing apoptosis in NIH3T3 fibroblast cells. Treatment with nanosilver induced the release of cytochrome c into the cytosol and translocation of Bax to mitochondria, indicating that nanosilver-mediated apoptosis is mitochondria-dependent. Nanosilver-induced apoptosis was associated with the generation of reactive oxygen species (ROS) and JNK activation, and inhibition of either ROS or JNK attenuated nanosilver-induced apoptosis. In nanosilver-resistant HCT116 cells, up-regulation of the anti-apoptotic proteins, Bcl-2 appeared to be associated with a diminished apoptotic response. Taken together, our results provide the first evidence for a molecular mechanism of nanosilver cytotoxicity, showing that nanosilver acts through ROS and JNK to induce apoptosis via the mitochondrial pathway.

A new secobutanolide, secoaggregatalactone A ( 1) was isolated from the leaves of Lindera aggregata. Results obtained from the cytotoxicity assay revealed that secoaggregatalactone A exhibited a noticeable cytotoxicity (EC (50) = 6.61 microg/mL; 22.1 microM) against the human hepatoma cell line (Hep G2 cell line). According to morphological observations, flow cytometric analysis, and DNA fragmentation analysis, it was proven that the cytotoxicity of secoaggregatalactone A on human cells was due to apoptosis. Moreover, based on the results from the protein expression assay and confocal laser scanning microscope observations, it is assumed that secoaggregatalactone A induced apoptosis through the mitochondria pathway by way of cleavage of Bit to release cytochrome C and activate caspases-9 and -3, and then degradation of PARP.

tNOX, a tumor-associated NADH oxidase, is a growth-related protein present in transformed cells. In this study, we employed RNA interference (RNAi)-mediated down-regulation of tNOX protein expression to explore the role of tNOX in regulating cell growth in human cervical adenocarcinoma (HeLa) cells. In this first reported use of RNAi to decrease tNOX expression, we found that HeLa cell growth was significantly inhibited by shRNA-knockdown of tNOX. Furthermore, cell migration and membrane association of Rac were decreased concomitantly with the reduction in tNOX protein expression. These results indicate that shRNA targeting of tNOX inhibits the growth of cervical cancer cells, and reduces cell migration via a decrease in the membrane association of Rac. We propose that tNOX is a potential upstream mediator of Rho activation that plays a role in regulating cell proliferation, migration, and invasion.

Previous reports have described a tumor-associated NADH oxidase (tNOX) and its continuous activation in transformed culture cells. Certain anticancer drugs have been shown to inhibit preferentially both the tNOX activity and the growth of transformed culture cells and the cytotoxicity is associated with the induction of apoptosis. To investigate the biological function of tNOX protein, we have raised polyclonal antisera against bacterial expressed tNOX protein and the antisera are able to recognize protein bands in transformed cells but not the non-transformed cells tested. With tNOX antisera treatment, the survival in transformed cell lines is decreased but not the non-transformed cells. In addition, tNOX antisera-induced cytotoxicity is accompanied by the induction of apoptosis. However, slightly higher amount of PARP cleavage and activation of caspase-9 are observed in tNOX antisera treated HCT116 cells. Further experiments have demonstrated the activation of JNK and phosphorylation of p53 by treatment. In addition, tNOX antisera treatment leads to an impressive increase in reactive oxygen species in COS cells but not the control sera. Our data suggest that (a) tNOX antisera treatment may inhibit the growth of transformed cells by inducing apoptosis and (b) the apoptotic mechanism might be through modulating ROS production and JNK pathway.

Capsaicin and the principal green tea catechin, (-)-epigallocatechin-3-gallate (EGCg), target tNOX, a tumor (cancer)-specific surface hydroquinone (NADH) oxidase with protein disulfide-thiol interchange activity (ECTO-NOX protein). Accordingly vector-forced over expression of tNOX in MCF-10A mammary epithelia or COS cells that lack tNOX or in COS cells that underexpress tNOX enhanced the susceptibility of growth and apoptosis to both EGCg and capsaicin. Additionally, the tNOX-transfected MCF-10A cells proliferated in Matrigel, a measure of invasiveness. In contrast, oligomeric antisense tNOX DNA abrogated growth inhibition by EGCg and capsaicin and reduced anchorage-dependent growth of HeLa (human cervical carcinoma) cells that naturally overexpress tNOX. The findings show cell surface expression of tNOX as both necessary and sufficient for the cellular anticancer activities attributed to both EGCg and capsaicin.

