Anchorage-independent Growth Research Articles

HGS Promotes Tumor Growth, Whereas the Coiled-Coil Domain and Its Oligopeptide of HGS Suppress It.

We previously isolated a cDNA clone for galactosylceramide expression factor 1, which is the rat homologue of hepatocyte-growth-factor-regulated tyrosine kinase substrate (HGS) and induces galactosylceramide expression and morphological changes in COS-7 cells, and reported that overexpression of HGS induced morphological changes in canine kidney epithelial MDCK cells. HGS is a component of the endosomal sorting complexes required for transport machinery that mediates endosomal multivesicle body formation. In this study, the overexpression of HGS induced epithelial-mesenchymal transition and caused transformation in MDCK cells, whereas the overexpression of a coiled-coil domain of HGS inhibited induction of epithelial-mesenchymal transition by HGF stimulation. The overexpression of HGS in mouse melanoma B16 cells and human colorectal cancer COLO205 cells promoted cancer characteristic anchorage-independent cell growth ability and tumor growth, whereas the overexpression of the coiled-coil domain of HGS in these cells suppressed them. The oligopeptide OP12-462 constituting the coiled-coil domain suppressed the anchorage-independent cell growth ability and tumor growth of COLO205 cells. The coiled-coil domain of HGS and OP12-462 are novel tumor growth inhibitors that do not directly destroy cancer cells but rather inhibit only the anchorage-independent cell growth ability of cancer cells.

MiR-146b overexpression promotes bladder cancer cell growth via the SMAD4/C-MYC/Cyclin D1 axis.

MiR-146b has been identified as being overexpressed in human bladder cancer (BCa) and implicated in promoting cancer cell invasion. However, its specific involvement in BCa cell growth remains unclear. In this study, we demonstrate that the downregulation of miR-146b significantly suppresses tumorigenic growth of human BCa cells both in vitro and in vivo by inducing G0/G1 cell cycle arrest. Specifically, miR-146b inhibition resulted in a significant reduction in colony formation (p < 0.05) and anchorage-independent growth in both UMUC3 and T24T cells, as measured by soft agar assays, with three independent replicates for each experiment. Notably, Cyclin D1 protein plays a crucial role in miR-146b-induced BCa cell proliferation, as confirmed by Western blotting (p < 0.05), with each experiment performed in triplicate. Mechanistic investigations reveal that miR-146b reduces mothers against decapentaplegic homolog 4 (SMAD4) mRNA stability by directly binding to its 3' untranslated region (3'-UTR), leading to decreased SMAD4 expression. This reduction in SMAD4 levels promotes cellular myelocytomatosis (C-MYC) transcription, which in turn enhances Cyclin D1 transcription, ultimately facilitating BCa cell proliferation. The findings unveil a novel regulatory axis involving SMAD4/C-MYC/Cyclin D1 in mediating the oncogenic role of miR-146b in BCa cells. Statistical significance was determined using Student's t-test, with p-values <0.05 considered significant. Together with its previously established function in BCa invasion, the results highlight the potential for developing miR-146b-based therapeutic strategies for treating human BCa patients.

Vitamin K-dependent gamma-carboxyglutamic acid protein 1 promotes pancreatic ductal adenocarcinoma progression through stabilizing oncoprotein KRAS and tyrosine kinase receptor EGFR.

