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.

Zinc suppresses stem cell properties of lung cancer cells through protein kinase C-mediated ß-catenin degradation.

Highly tumorigenic cancer stem cells (CSCs) residing in most cancers are responsible for cancer progression and treatment failure. Zinc is an element regulator of several cell functions; however, its role in regulation of stem cell program in lung cancer has not been demonstrated. The present study reveals for the first time that zinc can suppress stem cell properties of lung cancer cells. Such findings were proved in different lung cancer cell lines (H460, H23, and H292) and it was found that CSC markers (CD133 and ALDH1A1), stem cell-associated transcription factors (Oct4, Nanog, and Sox-2), and the ability to form tumor spheroid were dramatically suppressed by zinc treatments. Zinc was found to activate protein kinase C-α (PKCα) that further phosphorylated and mediated ß-catenin degradation through the ubiquitin-proteasomal pathway. Zinc was found to increase the ß-catenin-ubiquitin complex, which can be inhibited by a specific PKC inhibitor, bisindolylmaleimide I. Using specific reactive oxygen species detection and antioxidants, we have demonstrated that superoxide anions generated by zinc are a key upstream mechanism for PKCα activation leading to the subsequent suppression of stem cell features of lung cancer. Zinc increased cellular superoxide anions and the addition of superoxide anion scavenger prevented the activation of PKCα and ß-catenin degradation. These findings indicate a novel role for zinc regulation in the PKCα/ß-catenin pathway and explain an important mechanism for controlling of stem cell program in lung cancer cells.

Herbal root extracts in Ben-Cha-Moon-Yai remedy attenuated pain-like behaviors and inflammation through the opioid and prostaglandin systems.

ETHNOPHARMACOLOGICAL RELEVANCE: Ben-Cha-Moon-Yai (BMY) remedy used in Thai traditional medicine as an anti-inflammatory, analgesic, and antipyretic agent compromises five herbal root extracts of equal weights: Aegle marmelos (L.) Corrêa (AM), Oroxylum indicum (L.) Kurz (OI), Dimocarpus longan Lour. (DL), Dolichandrone serrulata (Wall. ex DC.) Seem. (DS), and Walsura trichostemon Miq. (WT). AIM OF THE STUDY: To assess the anti-nociceptive and anti-inflammatory effects of the root extracts of all five species of BMY in experimental animal (mouse) models to ensure the rational use of herbal products in Thai traditional medicine. MATERIALS AND METHODS: Root extracts prepared by ethanol and water extraction were used for the biological assays in animal models at five dose levels: 25, 50,100,200 & 400 mg/kg. The anti-nociceptive activity was evaluated based on hot-plate latency, duration of paw licking induced by formalin, and abdominal writhing induced by acetic acid. Carrageenan- and prostaglandin-induced paw oedema models were used to determine the anti-inflammatory activity. RESULTS: The oral administration of AM, DS and WT root extracts displayed significant analgesic effects in the hot-plate test, both phases (early and late) of formalin test and acetic-acid induced writhing test at different dose levels. OI and DL only produced significant analgesia in the late phase of the formalin test and writhing test. The pretreatment of animals with the non-selective opioid receptor antagonist naloxone, reverse AM, DS and WT induced-antinociceptive activity. In both carrageenan and prostaglandin-induced paw oedema tests, all five herbal plant root extracts significantly reduced paw oedema at 3 h or more at different dose levels. Rotarod test results showed no effects of five herbal plant root extracts on the balance and the motor coordination at the highest dose level evaluated (400 mg/kg). CONCLUSION: The root extracts of AM, DS, and WT possess both central and peripheral anti-nociceptive properties, while OI and DL possess only peripheral analgesic properties. All five root extracts own anti-inflammatory properties, which might be due to their activity on the prostaglandin system. Altogether these findings ensure the rational use of BMY remedy in Thai traditional medicine.

The SOX9-Aldehyde Dehydrogenase Axis Determines Resistance to Chemotherapy in Non-Small-Cell Lung Cancer.

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 the stem-like properties of NSCLC cells and increases their aldehyde dehydrogenase (ALDH) activity, which was identified to be 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 the 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.

SOX2Mediates Carbon Nanotube-Induced Fibrogenesis and Fibroblast Stem Cell Acquisition.

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-ß-induced fibrogenesis through its collagen- and FSC-inducing effects. Since many nanomaterials are known to induce TGF-ß, 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.

Acquisition of Cancer Stem Cell-like Properties in Human Small Airway Epithelial Cells after a Long-term Exposure to Carbon Nanomaterials.

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 break, and p53 downregulation, indicating 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 markers 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 the disease development.

Reactive oxygen species mediate cancer stem-like cells and determine bortezomib sensitivity via Mcl-1 and Zeb-1 in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an aggressive, incurable non-Hodgkin B-cell lymphoma with good initial response to therapy then subsequently relapse. Cancer stem cells (CSCs) are considered to be an underlying cause of these inevitable drug resistance and tumor regrowth, but how CSCs are regulated is largely unknown. We demonstrate here for the first time the existence of CSC-like subpopulations that are modulated by reactive oxygen species (ROS) in MCL cell lines and patient-derived primary cells in an inverse correlation with bortezomib (BTZ) sensitivity. Using various known donors and inhibitors of cellular superoxide (O2-), hydrogen peroxide (H2O2) and hydroxyl radical (OH), we unveil their distinct roles in the regulation of CSC-like subpopulations and thus MCL response to BTZ. O2- inhibits CSC-like cells and sensitizes BTZ-induced apoptosis, whereas H2O2 conversely enriches CSC-like cells and protects against apoptosis and OH has minimal effects. We further observed that an anti-apoptotic Mcl-1 and a transcription factor Zeb-1 are favorable targets of O2- and H2O2, respectively. Using small molecule inhibition, ectopic expression and CRISPR/Cas9-mediated gene manipulation, we verified the roles of Mcl-1 and Zeb-1 in CSC and apoptosis regulation by O2- and H2O2. Our findings provide a novel mechanistic insight into the significance of redox status of MCL cells in determining their drug response via CSC-like subpopulations, which are imperative to a better understanding of therapeutic resistance and relapse.

