Cardiac glycoside ouabain efficiently targets leukemic stem cell apoptotic machinery independent of cell differentiation status.

BACKGROUND: Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by an accumulation of immature leukemic myeloblasts initiating from leukemic stem cells (LSCs)-the subpopulation that is also considered the root cause of chemotherapy resistance. Repurposing cardiac glycosides to treat cancers has gained increasing attention and supporting evidence, but how cardiac glycosides effectively target LSCs, e.g., whether it involves cell differentiation, remains largely unexplored. METHODS: Digoxin, a user-designed digitoxigenin-α-L-rhamnoside (D6-MA), and ouabain were tested against various human AML-derived cells with different maturation phenotypes. Herein, we established two study models to specifically determine the effects of cardiac glycosides on LSC death and differentiation-one allowed change in dynamics of LSCs and leukemic progenitor cells (LPCs), while another maintained their undifferentiated status. Regulatory mechanisms underlying cardiac glycoside-induced cytotoxicity were investigated and linked to cell cycle distribution and apoptotic machinery. RESULTS: Primitive AML cells containing CD34+ LSCs/LPCs were very responsive to nanomolar concentrations of cardiac glycosides, with ouabain showing the greatest efficiency. Ouabain preferentially induces caspase-dependent apoptosis in LSCs, independent of its cell differentiation status, as evidenced by (i) the tremendous induction of apoptosis by ouabain in AML cells that acquired less than 15% differentiation and (ii) the higher rate of apoptosis in enriched LSCs than in LPCs. We sorted LSCs and LPCs according to their cell cycle distribution into G0/G1, S, and G2/M cells and revealed that G0/G1 cells in LSCs, which was its major subpopulation, were the top ouabain responders, indicating that the difference in ouabain sensitivity between LSCs and LPCs involved both distinct cell cycle distribution and intrinsic apoptosis regulatory mechanisms. Further, Mcl-1 and c-Myc, which were differentially expressed in LSCs and LPCs, were found to be the key apoptosis mediators that determined ouabain sensitivity in AML cells. Ouabain induces a more rapid loss of Mcl-1 and c-Myc in LSCs than in LPCs via the mechanisms that in part involve an inhibition of Mcl-1 protein synthesis and an induction of c-Myc degradation. CONCLUSIONS: Our data provide new insight for repurposing cardiac glycosides for the treatment of relapsed/refractory AML through targeting LSCs via distinct cell cycle and apoptosis machinery. Video Abstract.

An NLRP7-containing inflammasome mediates recognition of microbial lipopeptides in human macrophages.

Cytosolic pathogen- and damage-associated molecular patterns are sensed by pattern recognition receptors, including members of the nucleotide-binding domain and leucine-rich repeat-containing gene family (NLR), which cause inflammasome assembly and caspase-1 activation to promote maturation and release of the inflammatory cytokines interleukin-1ß (IL-1ß) and IL-18 and induction of pyroptosis. However, the contribution of most of the NLRs to innate immunity, host defense, and inflammasome activation and their specific agonists are still unknown. Here we describe identification and characterization of an NLRP7 inflammasome in human macrophages, which is induced in response to microbial acylated lipopeptides. Activation of NLRP7 promoted ASC-dependent caspase-1 activation, IL-1ß and IL-18 maturation, and restriction of intracellular bacterial replication, but not caspase-1-independent secretion of the proinflammatory cytokines IL-6 and tumor necrosis factor-α. Our study therefore increases our currently limited understanding of NLR activation, inflammasome assembly, and maturation of IL-1ß and IL-18 in human macrophages.

Theasaponin E1 Inhibits Platinum-Resistant Ovarian Cancer Cells through Activating Apoptosis and Suppressing Angiogenesis.

