[Show abstract][Hide abstract] ABSTRACT: A rapid increase in utility of engineered nanomaterials, including carbon nanotubes (CNTs), has raised a concern over their safety. Based on recent evidence from animal studies, pulmonary exposure of CNTs may lead to nanoparticle accumulation in the deep lung without effective clearance which could interact with local lung cells for a long period of time. Physicochemical similarities of CNTs to asbestos fibers may contribute to their asbestos-like carcinogenic potential after long-term exposure, which has not been well addressed. More studies are needed to identify and predict the carcinogenic potential and mechanisms for promoting their safe use. Our previous study reported a long-term in vitro exposure model for CNT carcinogenicity and showed that 6-month sub-chronic exposure of single-walled carbon nanotubes (SWCNT) causes malignant transformation of human lung epithelial cells. In addition, the transformed cells induced tumor formation in mice and exhibited an apoptosis resistant phenotype, a key characteristic of cancer cells. Although the potential role of p53 in the transformation process was identified, the underlying mechanisms of oncogenesis remain largely undefined. Here, we further examined the gene expression profile by using genome microarrays to profile molecular mechanisms of SWCNT oncogenesis. Based on differentially expressed genes, possible mechanisms of SWCNT-associated apoptosis resistance and oncogenesis were identified, which included activation of pAkt/p53/Bcl-2 signaling axis, increased gene expression of Ras family for cell cycle control, Dsh-mediated Notch 1, and downregulation of apoptotic genes BAX and Noxa. Activated immune responses were among the major changes of biological function. Our findings shed light on potential molecular mechanisms and signaling pathways involved in SWCNT oncogenic potential.
Nanoscale Research Letters 12/2015; 10(1):12. DOI:10.1186/s11671-014-0707-0 · 2.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cancer stem cells (CSCs) are a promising target for cancer therapy, particularly for metastatic lung cancers, but how CSCs are regulated is largely unknown. We identify two proteins, SLUG (encoded by SNAI2 gene) and SOX9, which are associated with advanced stage lung cancers and are implicated in the regulation of CSCs. Inhibition of either SLUG or SOX9 sufficiently inhibits CSCs in human lung cancer cells and attenuates experimental lung metastasis in a xenograft mouse model. Correlation between SLUG and SOX9 levels was observed remarkably, we therefore sought to explore their mechanistic relationship and regulation. SLUG, beyond its known function as an epithelial-mesenchymal transition transcription factor, was found to regulate SOX9 by controlling its stability via a post-translational modification process. SLUG interacts directly with SOX9 and prevents it from ubiquitin-mediated proteasomal degradation. SLUG expression and binding are necessary for SOX9 promotion of lung CSCs and metastasis in a mouse model. Together, our findings provide a novel mechanistic insight into the regulation of CSCs via SLUG-SOX9 regulatory axis, which represents a potential novel target for CSC therapy that may overcome cancer chemoresistance and relapse.Oncogene advance online publication, 21 September 2015; doi:10.1038/onc.2015.351.
[Show abstract][Hide abstract] ABSTRACT: Cisplatin is a commonly used drug for cancer treatment by crosslinking DNA, leading to apoptosis of cancer cells, resistance to cisplatin treatment often occurs, leading to relapse. Therefore, there is a need for the development of more effective treatment strategies that can overcome chemoresistance. Myricetin is a flavonoid from fruits and vegetables, showing anticancer activity in various cancer cells. In this study, we found myricetin exhibited greater cytotoxicity than cisplatin in two cisplatin-resistant ovarian cancer cell lines, OVCAR-3 and A2780/CP70, and it was less cytotoxic to the normal ovarian cell line IOSE-364. Myricetin selectively induced apoptosis in both cisplatin-resistant cancer cell lines, but did not induce apoptosis in the normal ovarian cell line. It induced both Bcl-2 family-dependent intrinsic and DR5 dependent extrinsic apoptosis in OVCAR-3 cells. P53, a multifunctional tumor suppressor, regulated apoptosis in OVCAR-3 cells through a Bcl-2 family protein-dependent pathway. Myricetin did not induce cell cycle arrest in either ovarian cancer cell line. Because of its potency and selectivity against cisplatin-resistant cancer cells, myricetin could potentially be used to overcome cancer chemoresistance against platinum-based therapy.