Ciprofloxacin Improves the Stemness of Human Dermal Papilla Cells.

Improvement in the expansion method of adult stem cells may augment their use in regenerative therapy. Using human dermal papilla cell line as well as primary dermal papilla cells as model systems, the present study demonstrated that ciprofloxacin treatment could prevent the loss of stemness during culture. Clonogenicity and stem cell markers of dermal papilla cells were shown to gradually decrease in the culture in a time-dependent manner. Treatment of the cells with nontoxic concentrations of ciprofloxacin could maintain both stem cell morphology and clonogenicity, as well as all stem cells markers. We found that ciprofloxacin exerted its effect through ATP-dependent tyrosine kinase/glycogen synthase kinase3ß dependent mechanism which in turn upregulated ß-catenin. Besides, ciprofloxacin was shown to induce epithelial-mesenchymal transition in DPCs as the transcription factors ZEB1 and Snail were significantly increased. Furthermore, the self-renewal proteins of Wnt/ß-catenin pathway, namely, Nanog and Oct-4 were significantly upregulated in the ciprofloxacin-treated cells. The effects of ciprofloxacin in preserving stem cell features were confirmed in the primary dermal papilla cells directly obtained from human hair follicles. Together, these results revealed a novel application of ciprofloxacin for stem cell maintenance and provided the underlying mechanisms that are responsible for the stemness in dermal papilla cells.

Ciprofloxacin mediates cancer stem cell phenotypes in lung cancer cells through caveolin-1-dependent mechanism.

Cancer stem cells (CSCs), a subpopulation of cancer cells with high aggressive behaviors, have been identified in many types of cancer including lung cancer as one of the key mediators driving cancer progression and metastasis. Here, we have reported for the first time that ciprofloxacin (CIP), a widely used anti-microbial drug, has a potentiating effect on CSC-like features in human non-small cell lung cancer (NSCLC) cells. CIP treatment promoted CSC-like phenotypes, including enhanced anchorage-independent growth and spheroid formation. The known lung CSC markers: CD133, CD44, ABCG2 and ALDH1A1 were found to be significantly increased, while the factors involving in epithelial to mesenchymal transition (EMT): Slug and Snail, were depleted. Also, self-renewal transcription factors Oct-4 and Nanog were found to be up-regulated in CIP-treated cells. The treatment of CIP on CSC-rich populations obtained from secondary spheroids resulted in the further increase of CSC markers. In addition, we have proven that the mechanistic insight of the CIP induced stemness is through Caveolin-1 (Cav-1)-dependent mechanism. The specific suppression of Cav-1 by stably transfected Cav-1 shRNA plasmid dramatically reduced the effect of CIP on CSC markers as well as the CIP-induced spheroid formation ability. Cav-1 was shown to activate protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) pathways in CSC-rich population; however, such an effect was rarely found in the main lung cancer cells population. These findings reveal a novel effect of CIP in positively regulating CSCs in lung cancer cells via the activation of Cav-1, Akt and ERK, and may provoke the awareness of appropriate therapeutic strategy in cancer patients.

Glycyrrhizic acid attenuates stem cell-like phenotypes of human dermal papilla cells.

BACKGROUND: Although the growth of unwanted hair or hirsutism is a harmless condition, many people find it bothersome and embarrassing. Maintaining stem cell features of dermal papilla cells is a critical biological process that keeps the high rate of hair growth. Glycyrrhizic acid has been reported to impair hair growth in some studies; however, its underlying mechanism has not yet been investigated. PURPOSE: This study aimed to explore the effect and underlying mechanism of glycyrrhizic acid on stemness of human dermal papilla cells. STUDY DESIGN/METHODS: The stem cell molecular markers, epithelial to mesenchymal markers and Wnt/ß-catenin-associated proteins of human dermal papilla cell line and primary human dermal papilla cells were analysed by western blot analysis and immunocytochemistry. RESULTS: The present study demonstrated that glycyrrhizic acid significantly depressed the stemness of dermal papilla cells in dose- and time-dependent manners. Clonogenicity and stem cell markers in the glycyrrhizic acid-treated cells were found to gradually decrease in the culture in a time-dependent manner. Our results demonstrated that glycyrrhizic acid exerted the stem cell suppressing effects through the interruption of ATP-dependent tyrosine kinase/glycogen synthase kinase3ß-dependent mechanism which in turn down-regulated the ß-catenin signalling pathway, coupled with decreased its down-stream epithelial-mesenchymal transition and self-renewal transcription factors, namely, Oct-4, Nanog, Sox2, ZEB1 and Snail. The effect of glycyrrhizic acid on the reduction of stem cell features was also observed in the primary dermal papilla cells directly obtained from human hair follicles. CONCLUSION: These results revealed a novel molecular mechanism of glycyrrhizic acid in regulation of dermal papilla cells and provided the evidence supporting the use of this compound in suppressing the growth of unwanted hair.