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Anthraquinone-based intercalating compounds, namely doxorubicin and mitoxantrone, have been used clinically based on their capacity to bind DNA and induce DNA damage. However, their applications have been limited by side effects and drug resistance. New-generation anthraquinone derivatives fused with different heterocycles have been chemically synthesized and screened for higher anticancer potency. Among the compounds reported in our previous study, 4,11-bis(2-(2-chloroacetamidine)ethylamino)anthra[2,3-b]thiophene-5,10-dione dihydrochloride (designated 2c) was found to be apoptotic, but the direct cellular target responsible for the cytotoxicity remained unknown. Here, we report the synthesis and anticancer properties of two other derivatives, 4,11-bis(2-(2-chloroacetamidine)ethylamino)naphtho[2,3-f]indole-5,10-dione dihydrochloride (2a) and 4,11-bis(2-(2-chloroacetamidine)ethylamino)-2-methylanthra[2,3-b]furan-5,10-dione dihydrochloride (2b). We sought to identify and validate the protein target(s) of these derivatives in oral cancer cells, using molecular docking simulations and cellular thermal shift assays (CETSA). Our CETSA results illustrate that these derivatives targeted the tumor-associated NADH oxidase (tNOX, ENOX2), and their direct binding downregulated tNOX in p53-functional SAS and p53-mutated HSC-3 cells. Interestingly, the compounds targeted and downregulated tNOX to reduce SIRT1 deacetylase activity and increase Ku70 acetylation, which triggers c-Flip ubiquitination and induces apoptosis in oral cancer cells. Together, our data highlight the potential value of these heteroarene-fused anthraquinones in managing cancer by targeting tNOX and augmenting apoptosis.

// 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.

Hepatocellular carcinoma (HCC) is the most frequent primary malignancy of the liver and is among the top three causes of cancer-associated death worldwide. However, the clinical use of chemotherapy for HCC has been limited by various challenges, emphasizing the urgent need for novel agents with improved anticancer properties. We recently synthesized and characterized a series of 4,11-diaminoanthra[2,3-b]furan-5,10-dione derivatives that exhibit potent apoptotic activity against an array of cancer cell lines, including variants with multidrug resistance. Their effect on liver cancer cells, however, was unknown. Here, we investigated three selected 4,11-diaminoanthra[2,3-b]furan-5,10-dione derivatives (compounds 1&ndash, 3) for their cytotoxicity and the underlying molecular mechanisms in wild-type or p53-deficient HCC cells. Cytotoxicity was determined by WST-1 assays and cell impedance measurements and apoptosis was analyzed by flow cytometry. The interaction between compounds and tumor-associated NADH oxidase (tNOX, ENOX2) was studied by cellular thermal shift assay (CETSA). We found that compound 1 and 2 induced significant cytotoxicity in both HepG2 and Hep3B lines. CETSA revealed that compounds 1 and 2 directly engaged with tNOX, leading to a decrease in the cellular NAD+/NADH ratio. This decreased the NAD+-dependent activity of Sirtuin 1 (SIRT1) deacetylase. In p53-wild-type HepG2 cells, p53 acetylation/activation was enhanced, possibly due to the reduction in SIRT1 activity, and apoptosis was observed. In p53-deficient Hep3B cells, the reduction in SIRT1 activity increased the acetylation of c-Myc, thereby reactivating the TRAIL pathway and, ultimately leading to apoptosis. These compounds thus trigger apoptosis in both cell types, but via different pathways. Taken together, our data show that derivatives 1 and 2 of 4,11-diaminoanthra[2,3-b]furan-5,10-diones engage with tNOX and inhibit its oxidase activity. This results in cytotoxicity via apoptosis through tNOX-SIRT1 axis to enhance the acetylation of p53 or c-Myc in HCC cells, depending on their p53 status.