Vitamin K-dependent γ-glutamic acid carboxylation (Gla) proteins are calcium-binding and membrane-associated, participating in coagulation, bone turnover, and cancer biology. The molecular function of transmembrane proline-rich Gla proteins (PRRGs) remains unexplored. Analysis of pancreatic ductal adenocarcinoma (PDAC) datasets, including transcription profiles, clinical data, and tissue microarrays, was conducted to evaluate PRRG1 expression and its clinical relevance. PDAC cell lines with overexpressed, knockdown, and mutated PRRG1 were developed to study biological functions and pathways using RNA-seq, co-immunoprecipitation with mass spectrometry, Western blotting, and immunofluorescence. In vivo xenograft and orthotopic models assessed PRRG1's impact on PDAC progression, with and without warfarin treatment. PRRG1 was significantly upregulated in PDAC compared to normal pancreas, correlating with poorer patient survival. PRRG1 knockdown reduced PDAC cell proliferation, anchorage-independent growth in vitro, and tumor growth in vivo. PRRG1 localized at the plasma membrane, interacted with the HECT E3 ligase NEDD4 via the C-terminal PPXY motif, and promoted NEDD4 self-ubiquitination, reducing its protein levels. PRRG1 knockdown elevated NEDD4, destabilizing the oncoprotein KRAS and receptor EGFR, and attenuating downstream signaling and macropinocytosis under nutrient deprivation. The vitamin K-dependent Gla modification of PRRG1 was crucial for its membrane localization and pro-tumorigenic effects, and was inhibited by low-dose warfarin, a clinical vitamin K antagonist. This study identifies PRRG1 as a key regulator of pro-tumorigenic signaling in PDAC, suggesting the potential of repurposing the anticoagulant warfarin as a therapeutic strategy. PRRG1 is identified as the transmembrane Gla protein mediating PDAC malignancy. PRRG1 recruits and induces self-ubiquitination of membrane-anchoring E3 ligase NEDD4. PRRG1 exerts a protective role toward KRAS and EGFR by inhibiting NEDD4. The anticoagulant warfarin can be utilized to inhibit PRRG1 and PDAC advancement.

Identification of Translocon-associated Protein Delta as An Oncogene in Human Colorectal Cancer Cells.

Identifying the roles of genes in cancer is critical in discovering potential genetic therapies for cancer care. Translocon-associated protein delta (TRAPδ), also known as signal sequence receptor 4 (SSR4), is a constituent unit in the TRAP/SSR complex that resides in the endoplasmic reticulum and plays a key role in transporting newly synthesized proteins into the endoplasmic reticulumn. However, its biological role in disease development remains unknown to date. This is the first study to identify the role of TRAPδ/SSR4 in colorectal cancer cells in vitro. Upon successful transient knockdown of TRAPδ/SSR4, we observed significant reduction of cell viability in all colorectal cancer cell lines tested. Both HCT 116 and SW480 cell lines were significantly arrested at S and G1 phases, while DLD-1 cells were significantly apoptotic. Moreover, TRAPδ/SSR4 stable knockdown HCT 116 and SW480 cells showed significantly lower viability, anchorage-independent growth, and increased S and G1 phase arrests. Overall, we conclude TRAPδ/SSR4 is a potential oncogene in human colorectal cancer cells.

The efficacy of an embryonic stem cell-based vaccine for lung cancer prevention depends on the undifferentiated state of the stem cells

Based on the antigenic similarity between tumor cells and embryonic stem cells (ESCs), several recent studies report the use of intact murine ESCs or exosomes from murine ESCs as cancer vaccines. Since the capacity for self-renewal is one of the most specialized properties shared between ESCs and a subset of tumor cells, cancer stem cells (CSCs), we investigated whether the undifferentiated state of murine ESCs is essential for the prophylactic effectiveness of an ESC-based vaccine. The undifferentiated state of ES-D3, a murine ESC line, was essential for their anchorage-independent growth potential. Importantly, differentiation of ES-D3 cells decreased their efficacy in preventing the outgrowth of implanted lung tumors. Furthermore, the long-term cancer-preventive potential of this vaccine was also inhibited by the differentiation of these cells. To examine the antigenicity of the ESC-derived vaccine, we performed combined affinity chromatography shotgun immunoproteomic experiments to identify antigens specific to the whole-cell ES vaccine as well as to the ESC-derived exosome vaccine. Our data demonstrate that antibodies against several lung cancer-associated keratin members were enriched in the serum of vaccinated mice. In summary, these data suggest that the tumor-preventing efficacy of ESC-based vaccine is reliant on the differentiation properties of these stem cells.

