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Oral submucous fibrosis (OSF) is a precancerous condition in the oral cavity, which is closely related to the myofibroblast conversion of buccal mucosal fibroblasts (BMFs) after chronic consumption of areca nut. Emerging evidence suggests pyroptosis, a form of programmed cell death that is mediated by inflammasome, is implicated in persistent myofibroblast activation and fibrosis. Besides, numerous studies have demonstrated the effects of non-coding RNAs on pyroptosis and myofibroblast activities. Herein, we aimed to target key long non-coding RNA PVT1 with natural compound, carvacrol, to alleviate pyroptosis and myofibroblast activation in OSF. We first identified PVT1 was downregulated in the carvacrol-treated fBMFs and then demonstrated that myofibroblast features and expression of pyroptosis makers were all reduced in response to carvacrol treatment. Subsequently, we analysed the expression of PVT1 and found that PVT1 was aberrantly upregulated in OSF specimens and positively correlated with several fibrosis markers. After revealing the suppressive effects of carvacrol on myofibroblast characterisitcs and pyroptosis were mediated by repression of PVT1, we then explored the potential mechanisms. Our data showed that PVT1 may serve as a sponge of microRNA(miR)-20a to mitigate the myofibroblast activation and pyroptosis. Altogether, these findings indicated that the anti-fibrosis effects of carvacrol merit consideration and may be due to the attenuation of pyroptosis and myofibroblast activation by targeting the PVT1/miR-20a axis., (© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

The antibiotic heliomycin (resistomycin), which is generated from Streptomyces 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 (CETSA) and molecular docking simulations to identify and validate that heliomycin and its water-soluble derivative, 4-(dimethylaminomethyl)heliomycin (designated compound 4-dmH) engaged and targeted with sirtuin-1 (SIRT1) in p53-functional SAS and p53-mutated HSC-3 oral cancer cells. We further addressed the cellular outcome of SIRT1 inhibition by these compounds and found that, in addition to SIRT1, the water-soluble 4-dmH preferentially targeted a tumor-associated NADH oxidase (tNOX, ENOX2). The direct binding of 4-dmH to tNOX decreased the oxidation of NADH to NAD + which diminished NAD + -dependent SIRT1 deacetylase activity, ultimately inducing apoptosis and significant cytotoxicity in both cell types, as opposed to the parental heliomycin-induced autophagy. 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, our in vitro and in vivo findings 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., Competing Interests: AI, YC, XC, CW, CC, CW, MC, AT, AS, PC No competing interests declared, (© 2023, Islam et al.)

Immunotherapy has emerged as a promising new treatment modality for head and neck cancer, offering the potential for targeted and effective cancer management. Squamous cell carcinomas pose significant challenges due to their aggressive nature and limited treatment options. Conventional therapies such as surgery, radiation, and chemotherapy often have limited success rates and can have significant side effects. Immunotherapy harnesses the power of the immune system to recognize and eliminate cancer cells, and thus represents a novel approach with the potential to improve patient outcomes. In the management of head and neck squamous cell carcinoma (HNSCC), important contributions are made by immunotherapies, including adaptive cell therapy (ACT) and immune checkpoint inhibitor therapy. In this review, we are focusing on the latter. Immune checkpoint inhibitors target proteins such as programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) to enhance the immune response against cancer cells. The CTLA-4 inhibitors, such as ipilimumab and tremelimumab, have been approved for early-stage clinical trials and have shown promising outcomes in terms of tumor regression and durable responses in patients with advanced HNSCC. Thus, immune checkpoint inhibitor therapy holds promise in overcoming the limitations of conventional therapies. However, further research is needed to optimize treatment regimens, identify predictive biomarkers, and overcome potential resistance mechanisms. With ongoing advancements in immunotherapy, the future holds great potential for transforming the landscape of oral tumor treatment and providing new hope for patients.

Colorectal cancer is the most common cancer that affects both sexes and has a poor prognosis due to aggressiveness and chemoresistance. Essential oils isolated from Calocedrus formosana (CF-EOs) have been shown to demonstrate anti-termite, antifungal, anti-mosquito, and anti-microbial activities. However, the anticancer effects of CF-EOs are not yet fully understood. Therefore, the present study aimed to explore the molecular mechanism underlying CF-EOs-mediated anti-proliferative activity in colon cancer cells. Here, cell impedance measurements showed that CF-EOs inhibit proliferation in colon cancer cells with wild-type or mutant p53. Flow cytometry revealed that CF-EOs at 20, 50 µg/mL significantly induced ROS generation and autophagy in both HCT116 p53-wt and HCT116 p53-null cell lines, whereas pretreatment with the ROS scavenger N-acetyl cysteine (NAC) markedly attenuated these changes. CF-EOs also induced apoptosis at 50 µg/mL in both lines, as determined by flow cytometry. Protein analysis showed that CF-EOs markedly induced apoptosis markers, including Trail, cleaved caspase-3, cleaved caspase-9, and cleaved PARP, as well as autophagy markers, such as the levels of ULK1, Atg5, Atg6, Atg7, and the conversion of LC3-I to LC3-II. CF-EOs were further found to inhibit the activity and expression of the NAD + -dependent deacetylase SIRT1 to increase the levels of acetylated p53 (Ac-p53) in p53-wt cells and acetylated c-Myc (Ac-c-Myc) in p53-null cells, ultimately inducing apoptosis in both lines. Interestingly, suppression of SIRT1 by CF-EOs enhanced the acetylation of ULK1, which in turn prompted ROS-dependent autophagy in colon cancer cells. The induction of apoptosis and autophagy by CF-EOs suggests that they may have potential as a promising new approach for treating cancer. Collectively, our results suggest that essential oils isolated from Calocedrus formosana act as a promising anticancer agent against colon cancer cells by targeting SIRT1 to induce ROS-mediated autophagy and apoptosis.