Novel therapeutic strategies for ovarian cancer treatment are in critical need due to the chemoresistance and adverse side effects of platinum-based chemotherapy. Theasaponin E1 (TSE1) is an oleanane-type saponin from Camellia sinensis seeds. Its apoptosis-inducing, cell cycle arresting and antiangiogenesis activities against platinum-resistant ovarian cancer cells were elucidated in vitro and using the chicken chorioallantoic membrane (CAM) assay. The results showed that TSE1 had more potent cell growth inhibitory effects on ovarian cancer OVCAR-3 and A2780/CP70 cells than cisplatin and was lower in cytotoxicity to normal ovarian IOSE-364 cells. TSE1 significantly induced OVCAR-3 cell apoptosis via the intrinsic and extrinsic apoptotic pathways, slightly arresting cell cycle at the G2/M phase, and obviously inhibited OVCAR-3 cell migration and angiogenesis with reducing the protein secretion and expression of vascular endothelial growth factor (VEGF). Western bolt assay showed that Serine/threonine Kinase (Akt) signaling related proteins including Ataxia telangiectasia mutated kinase (ATM), Phosphatase and tensin homolog (PTEN), Akt, Mammalian target of rapamycin (mTOR), Ribosome S6 protein kinase (p70S6K) and e IF4E-binding protein 1(4E-BP1) were regulated, and Hypoxia inducible factor-1α (HIF-1α) protein expression was decreased by TSE1 in OVCAR-3 cells. Moreover, TSE1 treatment potently downregulated protein expression of the Notch ligands including Delta-like protein 4 (Dll4) and Jagged1, and reduced the protein level of the intracellular domain (NICD) of Notch1. Combination treatment of TSE1 with the Notch1 signaling inhibitor tert-butyl (2S)-2-[[(2S)-2-[[2-(3,5-difluorophenyl)acetyl]amino]propanoyl]amino]-2-phenylacetate (DAPT), or the Akt signaling inhibitor wortmannin, showed a stronger inhibition toward HIF-1α activation compared with single compound treatment. Taken together, TSE1 might be a potential candidate compound for improving platinum-resistant ovarian cancer treatment via Dll4/Jagged1-Notch1-Akt-HIF-1α axis.

A novel TRPM7/O-GlcNAc axis mediates tumour cell motility and metastasis by stabilising c-Myc and caveolin-1 in lung carcinoma.

BACKGROUND: Calcium is an essential signal transduction element that has been associated with aggressive behaviours in several cancers. Cell motility is a prerequisite for metastasis, the major cause of lung cancer death, yet its association with calcium signalling and underlying regulatory axis remains an unexplored area. METHODS: Bioinformatics database analyses were employed to assess correlations between calcium influx channels and clinical outcomes in non-small cell lung cancer (NSCLC). Functional and regulatory roles of influx channels in cell migration and invasion were conducted and experimental lung metastasis was examined using in vivo live imaging. RESULTS: High expression of TRPM7 channel correlates well with the low survival rate of patients and high metastatic potential. Inhibition of TRPM7 suppresses cell motility in various NSCLC cell lines and patient-derived primary cells and attenuates experimental lung metastases. Mechanistically, TRPM7 acts upstream of O-GlcNAcylation, a post-translational modification and a crucial sensor for metabolic changes. We reveal for the first time that caveolin-1 and c-Myc are favourable molecular targets of TRPM7/O-GlcNAc that regulates NSCLC motility. O-GlcNAcylation of caveolin-1 and c-Myc promotes protein stability by interfering with their ubiquitination and proteasomal degradation. CONCLUSIONS: TRPM7/O-GlcNAc axis represents a potential novel target for lung cancer therapy that may overcome metastasis.

Interleukin-12 elicits a non-canonical response in B16 melanoma cells to enhance survival.