Epidermal growth factor receptor ligands enriched in follicular fluid exosomes promote oncogenesis of fallopian tube epithelial cells

BackgroundIncessant ovulation is the main etiologic factor of ovarian high-grade serous carcinomas (HGSC), which mostly originate from the fallopian tube epithelium (FTE). Receptor tyrosine kinase (RTK) ligands essential for follicle development and ovulation wound repair were abundant in the follicular fluid (FF) and promoted the transformation of FTE cells. This study determined whether RTK ligands are present in FF exosomes and whether epidermal growth factor receptor (EGFR) signaling is essential for oncogenic activity.MethodsThe FF of women undergoing in vitro fertilization was fractionated based on the richness of exosomes and tested for transformation toward FTE cells under different RTK inhibitors. EGFR ligands in FF exosomes were identified, and downstream signaling proteins in FTE cells were characterized.ResultsThe transforming activity of FF was almost exclusively enriched in exosomes, which possess a high capacity to induce anchorage-independent growth, clonogenicity, migration, invasion, and proliferation of FTE cells. EGFR inhibition abolished most of these activities. FF and FF exosome exposure markedly increased EGFR phosphorylation and the downstream signal proteins, including AKT, MAPK, and FAK. Multiple EGF family growth factors, such as amphiregulin, epiregulin, betacellulin, and transforming growth factor-alpha, were identified in FF exosomes.ConclusionsOur results demonstrate that FF exosomes serve as carriers of EGFR ligands as well as ligands of other RTKs that mediate the transformation of FTE cells and underscore the need to further explore the content and roles of FF exosomes in HGSC development.

EFFECT OF ADHESIVE LLC CELL PRETREATMENT BY OXAMATE ON THE SURVIVAL INDEXES AFTER TRANSITION TO DE-ADHESIVE GROWTH.

The ability to metabolic reprogramming is a distinctive feature of metastatically active tumor cells. A classic example of metabolic reprogramming, characteristic of almost all malignant cells, is aerobic glycolysis. Therefore, inhibition of glycolysis in tumor cells is considered a promising strategy for antitumor therapy. To generate Lewis lung carcinoma (LLC) cell subpopulation after pretreatment by a lactate dehydrogenase (LDH) inhibitor - oxamate in adhesive growth conditions, and then to study the metabolism of this subpopulation in the anchorage-independent growth conditions. LLC cells were cultured without oxamate or with 17 mM oxamate in the adhesive growth conditions with the following transition to the anchorage-independent growth conditions without oxamate. A distribution of LLC cells by cell cycle phases, apoptosis rate, levels of reactive oxygen species (ROS), E-cadherin, and vimentin were determined by flow cytometry. Glucose consumption and lactate production were determined by spectrophotometry. 48-h oxamate treatment in adhesive growth conditions resulted in a 30% decrease of the total number of LLC cells compared to the control. In 72 h after the transfer of both oxamate-treated and control cells into the anchorage-independent growth condition without oxamate, the number of viable cells pretreated with oxamate was reduced by 17% (p < 0.05) compared to the control cells. However, the distribution of cells by cell cycle phases did not differ. In cells pre-treated with oxamate, the rate of glucose consumption decreased by 20% (p < 0.05), ROS generation was reduced by 17%, vimentin expression decreased by 10% while the rate of lactate production was the same in oxamate-pretreated and control cells. The cytostatic effect of oxamate demonstrated in adhesive growth conditions persisted for 72 h in the anchorage-independent growth conditions. The absence of differences in the cell cycle phase distribution and a decrease in the ROS generation may indicate the initial stage of overcoming the cytostatic effect of oxamate after 72 h of culturing LLC cells in anchorage- independent growth conditions.

Establishment of two novel organoid lines from patients with combined hepatocellular cholangiocarcinoma.

