Water-Soluble Heliomycin Derivatives to Target i-Motif DNA.

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.

MicroRNA-708 activation by glucocorticoid receptor agonists regulate breast cancer tumorigenesis and metastasis via downregulation of NF-κB signaling.

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 IKKß 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.

miR-1246 as a therapeutic target in oral submucosa fibrosis pathogenesis.

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.

Glabridin inhibits the activation of myofibroblasts in human fibrotic buccal mucosal fibroblasts through TGF-ß/smad signaling.

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-ß 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-ß/Smad2 signaling pathway. Glabridin also prevented the arecoline-increased myofibroblast activities, and could serve as a natural anti-fibrosis compound for OSF.

Biocompatibility assessment of nanomaterials for environmental safety screening.

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.

Combined Use of Zoledronic Acid Augments Ursolic Acid-Induced Apoptosis in Human Osteosarcoma Cells through Enhanced Oxidative Stress and Autophagy.

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.

Capsaicin Inhibits Multiple Bladder Cancer Cell Phenotypes by Inhibiting Tumor-Associated NADH Oxidase (tNOX) and Sirtuin1 (SIRT1).

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 Inhibited Aggressive Phenotypes through Downregulation of Tumor-Associated NADH Oxidase (tNOX) by POU Domain Transcription Factor POU3F2.

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.

Extensive evaluations of the cytotoxic effects of gold nanoparticles.

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.

The Use of Immune Regulation in Treating Head and Neck Squamous Cell Carcinoma (HNSCC).

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.

Calocedrus formosana Essential Oils Induce ROS-Mediated Autophagy and Apoptosis by Targeting SIRT1 in Colon Cancer Cells.

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.

Water-soluble 4-(dimethylaminomethyl)heliomycin exerts greater antitumor effects than parental heliomycin by targeting the tNOX-SIRT1 axis and apoptosis in oral cancer cells.

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.

Chemotherapeutic agents enhance cell migration and epithelial-to-mesenchymal transition through transient up-regulation of tNOX (ENOX2) protein.

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

Phosphorylation of serine-504 of tNOX (ENOX2) modulates cell proliferation and migration in cancer cells.

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

Bis(chloroacetamidino)-Derived Heteroarene-Fused Anthraquinones Bind to and Cause Proteasomal Degradation of tNOX, Leading to c-Flip Downregulation and 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.

Antibiotic heliomycin and its water-soluble 4-aminomethylated derivative provoke cell death in T24 bladder cancer cells by targeting sirtuin 1 (SIRT1).

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.

Inhibitory effect of human breast cancer cell proliferation via p21-mediated G1 cell cycle arrest by araliadiol isolated from Aralia cordata Thunb.

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

Capsaicin exerts therapeutic effects by targeting tNOX-SIRT1 axis and augmenting ROS-dependent autophagy in melanoma cancer cells.

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 (ITDRFCETSA), 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.

Capsaicin acts through tNOX (ENOX2) to induce autophagic apoptosis in p53-mutated HSC-3 cells but autophagy in p53-functional SAS oral cancer 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 (ITDRFCETSA). 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.

Disturbed mitotic progression and genome segregation are involved in cell transformation mediated by nano-TiO2 long-term exposure.

Titanium dioxide (TiO2) nano-particles (<100 nm in diameter) have been of interest in a wide range of applications, such as in cosmetics and pharmaceuticals because of their low toxicity. However, recent studies have shown that TiO2 nano-particles (nano-TiO2) induce cytotoxicity and genotoxicity in various lines of cultured cells as well as tumorigenesis in animal models. The biological roles of nano-TiO2 are shown to be controversial and no comprehensive study paradigm has been developed to investigate their molecular mechanisms. In this study, we showed that short-term exposure to nano-TiO2 enhanced cell proliferation, survival, ERK signaling activation and ROS production in cultured fibroblast cells. Moreover, long-term exposure to nano-TiO2 not only increased cell survival and growth on soft agar but also the numbers of multinucleated cells and micronucleus (MN) as suggested in confocal microscopy analysis. Cell cycle phase analysis showed G2/M delay and slower cell division in long-term exposed cells. Most importantly, long-term TiO2 exposure remarkably affected mitotic progression at anaphase and telophase leading to aberrant multipolar spindles and chromatin alignment/segregation. Moreover, PLK1 was demonstrated as the target for nano-TiO2 in the regulation of mitotic progression and exit. Notably, a higher fraction of sub-G1 phase population appeared in TiO2-exposed cells after releasing from G2/M synchronization. Our results demonstrate that long-term exposure to nano-TiO2 disturbs cell cycle progression and duplicated genome segregation, leading to chromosomal instability and cell transformation.