BACKGROUND: Oncogenesis rewires signaling networks to confer a fitness advantage to malignant cells. For instance, the B16F0 melanoma cell model creates a cytokine sink for Interleukin-12 (IL-12) to deprive neighboring cells of this important anti-tumor immune signal. While a cytokine sink provides an indirect fitness advantage, does IL-12 provide an intrinsic advantage to B16F0 cells? METHODS: Acute in vitro viability assays were used to compare the cytotoxic effect of imatinib on a melanoma cell line of spontaneous origin (B16F0) with a normal melanocyte cell line (Melan-A) in the presence of IL-12. The results were analyzed using a mathematical model coupled with a Markov Chain Monte Carlo approach to obtain a posterior distribution in the parameters that quantified the biological effect of imatinib and IL-12. Intracellular signaling responses to IL-12 were compared using flow cytometry in 2D6 cells, a cell model for canonical signaling, and B16F0 cells, where potential non-canonical signaling occurs. Bayes Factors were used to select among competing signaling mechanisms that were formulated as mathematical models. Analysis of single cell RNAseq data from human melanoma patients was used to explore generalizability. RESULTS: Functionally, IL-12 enhanced the survival of B16F0 cells but not normal Melan-A melanocytes that were challenged with a cytotoxic agent. Interestingly, the ratio of IL-12 receptor components (IL12RB2:IL12RB1) was increased in B16F0 cells. A similar pattern was observed in human melanoma. To identify a mechanism, we assayed the phosphorylation of proteins involved in canonical IL-12 signaling, STAT4, and cell survival, Akt. In contrast to T cells that exhibited a canonical response to IL-12 by phosphorylating STAT4, IL-12 stimulation of B16F0 cells predominantly phosphorylated Akt. Mechanistically, the differential response in B16F0 cells is explained by both ligand-dependent and ligand-independent aspects to initiate PI3K-AKT signaling upon IL12RB2 homodimerization. Namely, IL-12 promotes IL12RB2 homodimerization with low affinity and IL12RB2 overexpression promotes homodimerization via molecular crowding on the plasma membrane. CONCLUSIONS: The data suggest that B16F0 cells shifted the intracellular response to IL-12 from engaging immune surveillance to favoring cell survival. Identifying how signaling networks are rewired in model systems of spontaneous origin can inspire therapeutic strategies in humans. Interleukin-12 is a key cytokine that promotes anti-tumor immunity, as it is secreted by antigen presenting cells to activate Natural Killer cells and T cells present within the tumor microenvironment. Thinking of cancer as an evolutionary process implies that an immunosuppressive tumor microenvironment could arise during oncogenesis by interfering with endogenous anti-tumor immune signals, like IL-12. Previously, we found that B16F0 cells, a cell line derived from a spontaneous melanoma, interrupts this secreted heterocellular signal by sequestering IL-12, which provides an indirect fitness advantage. Normally, IL-12 signals via a receptor comprised of two components, IL12RB1 and IL12RB2, that are expressed in a 1:1 ratio and activates STAT4 as a downstream effector. Here, we report that B16F0 cells gain an intrinsic advantage by rewiring the canonical response to IL-12 to instead initiate PI3K-AKT signaling, which promotes cell survival. The data suggest a model where overexpressing one component of the IL-12 receptor, IL12RB2, enables melanoma cells to shift the functional response via both IL-12-mediated and molecular crowding-based IL12RB2 homodimerization. To explore the generalizability of these results, we also found that the expression of IL12RB2:IL12RB1 is similarly skewed in human melanoma based on transcriptional profiles of melanoma cells and tumor-infiltrating lymphocytes. Additional file 6: Video abstract. (MP4 600 kb).

Carbon nanotube filler enhances incinerated thermoplastics-induced cytotoxicity and metabolic disruption in vitro.

BACKGROUND: Engineered nanomaterials are increasingly being incorporated into synthetic materials as fillers and additives. The potential pathological effects of end-of-lifecycle recycling and disposal of virgin and nano-enabled composites have not been adequately addressed, particularly following incineration. The current investigation aims to characterize the cytotoxicity of incinerated virgin thermoplastics vs. incinerated nano-enabled thermoplastic composites on two in vitro pulmonary models. Ultrafine particles released from thermally decomposed virgin polycarbonate or polyurethane, and their carbon nanotube (CNT)-enabled composites were collected and used for acute in vitro exposure to primary human small airway epithelial cell (pSAEC) and human bronchial epithelial cell (Beas-2B) models. Post-exposure, both cell lines were assessed for cytotoxicity, proliferative capacity, intracellular ROS generation, genotoxicity, and mitochondrial membrane potential. RESULTS: The treated Beas-2B cells demonstrated significant dose-dependent cellular responses, as well as parent matrix-dependent and CNT-dependent sensitivity. Cytotoxicity, enhancement in reactive oxygen species, and dissipation of ΔΨm caused by incinerated polycarbonate were significantly more potent than polyurethane analogues, and CNT filler enhanced the cellular responses compared to the incinerated parent particles. Such effects observed in Beas-2B were generally higher in magnitude compared to pSAEC at treatments examined, which was likely attributable to differences in respective lung cell types. CONCLUSIONS: Whilst the effect of the treatments on the distal respiratory airway epithelia remains limited in interpretation, the current in vitro respiratory bronchial epithelia model demonstrated profound sensitivity to the test particles at depositional doses relevant for occupational cohorts.

Substrate Stiffness-Dependent Carbon Nanotube-Induced Lung Fibrogenesis.