Combined hepatocellular cholangiocarcinoma (cHCC-CCA) is a unique subtype of primary liver cancer displaying both hepatocytic and cholangiocytic differentiation. The development of effective treatments for cHCC-CCA remains challenging because of its high heterogeneity and lack of a suitable model system. Using a three-dimensional culture system, we successfully established two novel cHCC-CCA organoid lines from patients undergoing surgical resection for primary liver cancer. cHCC-CCA organoid lines were authenticated by fingerprint analysis, and their morphology, growth kinetics, and anchorage-independent growth were also characterized. Hematoxylin and eosin staining and immunohistochemical analysis showed that the cHCC-CCA organoids preserved the growth pattern, differentiation grade, and phenotypic characteristics of their parental tumors. Whole-exome sequencing demonstrated that patient-derived cHCC-CCA organoid lines retained the genetic alterations identified in their original tumors. Subcutaneous tumors developed in immunodeficient mice after injection of cHCC-CCA organoids. Histologically, the xenografts recapitulated the features of the original cHCC-CCA tumors, harboring both HCC and intrahepatic cholangiocarcinoma components within the same tumor. The establishment of patient-derived cHCC-CCA organoid lines with high tumorigenicity provides a valuable resource for the mechanistic investigation and drug development of this disease.

MiR-198 targets TOPORS: implications for oral squamous cell carcinoma pathogenesis.

miRNAs play a critical role in the progression of various diseases, including oral squamous cell carcinoma (OSCC), which represents a major health concern and is one of the leading causes for new cancer cases worldwide. The miRNA dysregulation causes havoc and could be attributed to various factors, with epigenetic silencing of tumor suppressor genes being a major contributor to tumorigenesis. In this study, we have explored the tumor suppressive role of miR-198 in OSCC. The tumor suppressive effect of miR-198 is established using miRNA analysis in OSCC cell lines, patient samples and xenograft nude mice model. The relationship between the miR-198 and TOPORS is explored using bioinformatics analyses, qRT-PCR, dual-luciferase reporter assay, Western blotting and cancer hall marks assays. The hypermethylation of the MIR198 promoter is confirmed using bisulfite sequencing PCR. We have found miR-198 to be upregulated in OSCC cells treated with 5-Azacytidine, a known DNA methyltransferase inhibitor. Upregulation of miR-198 in 5-Azacytidine treated OSCC cells appears to be due to methylation of the MIR198 promoter. Using bioinformatics analysis and dual-luciferase reporter assay, we have identified TOPORS (TOP1 binding arginine/serine rich protein, E3 ubiquitin ligase) as a novel gene target for miR-198. miR-198-mediated repression of TOPORS decreases cell proliferation and anchorage-independent growth and enhances apoptosis of OSCC cells, which is dependent on the presence of the 3'UTR in TOPORS. An inverse correlation between the expression levels of miR-198 and TOPORS is observed in OSCC patient samples, highlighting the biological relevance of their interaction. Delivery of a synthetic miR-198 mimic to OSCC cells results in a significant decrease in xenograft size in nude mice, potentiating its use in therapeutics. These results suggest that miR-198 is epigenetically silenced in OSCC, which promotes tumor growth, in part, by upregulating the levels of TOPORS.

Mycobacteria Treatment Inhibits Bladder Cancer Cell Migration, Invasion, and Anchorage-Independent Growth.

Bladder cancer (BC) is a highly recurrent and invasive malignancy, with Mycobacterium bovis BCG serving as the primary immunotherapy, particularly for non-muscle-invasive bladder cancer (NMIBC). However, the mechanisms underlying BCG's antitumor effects and the potential of non-tuberculous mycobacteria like Mycobacterium brumae remain unclear. This study investigates the antitumor effects of M. bovis BCG and M. brumae on BC cell migration, invasion, and anchorage-independent growth. BC cell lines representing different stages of tumor differentiation were treated with either M. bovis BCG or M. brumae. Cell migration was assessed through wound healing and transwell assays, invasiveness by transwell invasion assays, MMP-9 production by gelatin zymography, and anchorage-independent growth via soft agar colony formation. Both mycobacteria inhibited individual cell migration across all BC lines, while collective migration was only reduced in intermediate-grade cells. Both treatments also reduced invasiveness, associated with decreased MMP-9 production. Furthermore, M. brumae inhibited anchorage-independent growth across all BC lines, while M. bovis BCG had a more selective effect, primarily inhibiting growth in high-grade cells. In conclusion, both mycobacteria reduce migration, invasion, and anchorage-independent growth of BC cells, with their effectiveness varying by species and tumor differentiation grade.

