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The antibiotic heliomycin (resistomycin), which is generated fromStreptomyces resistomycificus, has multiple activities, including anticancer effects. Heliomycin was first described in the 1960s, but its clinical applications have been hindered by extremely low solubility. A series of 4-aminomethyl derivatives of heliomycin were synthesized to increase water solubility; studies showed that they had anti-proliferative effects, but the drug targets remained unknown. In this study, we conducted cellular thermal shift assays and molecular docking simulations to identify and validate the intracellular targets of heliomycin and its water-soluble derivative, 4-(dimethylaminomethyl)heliomycin (designated compound 4-dmH), in p53-functional SAS and p53-mutated HSC-3 oral cancer cells. Consistent with ourin silicostudies, our cellular thermal shift assays (CETSA) revealed that, in addition to SIRT1, the water-soluble 4-dmH preferentially targeted a tumor-associated NADH oxidase called tNOX or ENOX2. The direct binding of 4-dmH to tNOX inhibited the activity of tNOX and enhanced its ubiquitin-proteasomal protein degradation in both SAS and HSC-3 cells. Moreover, the inhibition of tNOX by 4-dmH decreased the oxidation of NADH to NAD+which diminished NAD+-dependent SIRT1 deacetylase activity, ultimately inducing apoptosis and significant cytotoxicity in both cell types. We also observed that tNOX and SIRT1 were both upregulated in tumor tissues of oral cancer patients compared to adjacent normal tissues, suggesting their clinical relevance. Finally, the better therapeutic efficacy of 4-dmH was confirmed in tumor-bearing mice, which showed greater tNOX and SIRT1 downregulation and tumor volume reduction when treated with 4-dmH compared to heliomycin. Taken together, ourin vitroandin vivofindings suggest that the multifaceted properties of water-soluble 4-dmH enable it to offer superior antitumor value compared to parental heliomycin, and indicated that it functions through targeting the tNOX-NAD+-SIRT1 axis to induce apoptosis in oral cancer cells.

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.

Bladder cancer is one of the most frequent cancers among males, and a poor survival rate reflects problems with aggressiveness and chemo-resistance. Accumulating evidence indicates that SIRT1 is involved in bladder cancer tumorigenesis and is positively associated with chemo-resistance and poor prognosis. We recently synthesized water-soluble chemical derivatives of heliomycin, an antibiotic from Streptomyces resistomycificus, and demonstrated that they possess anticancer properties. In this present study, we used the cellular thermal shift assay (CETSA) in T24 bladder cancer cells to show that heliomycin (designated compound (H1)) and its 4-(tert-butylamino)methyl derivative (HD2) directly engaged with SIRT1 in the native cellular environment, whereas another derivative (HD3) did not. Upon binding, heliomycin downregulated SIRT1 protein expression without altering its transcript level, and subsequently induced autophagy. Interestingly, the derivative (HD2) triggered apoptosis. The interaction between SIRT1 protein and heliomycin or its derivatives was also speculated by a molecular docking simulation, suggesting heliomycin (H1) and derivative (HD2) acting with the different binding modes to SIRT1. Given the increased water-solubility, hydrogen bonds were found on Ala262 and Ile347 residues in the docked complex of derivative (HD2) to produce more steady interaction and initiate signaling pathways that were not observed in the case of heliomycin. Meanwhile, it is evident that derivative (HD3) did not engage with SIRT1 by CETSA or molecular docking studies, nor did it downregulate SIRT1 expression. Taken together, these findings clearly show that SIRT1 is targeted and downregulated by heliomycin and its water-soluble 4-aminomethylated derivative (HD2) possibly through autophagic and/or proteasomal degradation, leading to cell death and growth suppression of T24 bladder cancer cells.

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.