Most living tissues exhibit the specific stiffness, which has been known to have profound influence on cell behaviors, yet how the stiffness affects cellular responses to engineered nanomaterials has not been elucidated. Particularly, discrepancies exist between in vitro and in vivo nanotoxicological studies. Here, we investigated the effects of substrate stiffness on the fibrogenic responses of normal human lung fibroblasts (NHLFs) to multiwalled carbon nanotubes (MWCNTs). NHLFs were grown on polyacrylamide (PAAm) hydrogels with the stiffness comparable to that of human normal and fibrotic lung tissues, and treated with MWCNTs for various time. The fibrogenic responses, including cell proliferation, reactive oxygen species production, and collagen I expression, of NHLFs to MWCNTs were observed to be regulated by substrate stiffness in a time-dependent manner. NHLFs generally were rounded on soft hydrogels and required a long treatment time to exhibit fibrogenic responses, while on stiff hydrogels the cells were well-spread with defined stress fibers and short-time MWCNTs treatment sufficiently induced the fibrogenic responses. Mechanistic studies showed that MWCNTs induced fibrogenesis of NHLFs through promoting expression and phosphorylation of focal adhesion kinase (FAK), while attenuating intracellular tension in the cells on stiff gels could increase MWCNTs uptake and thus elevate the induced fibrogenic responses. Moreover, we proposed a time-stiffness superposition principle to describe the equivalent effects of treatment time and substrate stiffness on nanomaterials-induced fibrogenesis, which suggested that increasing substrate stiffness expedited fibrogenesis and shed light on the rational design of in vitro models for nanotoxicological study.

Standardized Saponin Extract from Baiye No.1 Tea (Camellia sinensis) Flowers Induced S Phase Cell Cycle Arrest and Apoptosis via AKT-MDM2-p53 Signaling Pathway in Ovarian Cancer Cells.

Ovarian cancer is considered to be one of the most serious malignant tumors in women. Natural compounds have been considered as important sources in the search for new anti-cancer agents. Saponins are characteristic components of tea (Camellia sinensis) flower and have various biological activities, including anti-tumor effects. In this study, a high purity standardized saponin extract, namely Baiye No.1 tea flower saponin (BTFS), which contained Floratheasaponin A and Floratheasaponin D, were isolated from tea (Camellia sinensis cv. Baiye 1) flowers by macroporous resin and preparative liquid chromatography. Then, the component and purity were detected by UPLC-Q-TOF/MS/MS. This high purity BTFS inhibited the proliferation of A2780/CP70 cancer cells dose-dependently, which is evidenced by the inhibition of cell viability, reduction of colony formation ability, and suppression of PCNA protein expression. Further research found BTFS induced S phase cell cycle arrest by up-regulating p21 proteins expression and down-regulating Cyclin A2, CDK2, and Cdc25A protein expression. Furthermore, BTFS caused DNA damage and activated the ATM-Chk2 signaling pathway to block cell cycle progression. Moreover, BTFS trigged both extrinsic and intrinsic apoptosis-BTFS up-regulated the expression of death receptor pathway-related proteins DR5, Fas, and FADD and increased the ratio of pro-apoptotic/anti-apoptotic proteins of the Bcl-2 family. BTFS-induced apoptosis seems to be related to the AKT-MDM2-p53 signaling pathway. In summary, our results demonstrate that BTFS has the potential to be used as a nutraceutical for the prevention and treatment of ovarian cancer.

Nitric oxide promotes cancer cell dedifferentiation by disrupting an Oct4:caveolin-1 complex: A new regulatory mechanism for cancer stem cell formation.

Cancer stem cells (CSCs) are unique populations of cells that can self-renew and generate different cancer cell lineages. Although CSCs are believed to be a promising target for novel therapies, the specific mechanisms by which these putative therapeutics could intervene are less clear. Nitric oxide (NO) is a biological mediator frequently up-regulated in tumors and has been linked to cancer aggressiveness. Here, we search for targets of NO that could explain its activity. We find that it directly affects the stability and function of octamer-binding transcription factor 4 (Oct4), known to drive the stemness of lung cancer cells. We demonstrated that NO promotes the CSC-regulatory activity of Oct4 through a mechanism that involves complex formation between Oct4 and the scaffolding protein caveolin-1 (Cav-1). In the absence of NO, Oct4 forms a molecular complex with Cav-1, which promotes the ubiquitin-mediated proteasomal degradation of Oct4. NO promotes Akt-dependent phosphorylation of Cav-1 at tyrosine 14, disrupting the Cav-1:Oct4 complex. Site-directed mutagenesis and computational modeling studies revealed that the hydroxyl moiety at tyrosine 14 of Cav-1 is crucial for its interaction with Oct4. Both removal of the hydroxyl via mutation to phenylalanine and phosphorylation lead to an increase in binding free energy (ΔGbind) between Oct4 and Cav-1, destabilizing the complex. Together, these results unveiled a novel mechanism of CSC regulation through NO-mediated stabilization of Oct4, a key stem cell transcription factor, and point to new opportunities to design CSC-related therapeutics.