International Journal of Oncology 08/2015; 47:1494-1502. DOI:10.3892/ijo.2015.3133 · 3.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 one hour 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. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Journal of Cellular Physiology 08/2015; DOI:10.1002/jcp.25129 · 3.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cell migration is one of the crucial steps in many physiological and pathological processes, including cancer development. Our recent studies have shown that carbon nanotubes (CNTs), similarly to asbestos, can induce accelerated cell growth and invasiveness that contribute to their mesothelioma pathogenicity. Malignant mesothelioma is a very aggressive tumor that develops from cells of the mesothelium, and is most commonly caused by exposure to asbestos. CNTs have a similar structure and mode of exposure to asbestos. This has raised a concern regarding the potential carcinogenicity of CNTs, especially in the pleural area which is a key target for asbestos-related diseases. In this paper, a static microfluidic gradient device was applied to study the migration of human pleural mesothelial cells which had been through a long-term exposure (4 months) to subcytotoxic concentration (0.02 µg cm−2) of single-walled CNTs (SWCNTs). Multiple migration signatures of these cells were investigated using the microfluidic gradient device for the first time. During the migration study, we observed that cell morphologies changed from flattened shapes to spindle shapes prior to their migration after their sensing of the chemical gradient. The migration of chronically SWCNT-exposed mesothelial cells was evaluated under different fetal bovine serum (FBS) concentration gradients, and the migration speeds and number of migrating cells were extracted and compared. The results showed that chronically SWCNT-exposed mesothelial cells are more sensitive to the gradient compared to non-SWCNT-exposed cells. The method described here allows simultaneous detection of cell morphology and migration under chemical gradient conditions, and also allows for real-time monitoring of cell motility that resembles in vivo cell migration. This platform would be much needed for supporting the development of more physiologically relevant cell models for better assessment and characterization of the mesothelioma hazard posed by nanomaterials.
Journal of Micromechanics and Microengineering 07/2015; 25(7). DOI:10.1088/0960-1317/25/7/075010 · 1.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: AF1q is an MLL fusion partner that was identified from acute myeloid leukemia (AML) patients with t (1; 11) (q21; q23) chromosomal abnormality. The function of AF1q is not yet fully known, however, elevated AF1q expression is associated with poor clinical outcomes in various malignancies. Here, we show that AF1q specifically binds to T-cell-factor-7 (TCF7) in the Wnt signaling pathway and results in transcriptional activation of CD44 as well as multiple downstream targets of the TCF7/LEF1. In addition, enhanced AF1q expression promotes breast cancer cell proliferation, migration, mammosphere formation, and chemo-resistance. In xenograft models, enforced AF1q expression in breast cancer cells also promotes liver metastasis and lung colonization. In a cohort of 63 breast cancer patients, higher percentages of AF1q-positive cancer cells in primary sites were associated with significantly poorer overall survival (OS), disease-free survival (DFS), and brain metastasis-free survival (b-MFS). Using paired primary/metastatic samples from the same patients, we demonstrate that AF1q-positive breast cancer cells become dynamically dominant in the metastatic sites compared to the primary sites. Our findings indicate that breast cancer cells with a hyperactive AF1q/TCF7/CD44 regulatory axis in the primary sites may represent "metastatic founder cells" which have invasive properties.