Background: Although considered a rare form of skin cancer, malignant melanoma has steadily increased internationally and is a main cause of cancer-associated death worldwide. The treatment options for malignant melanoma are very limited. Accumulating data suggest that the natural compound, capsaicin, exhibits preferential anticancer properties to act as a nutraceutical agent. Here, we explored the underlying molecular events involved in the inhibitory effects of capsaicin on the growth of melanoma cells.Methods: The cellular thermal shift assay (CETSA) and isothermal dose response fingerprint (ITDRFCETSA) were utilized to validate the binding of capsaicin with the tumor-associated NADH oxidase, tNOX (ENOX2) in melanoma cells. We also assessed the cellular impact of capsaicin-targeting of tNOX on A375 cells by flow cytometry and protein analysis. The essential role of tNOX in tumor- and melanoma-growth limiting abilities of capsaicin was evaluated in C57BL/6 mice.Results: Our data show that capsaicin directly targets cellular tNOX to inhibit its enzymatic activity and enhance protein degradation capacity. The inhibition of tNOX by capsaicin is accompanied by the attenuation of SIRT1, a NAD+-dependent deacetylase that enhances ULK1 acetylation to induce ROS-dependent autophagy in melanoma cells. Capsaicin treatment of mice implanted with melanoma cancer cells suppressed tumor growth by down-regulating tNOX and SIRT1, which was also seen in an in vivo xenograft study with tNOX-depleted melanoma cells. Conclusions: Together, our findings suggest that tNOX expression is important for the growth of melanoma cancer cells both in vitro and in vivo, and that inhibition of the tNOX-SIRT1 axis contributes to inducting cytotoxic ROS-dependent autophagy in melanoma cells.

Despite the progress that has been made in diagnosing and treating oral cancers, they continue to have a poor prognosis, with a 5-year overall survival rate of approximately 50%. We have intensively studied the anticancer properties of capsaicin (a burning constituent of chili pepper), mainly focusing on its apoptotic properties. Here, we investigated the interplay between apoptosis and autophagy in capsaicin-treated oral cancer cells with either functional or mutant p53. Cytotoxicity was determined by cell impedance measurements and WST-1 assays, and cell death was analyzed by flow cytometry. The interaction between capsaicin and tumor-associated NADH oxidase (tNOX, ENOX2) was studied by cellular thermal shift assay (CETSA) and isothermal dose-response fingerprint curves (ITDRF(CETSA)). Our CETSA data suggested that capsaicin directly engaged with tNOX, resulting in its degradation through the ubiquitin-proteasome and the autophagy-lysosome systems. In p53-functional SAS cells, capsaicin induced significant cytotoxicity via autophagy but not apoptosis. Given that tNOX catalyzes the oxidation of NADH, the direct binding of capsaicin to tNOX also inhibited the NAD(+)-dependent activity of sirtuin 1 (SIRT1) deacetylase, we found that capsaicin-induced autophagy involved enhanced acetylation of ULK1, which is a key player in autophagy activation, possibly through SIRT1 inhibition. In p53-mutated HSC-3 cells, capsaicin triggered both autophagy and apoptosis. In this case, autophagy occurred before apoptosis: during this early stage, autophagy seemed to inhibit apoptosis; at a later stage, in contrast, autophagy appeared to be essential for the induction of apoptosis. Western blot analysis revealed that the reduction in tNOX and SIRT1 associated with enhanced ULK1 acetylation and c-Myc acetylation, which in turn, reactivated the TRAIL pathway, ultimately leading to apoptosis. Taken together, our data highlight the potential value of leveraging capsaicin and tNOX in therapeutic strategies against oral cancer.

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.

We have studied the chemopreventive property of capsaicin, a major active component in chili pepper, and found that it exhibited apoptotic activity against various lines of cancer cells. Interestingly, accumulating data has revealed that, in addition to cytotoxicity, capsaicin also plays regulatory role on cell migration and invasion. However, its effect on cell migration is paradoxical and not completely understood. Here, we set out to elucidate the molecular events underlying capsaicin-inhibited cell migration in bladder cancer cells. Our results show that the capsaicin-reduced cell migration was associated with down-regulation of sirtuin 1 (SIRT1) deacetylase, possibly through proteasome-mediated protein degradation. More importantly, we employed a cellular thermal shift assay (CETSA) to demonstrate that there was a direct binding between capsaicin and SIRT1. The engagement with capsaicin and protein degradation diminished the deacetylase of SIRT1, which in turn, enhanced acetylation of cortactin and β-catenin to decrease MMP-2 and MMP-9 activation, resulting in cell migration impairment in bladder cancer cells.