Disulfide-thiol interchange proteins with hydroquinone (NADH) oxidase activities (designated NOX for plasma membrane-associated NADH oxidases) occur as extrinsic membrane proteins associated with the plasma membrane at the outer cell surface. The cancer-associated NOX protein, designated tNOX, has been cloned. The 34-kDa plasma membrane-associated form of the protein contains no strongly hydrophobic regions and is not transmembrane. No myristoylation or phosphatidylinositol anchor motifs were discovered. Evidence for lack of involvement of a glycosylphosphatidylinositol-linkage was derived from the inability of treatment with a phosphatidylinositol-specific phospholipase C or with nitrous acid at low pH to release the NOX protein from the surface of HeLa cells or from plasma membranes isolated from HeLa cells. Binding of NOX protein to the plasma membrane via amino acid side chain modification or by attachment of fatty acids also is unlikely based on use of specific fatty acid antisera to protein bound fatty acids and as a result of binding to the cancer cell surface of a truncated form of recombinant tNOX. Incubation of cells or plasma membranes with 0.1 M sodium acetate, pH 5, at 37°C for 1 h, was sufficient to release tNOX from the HeLa cell surface. Release was unaffected by protease inhibitors or divalent ions and was not accelerated by addition of cathepsin D. The findings suggest dissociable receptor binding as a possible basis for their plasma membrane association.

We have described a drug-responsive form of a cell surface NADH oxidase (hydroquinone oxidase) of cancer cells (tNOX) that exhibits unusual characteristics including resistance to proteases, resistance to cyanogen bromide digestion, and an ability to form amyloid filaments closely resembling those of spongiform encephalopathies and all of which are characteristics of PrP(sc) (PrP(res)), the presumed infective and proteinase K resistant particle of the scrapie prion. The tNOX protein from the HeLa cell surface copurified with authentic glyceraldehyde-3-phosphate dehydrogenase (muscle form) (GAPDH). Surprisingly, the tNOX-associated muscle GAPDH also was proteinase K resistant. In this paper, we show that combination of authentic rabbit muscle GAPDH with tNOX renders the GAPDH resistant to proteinase K digestion. This property, that of converting the normal form of a protein into a likeness of itself, is one of the defining characteristics of the group of proteins designated as prions.

Cell membrane redox systems carry electrons from intracellular donors and transport them to extracellular acceptors. This phenomenon appears to be universal. Numerous reviews have emphasized not only the bioenergetic mechanisms of redox systems but also the antioxidant defense mechanisms in which they participate. Moreover, significant progress has been made in the modulation of the membrane redox systems on cell proliferation. Because membrane redox systems play a key role in the regulation of cell growth, they need to be somehow linked into the signaling pathways resulting in either controlled or unregulated growth by both internal and external signals. Ultimately, these sequential events lead to either normal cell proliferation or cancer cell formation. However, much less is known about the involvement of membrane redox in transformation or tumorgenesis. In this review, the facts and ideas are summarized concerning the redox systems and tumorgenesis in several aspects, such as the regulation of cell growth and the effect on cell differentiation and on signaling pathways. In addition, information on a unique tumor-associated nicotinamide adenine dinucleotide (NADH) oxidase (tNOX) protein is reviewed.

A hormone- and growth factor-stimulated NADH oxidase of the mammalian plasma membrane, constitutively activated in transformed cells, was inhibited preferentially in HeLa, ovarian carcinoma, mammary adenocarcinoma, and HL-60 cells, all of human origin, by the naturally occurring quinone analog capsaicin (8-methyl-N-vanillyl-6-noneamide), compared with plasma membranes from human mammary epithelial, rat liver, normal rat kidney cells, or HL-60 cells induced to differentiate with dimethyl sulfoxide. With cells in culture, capsaicin preferentially inhibited growth of HeLa, ovarian carcinoma, mammary adenocarcinoma, and HL-60 cells but was largely without effect on the mammary epithelial cells, rat kidney cells, or HL-60 cells induced to differentiate with dimethyl sulfoxide. Inhibited cells became smaller and cell death was accompanied by a condensed and fragmented appearance of the nuclear DNA, as revealed by fluorescence microscopy with 4',6-diamidino-2-phenylindole, suggestive of apoptosis. The findings correlate capsaicin inhibition of cell surface NADH oxidase activity and inhibition of growth that correlate with capsaicin-induced apoptosis.