Octamer-binding Transcription Factor Research Articles

Octamer-binding transcription factor 4-positive circulating tumor cell predicts worse treatment response and survival in advanced cholangiocarcinoma patients who receive immune checkpoint inhibitors treatment

BackgroundOctamer-binding transcription factor 4-positive circulating tumor cell (OCT4+CTC) exhibits high stemness and invasive potential, which may influence the efficacy of immune checkpoint inhibitors (ICI). This study aimed to assess the prognostic role of OCT4+CTC in advanced cholangiocarcinoma (CCA) patients who received ICI treatment.MethodsIn total, 40 advanced CCA patients who received ICI treatment were included, and CTC and OCT4 counts were detected via a Canpatrol system and an RNA in situ hybridization method before ICI treatment. Patients were subsequently divided into none CTC, OCT4−CTC, and OCT4+CTC groups. Patients were followed up for a median of 10.4 months.ResultsThe percentages of patients in none CTC, OCT4−CTC, and OCT4+CTC groups were 25.0%, 30.0%, and 45.0%, respectively. The proportion of patients with lymph node metastasis was highest in OCT4+CTC group, followed by none CTC group, and lowest in OCT4−CTC group (P = 0.025). The objective response rate (ORR) was lowest in OCT4+CTC group, moderate in OCT4−CTC group, and highest in none CTC group (P = 0.009), while disease control rate was not different among three groups (P = 0.293). In addition, progression-free survival (PFS) (P < 0.001) and overall survival (OS) (P = 0.001) were shorter in the OCT4+CTC group than in none CTC & OCT4−CTC group. Moreover, OCT4+CTC (versus none CTC) was independently linked with poorer PFS [hazard ratio (HR) = 6.752, P = 0.001] and OS (HR = 6.674, P = 0.003) in advanced CCA patients.ConclusionOCT4+CTC relates to lymph node metastasis and shows a good predictive value for poor treatment response and survival in advanced CCA patients who receive ICI treatment.

Abstract 565: PDE7 modulates EMT, cells morphology and mitochondrial cristae in high grade serous ovarian cancer

Abstract Background: Due to the acquisition of chemoresistant phenotype by epithelial ovarian cancer (EOC) cells and, consequently, disease relapse, the identification of novel therapeutic strategies is urgent. Herein, we targeted phosphodiesterase 7A (PDE7A) in EOC cells, based on data derived from RNA-sequencing assays comparing high-grade serous ovarian carcinoma (HGSOC) and fallopian tube samples. Methods: To understand the role of PDE inhibition, we evaluated the effects of its inhibitor BRL50481 alone or in combination with paclitaxel (PTX) on untreated A2780 EOC cells and multi-resistant high grade serous ovarian cancer (HGSOC) OVCAR3 cells. To do so, we pursued with the: validation of data obtained by high throughput RNA sequencing assays using qRT-PCR; evaluation of PDE7 expression in normal and EOC cells, as well as in normal Fallopian tube cells by qRT-PCR; immunoblotting; metabolic cell viability (MCV) by MTT assays; cytokine dosage by qRT-PCR and ELISA; scanning electron microscopy; transmission electron microscopy; statistical analysis (GraphPad software). Results: Drug-sensitive A2780 cells, but not OVCAR3 cells, exhibited lower MCV when treated with the PDE7 inhibitor; however, the combinatory therapy decreased MCV in both cell lines. Of clinical relevance, pre-treatment of A2780 and OVCAR3 with the PDE7 inhibitor BRL50481 followed by PTX lowered its IC50 by 103- and 625-fold, respectively. While the phosphatidylinositol 3-kinase (PI3K)/protein kinase B(AKT)/mammalian target of rapamycin (mTOR) pathway was inhibited in both cell lines, the pro-apoptotic protein Bcl-2 Associated X-protein (BAX) was overexpressed in A2780 cells only. Analysis of mRNA and protein expression in OVCAR3 pretreated with BRL50481 showed a 2-fold increase in IL-6 cytokine expression. Pre-treatment of OVCAR3 with BRL50481 followed by the combination of BRL50481 and PTX inhibited the expression of vimentin and Octamer-binding transcription factor 4 (OCT4) by 2-fold. BRL50481 promoted changes in EOC cells morphology and mitochondrial cristae. Conclusions: Altogether, our data led us to postulate that inhibition of PDE7 in EOC cells, specifically in those presenting the chemoresistant phenotype, is a promising strategy to fight this yet highly lethal disease. Citation Format: Leticia B. A. Rangel, Nayara Gusmão Tessarollo, Isabella dos Santos Guimarães, Diandra Zipinotti dos Santos, Taciane Barbosa Henriques, Marcele Lorentz de Souza, Leide Laura Figueiredo Maciel, João Carlos Aquino Almeida, Tatiana Massariol Pimenta, Josiany Carlos de Souza, Ranna Batista Wanzeler, Barbara Silva Martins, Solenny Maria Silva Butzene, Jose Matheus Simões Padilha. PDE7 modulates EMT, cells morphology and mitochondrial cristae in high grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 565.

