Last data update: Apr 22, 2024. (Total: 46599 publications since 2009)
Records 1-12 (of 12 Records) |
Query Trace: Rojanasakul LW [original query] |
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In vitro inflammation and toxicity assessment of pre- and post-incinerated organomodified nanoclays to macrophages using high-throughput screening approaches
Stueckle TA , Jensen J , Coyle JP , Derk R , Wagner A , Dinu CZ , Kornberg TG , Friend SA , Dozier A , Agarwal S , Gupta RK , Rojanasakul LW . Part Fibre Toxicol 2024 21 (1) 16 BACKGROUND: Organomodified nanoclays (ONC), two-dimensional montmorillonite with organic coatings, are increasingly used to improve nanocomposite properties. However, little is known about pulmonary health risks along the nanoclay life cycle even with increased evidence of airborne particulate exposures in occupational environments. Recently, oropharyngeal aspiration exposure to pre- and post-incinerated ONC in mice caused low grade, persistent lung inflammation with a pro-fibrotic signaling response with unknown mode(s) of action. We hypothesized that the organic coating presence and incineration status of nanoclays determine the inflammatory cytokine secretary profile and cytotoxic response of macrophages. To test this hypothesis differentiated human macrophages (THP-1) were acutely exposed (0-20 µg/cm(2)) to pristine, uncoated nanoclay (CloisNa), an ONC (Clois30B), their incinerated byproducts (I-CloisNa and I-Clois30B), and crystalline silica (CS) followed by cytotoxicity and inflammatory endpoints. Macrophages were co-exposed to lipopolysaccharide (LPS) or LPS-free medium to assess the role of priming the NF-κB pathway in macrophage response to nanoclay treatment. Data were compared to inflammatory responses in male C57Bl/6J mice following 30 and 300 µg/mouse aspiration exposure to the same particles. RESULTS: In LPS-free media, CloisNa exposure caused mitochondrial depolarization while Clois30B exposure caused reduced macrophage viability, greater cytotoxicity, and significant damage-associated molecular patterns (IL-1α and ATP) release compared to CloisNa and unexposed controls. LPS priming with low CloisNa doses caused elevated cathepsin B/Caspage-1/IL-1β release while higher doses resulted in apoptosis. Clois30B exposure caused dose-dependent THP-1 cell pyroptosis evidenced by Cathepsin B and IL-1β release and Gasdermin D cleavage. Incineration ablated the cytotoxic and inflammatory effects of Clois30B while I-CloisNa still retained some mild inflammatory potential. Comparative analyses suggested that in vitro macrophage cell viability, inflammasome endpoints, and pro-inflammatory cytokine profiles significantly correlated to mouse bronchioalveolar lavage inflammation metrics including inflammatory cell recruitment. CONCLUSIONS: Presence of organic coating and incineration status influenced inflammatory and cytotoxic responses following exposure to human macrophages. Clois30B, with a quaternary ammonium tallow coating, induced a robust cell membrane damage and pyroptosis effect which was eliminated after incineration. Conversely, incinerated nanoclay exposure primarily caused elevated inflammatory cytokine release from THP-1 cells. Collectively, pre-incinerated nanoclay displayed interaction with macrophage membrane components (molecular initiating event), increased pro-inflammatory mediators, and increased inflammatory cell recruitment (two key events) in the lung fibrosis adverse outcome pathway. |
Chemotherapy-induced PDL-1 expression in cancer-associated fibroblasts promotes chemoresistance in NSCLC
Heenatigala Palliyage G , Samart P , Bobbala S , Rojanasakul LW , Coyle J , Martin K , Callery PS , Rojanasakul Y . Lung Cancer 2023 181 107258 OBJECTIVES: A cure for cancer is out of reach for most patients due to chemoresistance. Cancer-associated fibroblasts (CAFs) play a vital role in cancer chemoresistance, but detailed understanding of the process particularly in chemoresistant lung cancer is lacking. In this study, we investigated programmed death-ligand 1 (PDL-1) as a potential biomarker for CAF-induced chemoresistance and evaluated its role and the underlying mechanisms of chemoresistance in non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: A systemic search of gene expression profiles of multiple tissues in NSCLC was carried out to determine the expression intensities of traditional fibroblast biomarkers and