Biased signaling by mutant EGFR underlies dependence on PKCα in lung adenocarcinoma

Biased signaling by mutant EGFR underlies dependence on PKCα in lung adenocarcinoma

Promoter hypermethylation-mediated downregulation of PAX6 promotes tumor growth and metastasis during the progression of liver cancer

BackgroundThe progression of liver cancer is a complicated process that involves genetic and epigenetic changes. Paired box 6 (PAX6) is a critical transcription factor for embryonic development. PAX6 is abnormally methylated in human cancer. The role of the PAX6 gene in the pathogenesis of hepatocellular carcinoma (HCC) is still unclear.MethodsTranscriptional silencing of PAX6 mediated by promoter methylation was confirmed using quantitative methylation-specific polymerase chain reaction (PCR) and reverse-transcription (RT)-PCR. Then we conducted gain-and-loss of function approaches to evaluate the function of PAX6 in HCC progression in vitro. Moreover, we designed xenograft mouse models to assess the effect of PAX6 on tumor growth and metastasis. Finally, we used RNA sequencing (RNA-seq) strategy and phenotypic rescue experiments to identify potential targets of PAX6 performing tumor-suppressive function.ResultsConstitutive expression of PAX6 suppressed anchorage-independent growth and cell invasion in vitro as well as tumor growth and metastasis in xenograft mouse models. In contrast, the inhibition of PAX6 using knockout and knockdown strategies increased tumor growth both in vitro and in vivo. Downregulation of PAX6 by doxycycline depletion partially reversed the malignant phenotypes of HCC cells induced by PAX6. Moreover, we identified E-cadherin (CDH1) and thrombospondin-1 (THBS1) as targets of PAX6. Ultimately, we demonstrated that the knockdown of CDH1 and overexpression of THBS1 in PAX6-expressing HCC cells partly reversed the tumor-suppressive effect.ConclusionPAX6 functions as a tumor suppressor partly through upregulation of CDH1 and downregulation of THBS1. Promoter hypermethylation-mediated suppression of PAX6 reduces the tumor suppressor function in the progression of liver cancer.

FBXO46 negatively regulates p53 activity by stabilizing Mdm2.

The tumor suppressor p53 plays a central role in suppressing tumor formation. Mouse double minute 2 homolog (Mdm2) serves as the principal ubiquitin E3 ligase responsible for the ubiquitination and subsequent degradation of p53. However, the regulatory mechanisms governing the Mdm2-p53 pathway are not comprehensively understood. Here, we report that F-box only protein 46 (FBXO46) directly binds to Mdm2 and inhibits its self-ubiquitination and degradation, leading to Mdm2 stabilization and subsequent Mdm2-mediated ubiquitination and degradation of p53. Functionally, FBXO46 promotes cell proliferation, accelerates G1/S cell cycle progression, and increases anchorage-independent cell growth by inhibiting p53. Collectively, these findings reveal a critical role for FBXO46 in controlling Mdm2 stability and establish FBXO46 as an important regulator of the Mdm2-p53 pathway.

PKR associates with 4.1R to promote anchorage-independent growth of hepatocellular carcinoma and lead to poor prognosis