Hepatocellular carcinoma (HCC) is the most frequent primary malignancy of the liver and is among the top three causes of cancer-associated death worldwide. However, the clinical use of chemotherapy for HCC has been limited by various challenges, emphasizing the urgent need for novel agents with improved anticancer properties. We recently synthesized and characterized a series of 4,11-diaminoanthra[2,3-b]furan-5,10-dione derivatives that exhibit potent apoptotic activity against an array of cancer cell lines, including variants with multidrug resistance. Their effect on liver cancer cells, however, was unknown. Here, we investigated three selected 4,11-diaminoanthra[2,3-b]furan-5,10-dione derivatives (compounds 1–3) for their cytotoxicity and the underlying molecular mechanisms in wild-type or p53-deficient HCC cells. Cytotoxicity was determined by WST-1 assays and cell impedance measurements and apoptosis was analyzed by flow cytometry. The interaction between compounds and tumor-associated NADH oxidase (tNOX, ENOX2) was studied by cellular thermal shift assay (CETSA). We found that compound 1 and 2 induced significant cytotoxicity in both HepG2 and Hep3B lines. CETSA revealed that compounds 1 and 2 directly engaged with tNOX, leading to a decrease in the cellular NAD+/NADH ratio. This decreased the NAD+-dependent activity of Sirtuin 1 (SIRT1) deacetylase. In p53-wild-type HepG2 cells, p53 acetylation/activation was enhanced, possibly due to the reduction in SIRT1 activity, and apoptosis was observed. In p53-deficient Hep3B cells, the reduction in SIRT1 activity increased the acetylation of c-Myc, thereby reactivating the TRAIL pathway and, ultimately leading to apoptosis. These compounds thus trigger apoptosis in both cell types, but via different pathways. Taken together, our data show that derivatives 1 and 2 of 4,11-diaminoanthra[2,3-b]furan-5,10-diones engage with tNOX and inhibit its oxidase activity. This results in cytotoxicity via apoptosis through tNOX-SIRT1 axis to enhance the acetylation of p53 or c-Myc in HCC cells, depending on their p53 status.

The antibiotic heliomycin (resistomycin) has been studied since the 1950s, however, its low solubility in aqueous media has restricted its systemic bioavailability and therapeutic potential. We recently synthesized a series of novel aminomethyl derivatives that are readily soluble in aqueous solution. In this study, we examined their antiproliferative properties and underlying molecular mechanisms in p53 wild-type and p53-mutated cancer cells. We continuously monitored the cellular impact of these compounds using the xCELLigence System, a label-free, real-time cell monitoring platform that measures electrical impedance, displaying results as cell index values. The results from the system showed that heliomycin and its derivatives exhibited different biological effects on tumor cells. To further our understanding of the interactions between the novel derivatives and cellular targets, we utilized the cellular thermal shift assay (CETSA). Our CETSA results revealed that some of the derivatives that were directly engaged with tNOX enhanced its thermal stability, which in turn resulted in a marked attenuation of the intracellular NAD+/NADH ratio. This attenuated ratio signifies a decrease in the NAD+-dependent activity of Sirtuin 1 (SIRT1) deacetylase and triggers apoptosis/autophagy, with varying amounts of decrease depending on the cellular environment. We have successfully transformed heliomycin into derivatives that are much more soluble, allowing the more efficient modulating of biological activities - a significant step closer towards finding a novel anti-cancer candidate. Citation Format: Pin Ju Chueh, Atikul Islam, Xiao Qi Chen, Shi Han Chiu, Alexander S. Tikhomirov, Andrey E. Shchekotikhin. Novel antitumor semi-synthetic heliomycin derivatives direct targeting tNOX-NAD+-SIRT1 axis to activate apoptosis/autophagy, as determined by the cellular thermal shift assay (CETSA) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6224.

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.

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.

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.

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.

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.

The identification of prognostic markers and establishing their value as therapeutic targets improves therapeutic efficacy against human cancers. Ribophorin II (RPN2) has been demonstrated to be a prognostic marker of human cancer, including breast and pancreatic cancers. The present study aimed to evaluate RPN2 expression in gastric cancer and to examine the possible correlation between RPN2 expression and the response of cells to clinical anticancer drugs, which has received little research attention at present. The gastric cancer AGS, TMC-1, SNU-1, TMK-1, SCM-1, MKN-45 and KATO III cell lines were used as a model to elucidate the role of RPN2 in the response of cells to six common chemotherapeutic agents, comprising oxaliplatin, irinotecan, doxorubicin, docetaxel, cisplatin and 5-fluorouricil. The functional role of RPN2 was assessed by silencing RPN2 using small interfering RNA (siRNA), and the cytotoxicity was determined by an MTS assay and analysis of apoptosis. Molecular events were evaluated by western blotting. All the anticancer drugs were found to exert a concentration-dependent decrease on the cell survival rate of each of the cell lines tested, although the RPN2 levels in the various cell lines were not directly correlated with responsiveness to clinical anticancer drugs, based on the calculated IC50 values. siRNA-mediated RPN2 downregulation enhanced cisplatin-induced apoptosis in AGS cells, but did not markedly decrease the cell survival rates of these cells in response to the tested drugs. Furthermore, RPN2 silencing in MKN-45 cells resulted in no additional increase in the cisplatin-induced apoptosis and survival rates. It was also found that RPN2 depletion increased anticancer drug-mediated cytotoxicity in gastric cancer cell lines. However, the predictive value of RPN2 expression in cancer therapy is questionable in gastric cancer models.

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.

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.

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.

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.