CAF-secreted protumorigenic cytokines. PDL-1 expression in CAFs was analyzed by ELISA, Western blotting, and flow cytometry. Human cytokine array was used to identify specific cytokines secreted from CAFs. Role of PDL-1 in NSCLC chemoresistance was assessed using CRISPR/Cas9 knockdown and various functional assays including MTT, cell invasion, sphere formation, and cell apoptosis. In vivo experiments were conducted using a co-implantation xenograft mouse model with live cell imaging and immunohistochemistry. RESULTS: We demonstrated that chemotherapy-stimulated CAFs promoted tumorigenic and stem cell-like properties of NSCLC cells, which contribute to their chemoresistance. Subsequently, we revealed that PDL-1 expression is upregulated in chemotherapy-treated CAFs and is associated with poor prognosis. Silencing PDL-1 expression suppressed CAFs' ability to promote stem cell-like properties and invasiveness of lung cancer cells, favoring chemoresistance. Mechanistically, an upregulation of PDL-1 in chemotherapy-treated CAFs led to an increase in hepatocyte growth factor (HGF) secretion, which stimulates cancer progression, cell invasion, and stemness of lung cancer cells, while inhibiting apoptosis. CONCLUSION: Our results show that PDL-1-positive CAFs modulate stem cell-like properties of NSCLC cells by secreting elevated HGF, thereby promoting chemoresistance. Our finding supports PDL-1 in CAFs as a chemotherapy response biomarker and as a drug delivery and therapeutic target for chemoresistant NSCLC. |
Variation in pentose phosphate pathway-associated metabolism dictates cytotoxicity outcomes determined by tetrazolium reduction assays
Coyle JP , Johnson C , Jensen J , Farcas M , Derk R , Stueckle TA , Kornberg TG , Rojanasakul Y , Rojanasakul LW . Sci Rep 2023 13 (1) 8220 Tetrazolium reduction and resazurin assays are the mainstay of routine in vitro toxicity batteries. However, potentially erroneous characterization of cytotoxicity and cell proliferation can arise if verification of baseline interaction of test article with method employed is neglected. The current investigation aimed to demonstrate how interpretation of results from several standard cytotoxicity and proliferation assays vary in dependence on contributions from the pentose phosphate pathway (PPP). Non-tumorigenic Beas-2B cells were treated with graded concentrations of benzo[a]pyrene (B[a]P) for 24 and 48 h prior to cytotoxicity and proliferation assessment with commonly used MTT, MTS, WST1, and Alamar Blue assays. B[a]P caused enhanced metabolism of each dye assessed despite reductions in mitochondrial membrane potential and was reversed by 6-aminonicotinamide (6AN)-a glucose-6-phosphate dehydrogenase inhibitor. These results demonstrate differential sensitivity of standard cytotoxicity assessments on the PPP, thus (1) decoupling "mitochondrial activity" as an interpretation of cellular formazan and Alamar Blue metabolism, and (2) demonstrating the implicit requirement for investigators to sufficiently verify interaction of these methods in routine cytotoxicity and proliferation characterization. The nuances of method-specific extramitochondrial metabolism must be scrutinized to properly qualify specific endpoints employed, particularly under the circumstances of metabolic reprogramming. |
SOX2 mediates carbon nanotube-induced fibrogenesis and fibroblast stem cell acquisition
Kiratipaiboon C , Voronkova M , Ghosh R , Rojanasakul LW , Dinu CZ , Chen YC , Rojanasakul Y . ACS Biomater Sci Eng 2020 6 (9) 5290-5304 Certain nanosized particles like carbon nanotubes (CNTs) are known to induce pulmonary fibrosis, but the underlying mechanisms are unclear, and efforts to prevent this disease are lacking. Fibroblast-associated stem cells (FSCs) have been suggested as a critical driver of fibrosis induced by CNTs by serving as a renewable source of extracellular matrix-producing cells; however, a detailed understanding of this process remains obscure. Here, we demonstrated that single-walled CNTs induced FSC acquisition and fibrogenic responses in primary human lung fibroblasts. This was indicated by increased expression of stem cell markers (e.g., CD44 and ABCG2) and fibrogenic markers (e.g., collagen and α-SMA) in CNT-exposed cells. These cells also showed increased sphere formation, anoikis resistance, and aldehyde dehydrogenase (ALDH) activities, which are characteristics of stem cells. Mechanistic