[Show abstract][Hide abstract] ABSTRACT: Galangin and myricetin are flavonoids isolated from vegetables and fruits which exhibit anti-proliferative activity in human cancer cells. In this study, their anti-angiogenic effects were investigated with in vitro (HUVEC) and in vivo (CAM) models, which showed that galangin and myricetin inhibited angiogenesis induced by OVCAR-3 cells. The molecular mechanisms through which galangin and myricetin suppress angiogenesis were also studied. It was observed that galangin and myricetin inhibited secretion of the key angiogenesis mediator vascular endothelial growth factor (VEGF) and decreased levels of p-Akt, p-p70S6K and hypoxia-inducible factor-1α (HIF-1α) proteins in A2780/CP70 and OVCAR-3 cells. Transient transfection experiments showed that galangin and myricetin inhibited secretion of VEGF by the Akt/p70S6K/HIF-1α pathway. Moreover, a novel pathway, p21/HIF-1α/VEGF, was found to be involved in the inhibitory effect of myricetin on angiogenesis in OVCAR-3 cells. These data suggest that galangin and myricetin might serve as potential anti-angiogenic agents in the prevention of ovarian cancers dependent on new blood vessel networks.
[Show abstract][Hide abstract] ABSTRACT: Pulmonary fibrosis is a progressive lung disease hallmarked by increased fibroblast proliferation, amplified levels of extracellular matrix deposition and increased angiogenesis. Although dysregulation of angiogenic mediators has been implicated in pulmonary fibrosis, the specific rate-limiting angiogenic markers involved and their role in the progression of pulmonary fibrosis remains unclear. We demonstrate that bleomycin treatment induces angiogenesis, and inhibition of the central angiogenic mediator VEGF using anti-VEGF antibody CBO-P11 significantly attenuates bleomycin-induced pulmonary fibrosis in vivo. Bleomycin-induced nitric oxide (NO) was observed to be the key upstream regulator of VEGF via the PI3k/Akt pathway. VEGF regulated other important angiogenic proteins including PAI-1 and IL-8 in response to bleomycin exposure. Inhibition of NO and VEGF activity significantly mitigated bleomycin-induced angiogenic and fibrogenic responses. NO and VEGF are key mediators of bleomycin-induced pulmonary fibrosis, and could serve as important targets against this debilitating disease. Overall, our data suggests an important role for angiogenic mediators in the pathogenesis of bleomycin-induced pulmonary fibrosis. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: The toxicity of engineered nanomaterials in biological systems depends on both the nanomaterial properties and the exposure duration. Herein we used a multi-tier strategy to investigate the relationship between user-characterized multi-walled carbon nanotubes (MWCNTs) exposure duration and their induced biochemical and biomechanical effects on model human lung epithelial cells (BEAS-2B). Our results showed that exposure to MWCNTs leads to time-dependent intracellular uptake and generation of reactive oxygen species (ROS), along with time-dependent gradual changes in cellular biomechanical properties. In particular, the amount of internalized MWCNTs followed a sigmoidal curve with the majority of the MWCNTs being internalized within 6 h of exposure; further, the sigmoidal uptake correlated with the changes in the oxidative levels and cellular biomechanical properties respectively. Our study provides new insights into the time-dependent induced toxicity caused by exposure to occupationally relevant doses of MWCNTs and could potentially help establish bases for early risk assessments of other nanomaterials toxicological profiles.