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.

Tumor-associated NADH oxidase (tNOX, ENOX2), which belongs to a family of growth-related NADH oxidases, was originally identified as a plasma membrane protein of rat hepatoma and is inhibited or downregulated by several anti-cancer drugs. The objective of this study was to evaluate the anti-tumor effects of tNOX used as an immunogen against Lewis lung cancer. Human tNOX was expressed in Escherichia coli, purified by His-Tag affinity chromatography, and emulsified with the adjuvant, ISA 201 VG. Immunological analyses of the generated tNOX vaccine were performed in mice. The results of ELISA and ELISpot were significantly higher in tNOX vaccine group compared to the control group. In vivo, we examined the anti-tumor effects of mice that received the tNOX vaccine via the intraperitoneal or subcutaneous routes. Mice were vaccinated three times at 2-week intervals, challenged at 2 weeks after the final vaccination, and terminated at 34 days post-challenge. Antibody titers, tumor volume and histopathological scores were used to evaluate the anti-tumor effects of the tNOX vaccine. Our results revealed that tNOX-vaccinated mice had significantly higher antibody titers than negative control (NC) and challenge control (CC) mice. When compared to the corresponding CC groups, the intraperitoneal and subcutaneous vaccination with tNOX showed a significantly smaller tumor mass volume (P < 0.05) and a significantly lower histological lesion score (P < 0.05), respectively. Our results demonstrate that the use of a xenogeneic tNOX as an immunogen in mice activates immune responses and anti-tumor effects against Lewis lung cancer.

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.

There is currently a significant need for effective methods aimed at diagnosing and screening for nanoparticle exposure. We previously investigated the toxicity of three different particle sized gold nanoparticles (AuNPs) toward different types of mammalian cells and explored a related gene expression profile by cDNA microarray analysis of AuNP-exposed MRC-5 cells. In this study, we sought to further identify genes that could be used as biomarkers for AuNP exposure. We used cDNA microarray analysis to obtain comprehensive gene expression profiles from A549 cells exposed to three different-sized AuNPs. A total of 409 genes were commonly up-regulated by the tested AuNPs; of them, 71 had previously been analyzed to be up-regulated in MRC-5 cells. Among the top-ranked 30 of these 71 up-regulated genes, based on the magnitude of induction, nine genes were confirmed to be transcriptionally induced in A549 cells by all three tested AuNPs, as assessed by quantitative real-time polymerase chain reaction (qPCR). Among them, TSC22D3, TRIB3, PCK2 and DDIT4 were the most sensitive to the three AuNPs, and showed dose-dependent changes in several human cell lines. qPCR and immunoblotting analyses revealed that the same concentrations of micro-Au and nano-TiO2 failed to elicit up-regulation of these four genes at the mRNA and protein levels in any tested cell lines. Although the definition and practical implementation of specific biomarkers for nanoparticles is still in its infancy, our data suggest that it may be possible to define reliable biomarkers for the diagnosis of nanomaterial exposure.

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.

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.

NADH oxidases at the external surface of plant and animal cells (ECTO-NOX proteins) exhibit stable and recurring patterns of oscillations with potentially clock-related, entrainable, and temperature-compensated period lengths of 24 min. To determine if ECTO-NOX proteins might represent the ultradian time keepers (pacemakers) of the biological clock, COS cells were transfected with cDNAs encoding tNOX proteins having a period length of 22 min or with C575A or C558A cysteine to alanine replacements having period lengths of 36 or 42 min. Here we demonstrate that such transfectants exhibited 22, 36, or 40 to 42 h circadian patterns in the activity of glyceraldehyde-3-phosphate dehydrogenase, a common clock-regulated protein, in addition to the endogenous 24 h circadian period length. The fact that the expression of a single oscillatory ECTO-NOX protein determines the period length of a circadian biochemical marker (60 X the ECTO-NOX period length) provides compelling evidence that ECTO-NOX proteins are the biochemical ultradian drivers of the cellular biological clock.