studies revealed sex-determining region Y-box 2 (SOX2), a self-renewal associated transcription factor, as a key driver of FSC acquisition and fibrogenesis. Upregulation and colocalization of SOX2 and COL1 were found in the fibrotic lung tissues of CNT-exposed mice via oropharyngeal aspiration after 56 days. The knockdown of SOX2 by gene silencing abrogated the fibrogenic and FSC-inducing effects of CNTs. Chromatin immunoprecipitation assays identified SOX2-binding sites on COL1A1 and COL1A2, indicating SOX2 as a transcription factor in collagen synthesis. SOX2 was also found to play a critical role in TGF-β-induced fibrogenesis through its collagen-and FSC-inducing effects. Since many nanomaterials are known to induce TGF-β, our findings that SOX2 regulate FSCs and fibrogenesis may have broad implications on the fibrogenic mechanisms and treatment strategies of various nanomaterial-induced fibrotic disorders. |
Carbon nanotube filler enhances incinerated thermoplastics-induced cytotoxicity and metabolic disruption in vitro
Coyle JP , Derk RC , Kornberg TG , Singh D , Jensen J , Friend S , Mercer R , Stueckle TA , Demokritou P , Rojanasakul Y , Rojanasakul LW . Part Fibre Toxicol 2020 17 (1) 40 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. |
Iron oxide nanoparticle-induced neoplastic-like cell transformation in vitro is reduced with a protective amorphous silica coating
Kornberg TG , Stueckle TA , Coyle J , Derk R , Demokritou P , Rojanasakul Y , Rojanasakul LW . Chem Res Toxicol 2019 32 (12) 2382-2397 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 mug/cm(2) administered a dose of nFe2O3 ( approximately 0.58 mug/cm(2) delivered dose), SiO2-nFe2O3 ( approximately 0.55 mug/cm(2) delivered dose), or gas metal arc mild steel welding fumes (GMA-MS, approximately 0.58 mug/cm(2) 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. |
SOX9-ALDH axis determines resistance to chemotherapy in non-small cell lung cancer
Voronkova MA , Rojanasakul LW , Kiratipaiboon C , Rojanasakul Y . Mol Cell Biol 2019 40 (2) Chemotherapy resistance and tumor relapse are the major contributors to low patient survival, and both have been largely attributed to cancer stem-like cells (CSCs) or tumor initiating cells (TICs). Moreover, most conventional therapies are not effective against CSCs, which necessitates the discovery of CSC-specific biomarkers and drug targets. Here, we demonstrated that the embryonic transcription factor SOX9 is an important regulator of acquired chemoresistance in non-small cell lung cancer (NSCLC). Our results show that SOX9 expression is elevated in NSCLC cells after treatment with the chemotherapeutic cisplatin and that overexpression of SOX9 correlates with worse overall survival in lung cancer patients. We further demonstrated that SOX9 knockdown increases cellular sensitivity to cisplatin, whereas its overexpression promotes drug resistance. Moreover, this transcription factor promotes stem-like properties of NSCLC cells and increases their ALDH activity, which was identified as the key mechanism of SOX9-induced chemoresistance. Finally, we showed that ALDH1A1 is a direct transcriptional target of SOX9 based on chromatin immunoprecipitation and luciferase reporter assays. Taken together, our novel findings on the role of SOX9-ALDH axis support the use of this CSC regulator as a prognostic marker of cancer chemoresistance and as a potential drug target for CSC therapy. |
Substrate stiffness-dependent carbon nanotube-induced lung fibrogenesis
Wang K , Shi L , Linthicum W , Man K , He X , Wen Q , Rojanasakul LW , Rojanasakul Y , Yang Y . Nano Lett 2019 19 (8) 5443-5451 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. |
Acquisition of cancer stem cell-like properties in human small airway epithelial cells after a long-term exposure to carbon nanomaterials