[Show abstract][Hide abstract] ABSTRACT: Despite its importance, the death rate of ovarian cancer has remained unchanged over the past five decades, demanding an improvement in prevention and treatment of this malignancy. With no known carcinogens, targeted prevention is currently unavailable, and efforts in early detection of this malignancy by screening biomarkers have failed. The inhibition of angiogenesis, also known as angioprevention, is a promising strategy to limit the growth of solid tumors, including ovarian cancers. Nobiletin, a polymethoxy flavonoid compound isolated from the tiansheng plant, has been shown to inhibit the growth of multiple types of human cancers. However, there are no reports involving the effect on nobiletin on human ovarian cancer. The present report shows that nobiletin potently decreases the viability of ovarian cancer cells in vitro. However, nobiletin does not affect the viability of normal ovarian epithelial cells at <40 µM. The antitumor activity of nobiletin was also observed in athymic mouse models and in chicken chorioallantoic membrane (CAM) models. The anti-neoplastic activity of nobiletin was due to its ability to inhibit angiogenesis. We also studied the molecular mechanisms by which nobiletin suppresses angiogenesis. We observed that nobiletin inhibits secretion of the key angiogenesis mediators, Akt, HIF-1α, NF-κB and vascular epithelial growth factor (VEGF) by ovarian cancer cells. Transient transfection experiments showed that nobiletin inhibits production of HIF-1α by downregulation of Akt. Such decreased levels of HIF-1α were responsible for nobiletin-induced suppression of VEGF. Our data suggest that nobiletin may be a promising anti-angiogenic agent relevant for therapy of ovarian cancers.
International Journal of Oncology 04/2015; 46:2629-2638. DOI:10.3892/ijo.2015.2946 · 3.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Adverse side effects and acquired resistance to conventional platinum based chemotherapy have become major impediments in ovarian cancer treatment, and drive the development of more selective anticancer drugs. Chaetoglobosin K (ChK) was shown to have a more potent growth inhibitory effect than cisplatin on two cisplatin-resistant ovarian cancer cell lines, OVCAR-3 and A2780/CP70, and was less cytotoxic to a normal ovarian cell line, IOSE-364, than to the cancer cell lines. Hoechst 33342 staining and Flow cytometry analysis indicated that ChK induced preferential apoptosis and G2 cell cycle arrest in both ovarian cancer cells respect to the normal ovarian cells. ChK induced apoptosis through a p53-dependent caspase-8 activation extrinsic pathway, and caused G2 cell cycle arrest via cyclin B1 by increasing p53 expression and p38 phosphorylation in OVCAR-3 and A2780/CP70 cells. DR5 and p21 might play an important role in determining the sensitivity of normal and malignant ovarian cells to ChK. Based on these results, ChK would be a potential compound for treating platinum- resistant ovarian cancer.
Cancer letters 01/2015; 356(2):418-33. DOI:10.1016/j.canlet.2014.09.023 · 5.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ovarian cancer is a disease that continues to cause mortality in female individuals worldwide. Ovarian cancer is challenging to treat due to emerging resistance to chemotherapy, therefore, the identification of effective novel chemotherapeutic agents is important. Polyphenols have demonstrated potential in reducing the risk of developing numerous types of cancer, as well reducing the risk of cancer progression, due to their ability to reduce cell viability and vascular endothelial growth factor (VEGF) expression. In the present study, eight phenolic compounds were screened in two human ovarian cancer cell lines (OVCAR‑3 and A2780/CP70) to determine their effect on proliferation suppression and VEGF protein secretion inhibition, in comparison to cisplatin, a conventional chemotherapeutic agent. The current study identified that 40 µM gallic acid (GA) exhibited the greatest inhibitory effect on OVCAR‑3 cell viability, compared with all of the phenolic compounds investigated. Similarly to cisplatin, baicalein, GA, nobiletin, tangeretin and baicalin were all identified to exhibit significant VEGF inhibitory effects from ELISA results. Furthermore, western blot analysis indicated that GA effectively decreased the level of the VEGF‑binding protein hypoxia‑inducible factor‑1α in the ovarian cancer cell line. Considering the results of the present study, GA appears to inhibit cell proliferation and, thus, is a potential agent for the treatment of ovarian cancer.