Effects of different concentrations of reversine to enhance conversion of dedifferentiated fat cells into mature cardiomyocytes

Background There is an essential need for cardiomyocyte regeneration among patients with heart failure. Transplantation of dedifferentiated fat (DFAT) cells may lead to an improvement of cardiomyocyte regeneration among heart failure patients. We believe that DFAT cells are promising candidate cell sources for cardiac regeneration. However, the pathway underlying how DFAT cells of the adipose lineage differentiate into mature cardiomyocytes isn’t fully understood. Methods We conducted an experimental laboratory study on isolated DFAT cells from adipose tissue of healthy adults. Then, we treated cells with different concentrations of reversine (10, 20 and 40 nM), and performed RNA extraction and cDNA synthesis. Next, we used a ceiling culture method based on the buoyancy properties of mature lipid-filled adipocytes. Stemness expression (Octamer-binding transcription factor 4 [Oct4], brachyury, Fetal liver kinase 1 [Flk-1]) was quantified by reverse transcription-quantitative (RT-q)PCR, while cardiomyocyte expression (Transcription factor GATA-4 [GATA4] and cardiac troponin T [cTnT]) was quantified by immunocytochemistry. Results ANOVA with Tukey’s post-hoc found that 10 nM reversine increased greater Flk-1 expression compared to the control group (MD: 5.037 + 0.998; p &lt; 0.001), but there were no significant changes among Oct4 (MD: 0.013 + 1.244; p = 0.99) and brachyury expression (MD: 0.157 + 0.084; p = 0.252). Kruskal-Wallis revealed that the expression of GATA4 (1.65 [0.41-1.98] to 0.015 [0.007-0.034]; p =0.017) reduced significantly from day 7 until day 21 and cTnT (5.07 [6.62-8.91] to 8.22 [6.81-9.40]; p= 0.001) increased significantly from day 7 until day 21. Conclusions Reversine could increase the expression of Flk-1, but it was unable to stimulate the expression of Oct4 and brachyury related to stem cell-ness. An optimal concentration of 10 nM reversine may have the greatest effect on enhancing the differentiation of DFAT cells into mature cardiomyocytes, as indicated by higher cTnT expression between cells.

Oct4 activates IL-17A to orchestrate M2 macrophage polarization and cervical cancer metastasis

BackgroundCervical cancer is a common malignant tumor in the female. Interleukin (IL)-17A is a proinflammatory factor and exerts a vital function in inflammatory diseases and cancers. M2 macrophage has been confirmed to promote tumor development. Nevertheless, it is not yet known whether IL-17A facilitates cervical cancer development by inducing M2 macrophage polarization. Therefore, this study was conducted to investigate the regulatory effect of IL-17A on M2 macrophage polarization and the underlying mechanism in cervical cancer development.MethodsRT-qPCR was utilized for testing IL-17A expression in cancer tissues and cells. Flow cytometry was applied to evaluate the M1 or M2 macrophage polarization. Cell proliferative, migratory, and invasive capabilities were measured through colony formation and transwell assays. ChIP and luciferase reporter assays were applied to determine the interaction between IL-17A and octamer-binding transcription factor 4 (OCT4).ResultsIL-17A expression and concentration were high in metastatic tissues and cells of cervical cancer. IL-17A was found to facilitate M2 macrophage polarization in cervical cancer. Furthermore, IL-17A facilitated the macrophage-mediated promotion of cervical cancer cell proliferative, migratory, and invasive capabilities. Mechanistic assays manifested that Oct4 binds to and transcriptionally activated IL-17A in cervical cancer cells. Furthermore, Oct4 promoted cervical cancer cell malignant phenotype and M2 macrophage polarization by activating the p38 pathway that, in turn, upregulated IL-17A. Additionally, in vivo experiments confirmed that Oct4 knockdown reduced tumor growth and metastasis. ConclusionOct4 triggers IL-17A to facilitate the polarization of M2 macrophages, which promotes cervical cancer cell metastasis.