Impacts of Organomodified Nanoclays and Their Incinerated Byproducts on Bronchial Cell Monolayer Integrity.

Incorporation of engineered nanomaterials (ENMs) into nanocomposites using advanced manufacturing strategies is set to revolutionize diverse technologies. Of these, organomodified nanoclays (ONCs; i.e., smectite clays with different organic coatings) act as nanofillers in applications ranging from automotive to aerospace and biomedical systems. Recent toxicological evaluations increased awareness that exposure to ONC can occur along their entire life cycle, namely, during synthesis, handling, use, manipulation, and disposal. Compared to other ENMs, however, little information exists describing which physicochemical properties contribute to induced health risk. This study conducted high content screening on bronchial epithelial cell monolayers for coupled high-throughput in vitro assessment strategies aimed to evaluate acute toxicity of a library of ONCs (all of prevalent use) prior to and after simulated disposal by incineration. Coating-, incineration status-, and time-dependent effects were considered to determine changes in the pulmonary monolayer integrity, cell transepithelial resistance, apoptosis, and cell metabolism. Results showed that after exposure to each ONC at its half-maximal inhibitory concentration (IC50) there is a material-induced toxicity effect with pristine nanoclay, for instance, displaying acute loss of monolayer coverage, resistance, and metabolism, coupled with increased number of apoptotic cells. Conversely, the other three ONCs tested displayed little loss of monolayer integrity; however, they exhibited differential coating-dependent increased apoptosis and up to 40-45% initial reduction in cell metabolism. Moreover, incinerated byproducts of ONCs exhibited significant loss of monolayer coverage and integrity, increased necrosis, with little evidence of monolayer re-establishment. These findings indicate that characteristics of organic coating type largely determine the mechanism of cytotoxicity and the ability of the monolayer to recover. Use of high content screening coupled with traditional in vitro assays proves to serve as a rapid pulmonary toxicity assessment tool to help define prevention by targeted physicochemical material properties design strategies.

Iron Oxide Nanoparticle-Induced Neoplastic-Like Cell Transformation in Vitro Is Reduced with a Protective Amorphous Silica Coating.

Iron oxide nanoparticles (IONP) have recently surged in production and use in a wide variety of biomedical and environmental applications. However, their potential long-term health effects, including carcinogenesis, are unknown. Limited research suggests IONP can induce genotoxicity and neoplastic transformation associated with particle dissolution and release of free iron ions. "Safe by design" strategies involve the modification of particle physicochemical properties to affect subsequent adverse outcomes, such as an amorphous silica coating to reduce IONP dissolution and direct interaction with cells. We hypothesized that long-term exposure to a specific IONP (nFe2O3) would induce neoplastic-like cell transformation, which could be prevented with an amorphous silica coating (SiO2-nFe2O3). To test this hypothesis, human bronchial epithelial cells (Beas-2B) were continuously exposed to a 0.6 µg/cm2 administered a dose of nFe2O3 (∼0.58 µg/cm2 delivered dose), SiO2-nFe2O3 (∼0.55 µg/cm2 delivered dose), or gas metal arc mild steel welding fumes (GMA-MS, ∼0.58 µg/cm2 delivered dose) for 6.5 months. GMA-MS are composed of roughly 80% iron/iron oxide and were recently classified as a total human carcinogen. Our results showed that low-dose/long-term in vitro exposure to nFe2O3 induced a time-dependent neoplastic-like cell transformation, as indicated by increased cell proliferation and attachment-independent colony formation, which closely matched that induced by GMA-MS. This transformation was associated with decreases in intracellular iron, minimal changes in reactive oxygen species (ROS) production, and the induction of double-stranded DNA damage. An amorphous silica-coated but otherwise identical particle (SiO2-nFe2O3) did not induce this neoplastic-like phenotype or changes in the parameters mentioned above. Overall, the presented data suggest the carcinogenic potential of long-term nFe2O3 exposure and the utility of an amorphous silica coating in a "safe by design" hazard reduction strategy, within the context of a physiologically relevant exposure scenario (low-dose/long-term), with model validation using GMA-MS.

Predicting Nanotube Fibrogenicity through Stem Cell-Mediated Fibroblast Focus and Spheroid Formation.