[Show abstract][Hide abstract] ABSTRACT: Pulmonary barrier function plays a pivotal role in protection from inhaled particles. However, some nano-scaled particles, such as carbon nanotubes (CNT), have demonstrated the ability to penetrate this barrier in animal models, resulting in an unusual, rapid interstitial fibrosis. To delineate the underlying mechanism and specific bio-effect of inhaled nanoparticles in respiratory toxicity, models of lung epithelial barriers are required that allow accurate representation of in vivo systems; however, there is currently a lack of consistent methods to do so. Thus, this work demonstrates a well-characterized in vitro model of pulmonary barrier function using Calu-3 cells, and provides the experimental conditions required for achieving tight junction complexes in cell culture, with trans-epithelial electrical resistance measurement used as a biosensor for proper barrier formation and integrity. The effects of cell number and serum constituents have been examined and we found that changes in each of these parameters can greatly affect barrier formation. Our data demonstrate that use of 5.0x104 Calu-3 cells/well in the Transwell cell culture system, with 10% serum concentrations in culture media is optimal for assessing epithelial barrier function. In addition, we have utilized CNT exposure to analyze the dose-, time-, and nanoparticle property- dependent alterations of epithelial barrier permeability as a means to validate this model. Such high throughput in vitro cell models of the epithelium could be used to predict the interaction of other nanoparticles with lung epithelial barriers to mimic respiratory behavior in vivo, thus providing essential tools and bio-sensing techniques that can be uniformly employed.
Sensing and Bio-Sensing Research 12/2014; 3. DOI:10.1016/j.sbsr.2014.12.002
[Show abstract][Hide abstract] ABSTRACT: Despite the intriguing potential of carbon nanotubes (CNTs) for biomedical applications, their implementation is currently being hindered by many uncertainties regarding their toxicity and fate inside the biological systems. Several reports have shown that CNTs toxicity can be attributed to their length, surface chemistry, aggregation and metal impurities, just to name a few. However, due to the various types of CNTs being synthesized every day along with the different surface functionalization techniques, there is no fundamental understanding of the toxicological and pharmacological profiles of cellular systems exposed to CNTs with different physico-chemical properties. In this research, we provide a comprehensive analysis of the cellular behavior of human lung cells post exposure to CNTs using a combination of electronic and cell-based techniques. Our analyses rely on a non-invasive electronic cell impedance sensing (ECIS) platform to provide real-time measurements of cell adhesion, cell-cell interactions as well as changes in cellular morphology upon exposure to CNTs with user-defined physical and chemical properties. The approach is complemented by standard microscopy techniques as well as conventional in vitro cellular-based assays, such as cell cycle analysis, cell proliferation and viability, in order to derive structure-function relations associated with the cytotoxic and apoptotic events induced by exposure to CNTs. Our results correlated the different physico-chemical properties of CNTs with the various biological responses and provided mechanistic insights into their Cyto- and genotoxic effects. Out study help define a novel platform for nanomaterials toxicity analysis and thus could expand the biomedical applications of CNTs and other nanomaterials.
[Show abstract][Hide abstract] ABSTRACT: Chronic exposure to single-walled carbon nanotubes (SWCNT) has been reported to induce apoptosis resistance of human lung epithelial cells. Since resistance to apoptosis is a foundation of neoplastic transformation and cancer development, we evaluated the apoptosis resistance characteristic of the exposed lung cells to understand the pathogenesis mechanism. Passage control and SWCNT-transformed human lung epithelial cells were treated with known inducers of apoptosis via the intrinsic (antimycin A and CDDP) or extrinsic (FasL and TNF-α) pathway and analyzed for apoptosis by DNA fragmentation, annexin-V expression, and caspase activation assays. Whole genome microarray was performed to aid the analysis of apoptotic gene signaling network. The SWCNT-transformed cells exhibited defective death receptor pathway in association with c-FLIP overexpression. Knockdown or chemical inhibition of c-FLIP abrogated the apoptosis resistance of SWCNT-transformed cells. Whole genome expression signature analysis confirmed these findings. This study is the first to demonstrate carbon nanotube-induced defective death receptor pathway and the role of c-FLIP in the process.
Published by Oxford University Press on behalf of the Society of Toxicology 2014. This work is written by US Government employees and is in the public domain in the US.