RNA-dependent protein kinase (PKR) may have a positive regulatory role in controlling tumor growth and progression in hepatocellular carcinoma (HCC). However, the downstream substrates and the molecular mechanism of PKR in the growth and progression of HCC have not been clarified. In this study, mass spectrometry analysis was performed with immunoprecipitated samples, and 4.1R was identified as a protein that binds to PKR. In transfected COS7 cells, an immunoprecipitation experiment showed that 4.1R binds to wild-type PKR, but not to a kinase-deficient mutant PKR, suggesting that PKR binds to 4.1R in a kinase activity-dependent manner. In HCC cell lines, HuH7 and HepG2, the expression level of 4.1R protein was shown to be regulated by protein expression and activation of PKR. Interestingly, high expression of 4.1R, as well as PKR, is associated with a worse prognosis in HCC. PKR increased HCC cell growth in both anchorage-dependent and anchorage-independent manners, whereas 4.1R was involved in HCC cell growth only in an anchorage-independent manner, not in an anchorage-dependent manner. The rescue experiment indicated that increased anchorage-independent growth of HCC cells by PKR might be caused by 4.1R. In conclusion, PKR associates with 4.1R and promotes anchorage-independent growth of HCC. The PKR-4.1R axis might be a new therapeutic target in HCC.

Hepatocellular carcinoma hosts cholinergic neural cells and tumoral hepatocytes harboring targetable muscarinic receptors

Hepatocellular carcinoma hosts cholinergic neural cells and tumoral hepatocytes harboring targetable muscarinic receptors

Zymogen granule protein 16B (ZG16B) is a druggable epigenetic target to modulate the mammary extracellular matrix.

High tissue density of the mammary gland is considered a pro-tumorigenic factor, hence suppressing the stimuli that induce matrix buildup carries the potential for cancer interception. We found that in non-malignant mammary epithelial cells the combination of the chemopreventive agents bexarotene (Bex) and carvedilol (Carv) suppresses the zymogen granule protein 16B (ZG16B, PAUF) through an interaction of ARID1A with a proximal enhancer. Bex + Carv also reduced ZG16B levels invivo in normal breast tissue and MDA-MB231 tumor xenografts. The relevance of ZG16B is underscored by ongoing clinical trials targeting ZG16B in pancreatic cancers, but its role in breast cancer development is unclear. In immortalized mammary epithelial cells, secreted recombinant ZG16B stimulated mitogenic kinase phosphorylation, detachment and mesenchymal characteristics, and promoted proliferation, motility and clonogenic growth. Highly concerted induction of specific laminin, collagen and integrin isoforms indicated a shift in matrix properties toward increased density and cell-matrix interactions. Exogenous ZG16B alone blocked Bex + Carv-mediated control of cell growth and migration, and antagonized Bex + Carv-induced gene programs regulating cell adhesion and migration. In breast cancer cells ZG16B induced colony formation and anchorage-independent growth, and stimulated migration in a PI3K/Akt-dependent manner. In contrast, Bex + Carv inhibited colony formation, reduced Ki67 levels, ZG16B expression and glucose uptake in MDA-MB231 xenografts. These data establish ZG16B as a druggable pro-tumorigenic target in breast cell transformation and suggest a key role of the matrisome network in rexinoid-dependent antitumor activity.

ISO-upregulated BECN1 specifically promotes LC3B-dependent autophagy and anticancer activity in invasive bladder cancer

ISO-upregulated BECN1 specifically promotes LC3B-dependent autophagy and anticancer activity in invasive bladder cancer

The Role of the Bone Morphogenetic Protein Antagonist Noggin in Nucleus Pulposus Intervertebral Disc Cells.

Low back pain (LBP) is a significant global health issue, contributing to disability and socioeconomic burdens worldwide. The degeneration of the human intervertebral disc (IVD) is a critical factor in the pathogenesis of LBP. Recent studies have emphasized the significance of a specific set of genes and extracellular matrix (ECM) in IVD health. In particular, Noggin has emerged as a critical gene due to its high expression levels in healthy nucleus pulposus cells (NPCs) observed in our previous research. In this study, it was hypothesized that decreased Noggin expression in NPCs is associated with IVD degeneration and contributes to LBP development. A lentivirus-mediated RNAi was applied to knock down Noggin expression in primary NPCs from six human donors. The NPCs after transduction were evaluated through cell viability analysis, XTT assay, and cell apoptosis analyses. After two weeks, a colony formation assay was used to examine the anchor-independent growth ability of transduced cells. At the transcript level, anabolic and catabolic markers were quantified using RT-qPCR. The results demonstrated that lentivirus-mediated downregulation of Noggin significantly inhibited cell proliferation, reduced cell viability, and suppressed colony formation, while inducing apoptosis in human NPCs in vitro. Notably, it disrupted cellular anabolic processes and promoted catabolic activity in human NPCs post-transduction. Our findings indicated that the degeneration of human IVD is possibly related to decreased Noggin expression in NPCs. This research provides valuable insights into the role of Noggin in IVD homeostasis and its implications in LBP treatment.