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., Competing Interests: None., (AJCR Copyright © 2022.)

Although considered a sporadic type of skin cancer, malignant melanoma has regularly increased internationally and is a major 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 effect of capsaicin on melanoma growth. The cellular thermal shift assay (CETSA), isothermal dose-response fingerprint curves (ITDRF CETSA ), and CETSA-pulse proteolysis were utilized to confirm the direct 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. Our data show that capsaicin directly engaged with cellular tNOX to inhibit its enzymatic activity and enhance protein degradation capacity. The inhibition of tNOX by capsaicin was accompanied by the attenuation of SIRT1, a NAD + -dependent deacetylase. The suppression of tNOX and SIRT1 then enhanced ULK1 acetylation and induced 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. Taken 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 ROS-dependent autophagy in melanoma cells., Competing Interests: None., (AJCR Copyright © 2021.)

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., Competing Interests: None., (AJCR Copyright © 2020.)

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 "target engagement" 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/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., (Copyright © 2019. Published by Elsevier B.V.)

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., Competing Interests: None.

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., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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., Competing Interests: None.

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., Competing Interests: The authors declare no conflict of interest.

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.

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.

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.

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.

Capsaicin has been reported to preferentially inhibit the activity of tumor-associated NADH oxidase (tNOX), which belongs to a family of growth-related plasma membrane hydroquinone oxidases in cancer/transformed cells. The inhibitory effect of capsaicin on tNOX is associated with cell growth attenuation and apoptosis. However, no previous study has examined the transcriptional regulation of tNOX protein expression. Bioinformatic analysis has indicated that the tNOX promoter sequence harbors a binding motif for POU3F2, which is thought to play important roles in neuronal differentiation, melanocytes growth/differentiation and tumorigenesis. In this study, we found that capsaicin-mediated tNOX downregulation and cell migration inhibition were through POU3F2. The protein expression levels of POU3F2 and tNOX are positively correlated, and that overexpression of POU3F2 (and the corresponding upregulation of tNOX) enhanced the proliferation, migration and invasion in AGS (human gastric carcinoma) cells. In contrast, knockdown of POU3F2 downregulates tNOX, and the cancer phenotypes are affected. These findings not only shed light on the molecular mechanism of the anticancer properties of capsaicin, but also the transcription regulation of tNOX expression that may potentially explain how POU3F2 is associated with tumorigenesis.

Objective: To compare the use of laparoscopy and laparotomy for hysterectomy in treating benign gynecological conditions in nurses and nonmedical working women., Materials and Methods: A nationwide population-based study was conducted using data from inpatient expenditures of Taiwan's National Health Insurance Research Datasets between 2008 and 2011. All women undergoing total hysterectomies via laparoscopy and laparotomy between the ages of 20 years and 65 years were identified. A generalized equation estimation model was used to compare the differences in laparoscopic hysterectomy (LH) and total abdominal hysterectomy (TAH) between the two groups., Results: There were 1226 nurses, and 36,624 nonmedical working women, serving as controls, in the present study. The LH rate, as compared to the TAH rate, was significantly higher among nurses than among controls (56% vs. 52%, p = 0.006). A multivariate analysis indicated that nurses were significantly more likely to undergo LH (odds ratio, 1.12; 95% confidence interval, 1.01-1.26) than TAH. Nurses were more often treated at medical centers or high-volume hospitals, and were more often treated by high-volume gynecologists., Conclusion: Compared to nonmedical working women, nurses are more likely to undergo LH rather than TAH. These differences may suggest the influence of medical knowledge and physician-patient interaction on the decision to undergo laparoscopy, in addition to patients' medical conditions., (Copyright © 2016. Published by Elsevier B.V.)

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.

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 IC 50 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.

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.

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.

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.

Plasma membranes of plant cells are characterized by a plant hormone (auxin)-responsive oxidation of NADH. The latter proceeds under argon. Also, when NADH oxidation is stimulated 50% by auxin addition, oxygen consumption is reduced by 40%. These findings are reconciled by direct assays using 5,5'-dithiobis-(2nitrobenzoic acid) (DTNB) (Ellman's reagent) that show protein disulfides to be electron acceptors for auxin-stimulated NADH oxidation. In the presence of an external reducing agent such as NADH, cysteine, or dithiothreitol, protein disulfides of the membrane are reduced with a concomitant stoichiometric increase in free thiols. In the absence of an external reducing agent, or in the presence of oxidized glutathione, DTNB-reactive thiols of the plasma membrane are decreased in the presence of auxins. Several auxin-reductant combinations were effective, but the same reductants plus chemically related and growth-inactive auxin analogs were not. A cell surface location of the affected thiols demonstrated with detergents and impermeant thiol reagents suggests that the protein may have a different physiological role than oxidation of NADH. For example, it may carry out some other role more closely related to the function of the auxin hormones in cell enlargement such as protein disulfide-thiol interchange.

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.