Fibroblast stem cells or stemlike cells (FSCs) are proposed to play a pivotal role in extracellular matrix (ECM) regeneration by serving as a key source of ECM-producing fibroblasts. We developed a mechanism-based in vitro model for fibrogenicity testing of nanomaterials based on their ability to induce FSCs. Using a FSC-enriched fibroblast focus model to mimic in vivo fibrogenic response, we demonstrated a dose-dependent increase in fibroblast focus formation and collagen production by primary lung fibroblasts treated with multiwalled carbon nanotubes (MWCNTs). The focus-forming cells exhibited stem properties as indicated by stem cell markers expression, sphere formation, and ALDH activity assays. Inhibition of ALDH activity diminished the focus and sphere formation as well as collagen production. In vivo animal studies supported the in vitro findings and indicated the potential utility of FSC-based assays as a rapid screening tool for fibrogenicity testing of nanomaterials. This study also unveils a novel mechanism of nanotube-induced fibrogenesis through ALDH-dependent FSC activation.

Theaflavin-3,3'-Digallate Enhances the Inhibitory Effect of Cisplatin by Regulating the Copper Transporter 1 and Glutathione in Human Ovarian Cancer Cells.

Ovarian cancer has the highest fatality rate among the gynecologic cancers. The side effects, high relapse rate, and drug resistance lead to low long-term survival rate (less than 40%) of patients with advanced ovarian cancer. Theaflavin-3,3'-digallate (TF3), a black tea polyphenol, showed less cytotoxicity to normal ovarian cells than ovarian cancer cells. We aimed to investigate whether TF3 could potentiate the inhibitory effect of cisplatin against human ovarian cancer cell lines. In the present study, combined treatment with TF3 and cisplatin showed a synergistic cytotoxicity against A2780/CP70 and OVCAR3 cells. Treatment with TF3 could increase the intracellular accumulation of platinum (Pt) and DNA-Pt adducts and enhanced DNA damage induced by cisplatin in both cells. Treatment with TF3 decreased the glutathione (GSH) levels and upregulated the protein levels of the copper transporter 1 (CTR1) in both cells, which led to the enhanced sensitivity of both ovarian cancer cells to cisplatin. The results imply that TF3 might be used as an adjuvant to potentiate the inhibitory effect of cisplatin against advanced ovarian cancer.

Detachment-induced E-cadherin expression promotes 3D tumor spheroid formation but inhibits tumor formation and metastasis of lung cancer cells.

The epithelial-to-mesenchymal transition is proposed to be a key mechanism responsible for metastasis-related deaths. Similarly, cancer stem cells (CSCs) have been proposed to be a key driver of tumor metastasis. However, the link between the two events and their control mechanisms is unclear. We used a three-dimensional (3D) tumor spheroid assay and other CSC-indicating assays to investigate the role of E-cadherin in CSC regulation and its association to epithelial-to-mesenchymal transition in lung cancer cells. Ectopic overexpression and knockdown of E-cadherin were found to promote and retard, respectively, the formation of tumor spheroids in vitro but had opposite effects on tumor formation and metastasis in vivo in a xenograft mouse model. We explored the discrepancy between the in vitro and in vivo results and demonstrated, for the first time, that E-cadherin is required as a component of a major survival pathway under detachment conditions. Downregulation of E-cadherin increased the stemness of lung cancer cells but had an adverse effect on their survival, particularly on non-CSCs. Such downregulation also promoted anoikis resistance and invasiveness of lung cancer cells. These results suggest that anoikis assay could be used as an alternative method for in vitro assessment of CSCs that involves dysregulated adhesion proteins. Our data also suggest that agents that restore E-cadherin expression may be used as therapeutic agents for metastatic cancers.

Anti-Tumor Effects of Cardiac Glycosides on Human Lung Cancer Cells and Lung Tumorspheres.

Lung cancer is a leading cause of cancer-related death in the United States. Although several drugs have been developed that target individual biomarkers, their success has been limited due to intrinsic or acquired resistance for the specific targets of such drugs. A more effective approach is to target multiple pathways that dictate cancer progression. Cardiac glycosides demonstrate such multimodal effects on cancer cell survival, and our aim was to evaluate the effect of two naturally occurring monosaccaridic cardiac glycosides-Convallatoxin and Peruvoside on lung cancer cells. Although both drugs had significant anti-proliferative effects on H460 and Calu-3 lung cancer cells, Convallatoxin demonstrated twofold higher activity as compared to Peruvoside using both viability and colony forming assays, suggesting a role for the aglycone region in dictating drug potency. The tumor suppressor p53 was found to be important for action of both drugs-p53-underexpressing cells were less sensitive as compared to p53-positive H460 cells. Further, assessment of p53-underexpressing H460 cells showed that drugs were able to arrest cells in the G0/G1 phase of the cell cycle in a dose-dependent manner. Both drugs significantly inhibited migration and invasion of cancer cells and decreased the viability of floating tumorspheres. An assessment of intracellular pathways indicated that both drugs were able to modulate proteins that are involved in apoptosis, autophagy, cell cycle, proliferation, and EMT. Our data suggest, a promising role for cardiac glycosides in lung cancer treatment, and provides impetus for further investigation of the anti-cancer potential of this class of drugs. J. Cell. Physiol. 232: 2497-2507, 2017. © 2016 Wiley Periodicals, Inc.