A simple, economical, and high-yield method for polyethylene glycol-based extraction of follicular and serum-derived extracellular vesicles.

The optimization of polyethylene glycol (PEG)-based extracellular vesicles (EVs) extraction from human follicular fluid (FF) and serum was investigated, and their functional analysis was confirmed. The PEG-based EV results were compared to the ExoQuick (ExoQ)-based EV. FF-EVs and serum-EVs were extracted by using different concentrations of PEG (8000). Nanoparticle tracking analysis was used to count the particles, and electron microscopy of EVs was performed for visualization. Exosomes were confirmed by the western blot analysis with exosome-specific markers. RNA and microRNA were extracted from exosomes and quantitative polymerase chain reaction analysis was performed. Fallopian tube epithelial (FTE) cells were used for the EV uptake experiment and an anchorage-independent growth test to confirm that extracted EVs harbor transformation activity. The PEG 8% enriched method produced the highest yield and the lowest carry-over protein. Salt containing PEG 8% produced a higher yield than nonsalted PEG 8%. Overnight enrichment increased four times and 18 times for PEG 8% and ExoQ-based EV extraction from FF. For serum EV, the same overnight enrichment moderately increased yield for both PEG 8% and ExoQ methods. Less carry-over protein resulted in more EV-promoted transformation activity. This study overcomes the time-consuming, expensive, laborious, and complicated machine-dependent EV extraction methods. The study highlights that longer incubation time is needed for EV extraction from FF. PEG 8000-based EV extraction provided a higher yield and less carry-over protein than ExoQ-based EV extraction.

Chronic exposure to hexavalent chromium induces esophageal tumorigenesis via activating the Notch signaling pathway.

Hexavalent chromium Cr(VI), as a well-established carcinogen, contributes to tumorigenesis for many human cancers, especially respiratory and digestive tumors. However, the potential function and relevant mechanism of Cr(VI) on the initiation of esophageal carcinogenesis are largely unknown. Here, immortalized human esophageal epithelial cells (HEECs) were induced to be malignantly transformed cells, termed HEEC-Cr(VI) cells, via chronic exposure to Cr(VI), which simulates the progress of esophageal tumorigenesis. In vitro and in vivo experiments demonstrated that HEEC-Cr(VI) cells obtain the ability of anchorage-independent growth, greater proliferative capacity, cancer stem cell properties, and the capacity to form subcutaneous xenografts in BALB/c nude mice when compared to their parental cells, HEECs. Additionally, HEEC-Cr(VI) cells exhibited weakened cell motility and enhanced cell adhesion. Interestingly, HEECs with acute exposure to Cr(VI) failed to display those malignant phenotypes of HEEC-Cr(VI) cells, suggesting that Cr(VI)‍-induced malignant transformation, but not Cr(VI) itself, is the cause for the tumor characteristics of HEEC-Cr(VI) cells. Mechanistically, chronic exposure to Cr(VI) induced abnormal activation of Notch signaling, which is crucial to maintaining the capacity for malignant proliferation and stemness of HEEC-Cr(VI) cells. As expected, N-‍[N-‍(3,5-difluorophenacetyl)‍-L-alanyl]‍-S-phenylglycine t-butyl ester (DAPT), an inhibitor for the Notch pathway, drastically attenuated cancerous phenotypes of HEEC-Cr(VI) cells. In conclusion, our study clarified the molecular mechanism underlying Cr(VI)‍-induced esophageal tumorigenesis, which provides novel insights for further basic research and clinical therapeutic strategies about Cr(VI)‍-associated esophageal cancer.