Toxic effect and mechanism of Tripterygium glycosides on ovarian germline stem cells of mice in vitro by Notch signaling pathway

The ovarian germline stem cells(OGSCs) cultured in the optimized culture system were used as the research object to observe the effect of Tripterygium glycosides(TG) on OGSCs and explore the mechanism of reproductive toxicity by the Notch signaling pathway. Cell counting kit-8(CCK-8) was used to observe the viability level of OGSCs in mice cultured in vitro by TG of 3.75, 7.5, and 15 μg·mL~(-1). Immunofluorescence technology and reverse transcription-polymerase chain reaction(RT-PCR) were used to detect the protein and gene expression level of OGSCs marker mouse vasa homologue(MVH) and octamer-binding transcription factor 4(Oct4) by TG of 3.75 μg·mL~(-1). RT-PCR detected the gene expression of neurogenic locus Notch homolog protein 1(Notch1), Hes family BHLH transcription factor 1(Hes1), and jagged canonical Notch ligand 1(Jagged1). The RNA was extracted for transcriptome analysis to analyze the mechanism of action of TG intervention on OGSCs. 3.75 μg·mL~(-1) of TG was combined with 40 ng·mL~(-1) Notch signaling pathway γ-secretagocin agonist jagged canonical notch ligand(Jagged) for administration. CCK-8 was used to detect the viability level of OGSCs. Double immunofluorescence technology was used to detect the protein co-expression of MVH with Hes1, Notch1, and Jagged1. The results showed that compared with the blank group, the TG administration group significantly inhibited the activity of OGSCs(P&lt;0.01 or P&lt;0.001). It could reduce the protein and gene expression of OGSC markers, namely MVH and Oct4(P&lt;0.05, P&lt;0.01, or P&lt;0.001). It could significantly inhibit the gene expression of Notch1, Hes1, and Jagged1(P&lt;0.001). Transcriptomic analysis showed that TG affected the growth and proliferation of OGSCs by intervening Jagged1, a ligand associated with the Notch signaling pathway. The experimental results showed that the combination of Notch signaling pathway γ-secretagorein agonist Jagged could significantly alleviate the decrease in OGSC viability induced by TG(P&lt;0.001) and significantly increased the OGSC viability compared with the TG group(P&lt;0.001). It also could significantly increase the co-expression of MVH/Jagged1, MVH/Hes1, and MVH/Notch1 proteins(P&lt;0.01 or P&lt;0.001). It suggested that TG play the role of γ-secretagorease inhibitors by downregulating the OGSC markers including MVH and Oct4 and Notch signaling pathway molecules such as Notch1, Hes1, and Jagged1, participate in the OGSC pathway, and mediate reproductive toxicity caused by the Notch signaling pathway.

Role of sodium/iodide symporter overexpression in inhibiting thyroid cancer cell invasion and stem cell maintenance by inhibiting the β-catenin/LEF-1 pathway

BackgroundIn thyroid cancers, a reduction in the expression of the sodium/iodide symporter (NIS) is observed concomitant with a diminution in cancer cell differentiation. The β-catenin/LEF-1 pathway emerges as a crucial regulatory pathway influencing the functional expression of NIS in human thyroid cancer cells. Further research is required to comprehensively elucidate the role of NIS overexpression in impeding the progression of thyroid cancer cells. MethodsHuman papillary thyroid carcinoma (PTC) cell lines, specifically PTC-1 and KTC-1, were subjected to Scratch and Transwell assays, colony formation, and tumor sphere formation tests to investigate invasion and migration, focusing on the impact of NIS overexpression. The assessment involved the use of western blot to analyze the expression levels of β-catenin, NIS, CD133, SRY-related HMG box2 (Sox2), lymphoid enhancer-binding factor 1 (LEF-1), NANOG, octamer-binding transcription factor 4 (Oct4), aldehyde dehydrogenase 1 family, member A1 (ALDH1A1), and epithelial cellular adhesion molecule (EpCAM). Statistical analysis was conducted using SPSS version 20.0, and the graphs were developed using GraphPad Prism 7 (GraphPad Software, Inc.). ResultsOur observations revealed that Nthy-ori-3-1 cell lines exhibited notably higher average expression levels of NIS, yet significantly lower levels of LEF-1 and β-catenin compared to PTC-1 and KTC-1 cell lines. Furthermore, the overexpression of β-catenin resulted in reduced binding of LEF-1 to NIF promotion but concurrently increased the expression of NIS. The downregulation of NIS markedly enhanced the expression of ALDH1A1, CD133, OCT4, Nanog, SOX2, and EpCam—all of which are targets within the Wnt/β-catenin signaling pathway. Conversely, the upregulation of NIS suppressed the expression of these proteins. Moreover, cells treated with β-catenin activators demonstrated an increased capability to form more spheroids and displayed heightened aggressiveness. Conversely, the NIS overexpression (OE) group exhibited suppressed abilities in invasion and colony formation. ConclusionThyroid cancer cells exhibit diminished expression of NIS, and the invasion and maintenance of stem cells in thyroid cancer cells were hindered by NIS OE through the inhibition of the β-catenin/LEF-1 pathway. Further research is warranted to comprehensively assess this outcome, which holds promise as a potential targeted treatment for thyroid cancer.