Kiratipaiboon C , Stueckle TA , Ghosh R , Rojanasakul LW , Chen YC , Dinu CZ , Rojanasakul Y . Environ Sci Nano 2019 6 (7) 2152-2170 Cancer stem cells (CSCs) are a key driver of tumor formation and metastasis, but how they are affected by nanomaterials is largely unknown. The present study investigated the effects of different carbon-based nanomaterials (CNMs) on neoplastic and CSC-like transformation of human small airway epithelial cells and determined the underlying mechanisms. Using a physiologically relevant exposure model (long-term/low-dose) with system validation using a human carcinogen, asbestos, we demonstrated that single-walled carbon nanotubes, multi-walled carbon nanotubes, ultrafine carbon black, and crocidolite asbestos induced particle-specific anchorage-independent colony formation, DNA-strand breaks, and p53 downregulation, indicating the genotoxicity and carcinogenic potential of CNMs. The chronic CNM-exposed cells exhibited CSC-like properties as indicated by 3D spheroid formation, anoikis resistance, and CSC marker expression. Mechanistic studies revealed specific self-renewal and epithelial-mesenchymal transition (EMT)-related transcription factors that are involved in the cellular transformation process. Pathway analysis of gene signaling networks supports the role of SOX2 and SNAI1 signaling in CNM-mediated transformation. These findings support the potential carcinogenicity of high aspect ratio CNMs and identified molecular targets and signaling pathways that may contribute to disease development. |
Predicting nanotube fibrogenicity through stem cell-mediated fibroblast focus and spheroid formation
He X , Kiratipaiboon C , Porter DW , Rojanasakul LW , Dinu CZ , Wang K , Yang Y , Rojanasakul Y . Nano Lett 2018 18 (10) 6500-6508 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. |
SOX9 regulates cancer stem-like properties and metastatic potential of single-walled carbon nanotube-exposed cells
Voronkova MA , Luanpitpong S , Rojanasakul LW , Castranova V , Dinu CZ , Riedel H , Rojanasakul Y . Sci Rep 2017 7 (1) 11653 Engineered nanomaterials hold great promise for the future development of innovative products but their adverse health effects are a major concern. Recent studies have indicated that certain nanomaterials, including carbon nanotubes (CNTs), may be carcinogenic. However, the underlying mechanisms behind their potential malignant properties remain unclear. In this study, we linked SOX9, a stem cell associated transcription factor, to the neoplastic-like properties of human lung epithelial cells chronically exposed to a low-dose of single-walled carbon nanotubes (SWCNTs). We found that SOX9 is upregulated in SWCNT-exposed cells, which is consistent with their abilities to induce tumor formation and metastasis in vivo. We therefore hypothesized that SOX9 overexpression may be responsible for the neoplastic-like phenotype observed in our model. Indeed, SOX9 knockdown inhibited anchorage-independent cell growth in vitro and lung colonization in vivo in a mouse xenograft model. SOX9 depletion also suppressed the formation of cancer stem-like cells (CSCs), as determined by tumor sphere formation and aldehyde dehydrogenase (ALDH) activity (Aldefluor) assays. Furthermore, SOX9 knockdown suppressed tumor metastasis and the expression of the stem cell marker ALDH1A1. Taken together, our findings provide a mechanistic insight into SWCNT-induced carcinogenesis and the role of SOX9 in CSC regulation and metastasis. |
Fas/FasL pathway-mediated alveolar macrophage apoptosis involved in human silicosis
Yao SQ , Rojanasakul LW , Chen ZY , Xu YJ , Bai YP , Chen G , Zhang XY , Zhang CM , Yu YQ , Shen FH , Yuan JX , Chen J , He QC . Apoptosis 2011 16 (12) 1195-204 In vitro and in vivo studies have demonstrated that lung cell apoptosis is associated with lung fibrosis; however the relationship between apoptosis of alveolar macrophages (AMs) and human silicosis has not been addressed. In the present study, AM apoptosis was determined in whole-lung lavage fluid from 48 male silicosis patients, 13 male observers, and 13 male healthy volunteers. The relationships between apoptosis index (AI) and silica exposure history, soluble Fas (sFas)/membrane-bound Fas (mFas), and caspase-3/caspase-8 were analyzed. AI, mFas, and caspase-3 were significantly higher in lung lavage fluids from silicosis patients than those of observers or healthy volunteers, but the level of sFas demonstrated a decreasing trend. AI was related to silica exposure, upregulation of mFas, and activation of caspase-3 and -8, as well as influenced by smoking status after adjusting for confounding factors. These results indicate that AM apoptosis could be used as a potential biomarker for human silicosis, and the Fas/FasL pathway may regulate this process. The present data from human lung lavage samples may help to understand the mechanism of silicosis and in turn lead to strategies for preventing or treating this disease. |
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