Mesothelin promotes epithelial-to-mesenchymal transition and tumorigenicity of human lung cancer and mesothelioma cells.

BACKGROUND: Lung cancer and pleural mesothelioma are two of the most deadly forms of cancer. The prognosis of lung cancer and mesothelioma is extremely poor due to limited treatment modalities and lack of understanding of the disease mechanisms. We have identified mesothelin as a potentially unique therapeutic target that as a specific advantage appears nonessential in most cell types. Mesothelin (MSLN), a plasma membrane differentiation antigen, is expressed at a high level in many human solid tumors, including 70% of lung cancer and nearly all mesotheliomas. However, the role of MSLN in the disease process and underlying mechanisms is largely unknown. METHODS: ShRNA knockdown and overexpression of MSLN were performed in human cancer cell lines and corresponding normal cells, respectively. Tumorigenic and metastatic effects of MSLN were examined by tumor sphere formation, migration, and invasion assays in vitro, as well as xenograft tumor assay in vivo. EMT and CSCs were detected by qPCR array, immunoblotting and flow cytometry. RESULTS: MSLN plays a key role in controlling epithelial-to-mesenchymal transition (EMT) and stem properties of human lung cancer and mesothelioma cells that control their tumorigenicity and metastatic potential. Firstly, MSLN was found to be highly upregulated in non-small cell lung cancer (NSCLC) patient tissues and in lung carcinoma and mesothelioma cell lines. Secondly, genetic knockdown of MSLN significantly reduced anchorage-independent cell growth, tumor sphere formation, cell adhesion, migration and invasion in vitro, as well as tumor formation and metastasis in vivo. Thirdly, ectopic overexpression of MSLN induced the malignant phenotype of non-cancerous cells, supporting its role as an oncogene. Finally, mechanistic studies revealed that knockdown of MSLN reversed EMT and attenuated stem cell properties, in addition to inhibiting tumor growth and metastasis. CONCLUSIONS: These results indicate an essential role of MSLN in controlling EMT and stem cell properties of human lung cancer and mesothelioma cells. Since EMT is an important process in tumor progression and metastasis, and MSLN is nonessential in most normal tissue, our findings on MSLN may provide new insights into the disease mechanisms and may aid in the development of novel targeted therapy for lung cancer and mesothelioma.

Induction of Slug by Chronic Exposure to Single-Walled Carbon Nanotubes Promotes Tumor Formation and Metastasis.

Carbon nanotubes (CNTs) represent a major class of engineered nanomaterials that are being used in diverse fields. However, their use has increasingly become a concern because of their carcinogenic potential. Accumulating evidence has demonstrated that certain types of CNTs are carcinogenic or tumor-promoting in animal models. However, the underlying molecular and cellular mechanisms are unclear. Here, we report that chronic exposure to single-walled (SW) CNTs results in the induction of Slug, a key transcription factor that induces an epithelial-mesenchymal transition (EMT), in human lung epithelial cells. We show that SWCNT-induced Slug upregulation plays a critical role in the aggressive phenotype of SWCNT-exposed cells, which includes increased cell migration, invasion, and anchorage-independent cell growth. Our in vivo studies also show that SWCNT-induced Slug upregulation and EMT activation play a pivotal role in tumor formation and metastasis. Our findings illustrate a direct link between CNT-induced Slug upregulation, EMT activation, and tumor formation and metastasis, and they highlight the potential of CNT-induced Slug upregulation as a target for future risk assessment and prevention of CNT-associated diseases.

Toxicity evaluations of nanoclays and thermally degraded byproducts through spectroscopical and microscopical approaches.

BACKGROUND: Montmorillonite is a type of nanoclay that originates from the clay fraction of the soil and is incorporated into polymers to form nanocomposites with enhanced mechanical strength, barrier, and flammability properties used for food packaging, automotive, and medical devices. However, with implementation in such consumer applications, the interaction of montmorillonite-based composites or derived byproducts with biological systems needs to be investigated. METHODS: Herein we examined the potential of Cloisite Na+ (pristine) and Cloisite 30B (organically modified montmorillonite nanoclay) and their thermally degraded byproducts' to induce toxicity in model human lung epithelial cells. The experimental set-up mimicked biological exposure in manufacturing and disposal areas and employed cellular treatments with occupationally relevant doses of nanoclays previously characterized using spectroscopical and microscopical approaches. For nanoclay-cellular interactions and for cellular analyses respectively, biosensorial-based analytical platforms were used, with induced cellular changes being confirmed via live cell counts, viability assays, and cell imaging. RESULTS: Our analysis of byproducts' chemical and physical properties revealed both structural and functional changes. Real-time high throughput analyses of exposed cellular systems confirmed that nanoclay induced significant toxic effects, with Cloisite 30B showing time-dependent decreases in live cell count and cellular viability relative to control and pristine nanoclay, respectively. Byproducts produced less toxic effects; all treatments caused alterations in the cell morphology upon exposure. CONCLUSIONS: Our morphological, behavioral, and viability cellular changes show that nanoclays have the potential to produce toxic effects when used both in manufacturing or disposal environments. GENERAL SIGNIFICANCE: The reported toxicological mechanisms prove the extensibility of a biosensorial-based platform for cellular behavior analysis upon treatment with a variety of nanomaterials.

Effects of titanium dioxide nanoparticles on human keratinocytes.

Titanium dioxide (TiO2) is a ubiquitous whitening compound widely used in topical products such as sunscreens, lotions and facial creams. The damaging health effects of TiO2 inhalation has been widely studied in rats, mice and humans showing oxidative stress increase, DNA damage, cell death and inflammatory gene upregulation in lung and throat cells; however, the effects on skin cells from long-term topical use of various products remain largely unknown. In this study, we assessed the effect of specific TiO2 nanoparticles (H2TiO7) on a human keratinocyte cell line (HaCaT). We performed a comparative analysis using three TiO2 particles varying in size (Fine, Ultrafine and H2TiO7) and analyzed their effects on HaCaTs. There is a clear dose-dependent increase in superoxide production, caspase 8 and 9 activity, and apoptosis in HaCaTs after treatment with all three forms of TiO2; however, there is no consistent effect on cell viability and proliferation with either of these TiO2 particles. While there is data suggesting UV exposure can enhance the carcinogenic effects of TiO2, we did not observe any significant effect of UV-C exposure combined with TiO2 treatment on HaCaTs. Furthermore, TiO2-treated cells showed minimal effects on VEGF upregulation and Wnt signaling pathway thereby showing no potential effect on angiogenesis and malignant transformation. Overall, we report here an increase in apoptosis, which may be caspase 8/Fas-dependent, and that the H2TiO7 nanoparticles, despite their smaller particle size, had no significant enhanced effect on HaCaT cells as compared to Fine and Ultrafine forms of TiO2.

Autophagy-Induced Apoptosis in Lung Cancer Cells by a Novel Digitoxin Analog.

We have synthesized a novel derivative of Digitoxin, termed "MonoD", which demonstrates cytotoxic effects in lung cancer cells with much higher potency as compared to Digitoxin. Our data show that within 1 h of MonoD treatment, H460 cells showed increased oxidative stress, increased formation of autophagic vacuoles, and increased expression of pro-autophagic markers Beclin-1 and LC3-II. Cells pretreated with MnTBAP, a superoxide scavenger not only lowered superoxide production, but also had lower levels of LC3-II and Beclin-1. Prolonged treatment with MonoD-induced apoptosis in lung cancer cells. We investigated MonoD-dependent regulation of Akt and Bcl2, proteins that are known regulators of both autophagy and apoptosis. Molecular and pharmacologic inhibitors of Bcl2 and Akt, when combined with MonoD, led to higher expression of LC3-II and Beclin-1 as compared to MonoD alone, suggesting a repressive effect for these proteins in MonoD-dependent autophagy. Pretreatment of cells with an autophagy inhibitor repressed the apoptotic potential of MonoD, confirming that early autophagic flux is important to drive apoptosis. Therapeutic entities such as MonoD that target multiple pathways such as autophagy and apoptosis may prove advantageous over current therapies that have unimodal basis for action and may drive sustained tumor regression, which is highly desirable. J. Cell. Physiol. 231: 817-828, 2016. © 2015 Wiley Periodicals, Inc.