Cancer stem cell biomarkers SOX2 and Oct4 in cervical cancer patients undergoing chemoradiotherapy.

Cancer stem cell biomarkers SRY (sex-determining region Y)-box 2 (SOX2) and octamer-binding transcription factor 4 (Oct4) account for radioresistance in cervical squamous cell cancers (CSCCs). Their clinical implications are limited and contradictory. In this prospective cohort study, we recruited patients with FIGO IB2-IVA CSCC treated with primary chemoradiotherapy on regular follow-up. Tissue biopsy specimens were evaluated for SOX2 and Oct4 expression by immunohistochemistry, quantified by a product of proportion and intensity scores. A total of 59 patients were included. Most had a moderately differentiated (81%), keratinizing (59%) CSCC, and ≥FIGO stage IIB disease (95%). SOX2 expression (high:low 21:38 patients) and Oct4 expression (high:low 4:55 patients) had a significant interrelation (p=0.005, odds ratio (95% CI) - 1.23 (1.004-1.520)). At a median follow-up of 36 months, the 3-year overall survival (OS) was 60% and 53% for low and high SOX2 expression (p=0.856), and 54% and 100% for low and high Oct4 expression (p=0.114). The 3-year disease-frese survival (DFS) was 65% and 50% in the low and high SOX2 expression (p=0.259), and 59% and 75% for low and high Oct4 expression (p=0.598). SOX2 expression was the only variable significantly associated with a lower OS and DFS on regression analysis. Our study demonstrated a trend toward improved OS and DFS with low SOX2 and high Oct4 expression in CSCC patients undergoing chemoradiotherapy.

Effect of Octamer-Binding Transcription Factor 4 Overexpression on the Neural Induction of Human Dental Pulp Stem Cells

Stem cell-based therapy is a potential alternative strategy for brain repair, with neural stem cells (NSC) presenting as the most promising candidates. Obtaining sufficient quantities of NSC for clinical applications is challenging, therefore alternative cell types, such as neural crest-derived dental pulp stem cells (DPSC), may be considered. Human DPSC possess neurogenic potential, exerting positive effects in the damaged brain through paracrine effects. However, a method for conversion of DPSC into NSC has yet to be developed. Here, overexpression of octamer-binding transcription factor 4 (OCT4) in combination with neural inductive conditions was used to reprogram human DPSC along the neural lineage. The reprogrammed DPSC demonstrated a neuronal-like phenotype, with increased expression levels of neural markers, limited capacity for sphere formation, and enhanced neuronal but not glial differentiation. Transcriptomic analysis further highlighted the expression of genes associated with neural and neuronal functions. In vivo analysis using a developmental avian model showed that implanted DPSC survived in the developing central nervous system and respond to endogenous signals, displaying neuronal phenotypes. Therefore, OCT4 enhances the neural potential of DPSC, which exhibited characteristics aligning with neuronal progenitors. This method can be used to standardise DPSC neural induction and provide an alternative source of neural cell types.Graphical

In vivo study of the effect of anlotinib on the stemness of the lenvatinib-resistant hepatocellular carcinoma cells and the underlying mechanisms

Abstract Background In vivo experiments were conducted to examine the effects of the targeted drug anlotinib on the stemness of hepatocellular carcinoma (HCC) cells and lenvatinib-resistant liver cancer cells and to explore the underlying molecular mechanisms. Methods A subcutaneous xenograft model of Hep3B-derived HCC was established in nude mice, which were randomly divided into 2 groups (n = 5 males per group): (1) intragastric administration of anlotinib (0.4 mg/kg) and (2) intragastric administration of normal saline. We constructed lenvatinib-resistant cell lines and randomly divided the mice into 3 groups (n = 5 males per group): (1) intragastric administration of anlotinib, (2) intragastric administration of lenvatinib, and (3) intragastric administration of normal saline. After 2 weeks of treatment, tumor tissues were harvested, and mRNA and proteins were isolated from the tissues. Changes in the expression of cancer stemness markers (epithelial cell adhesion molecule [EpCAM], CD13, CD90, aldehyde dehydrogenase 1 [ALDH1], CD44, and CD45), totipotency factors (sex-determining region Y-box 2 [Sox2], Nanog, octamer-binding transcription factor 4 [Oct4]), and genes related to the Notch signaling pathway were examined. Results Compared with that in the control group, tumor size and weight were reduced in nude mice treated with anlotinib. These differences were statistically significant in both the types of nude mice. Anlotinib affected stemness markers and totipotency factors by downregulating the expression of CD133, CD90, and G-protein–coupled receptor 5 (LGR5) and upregulating the expression of intercellular adhesion molecule 1 (ICAM-1) and Sox2. In addition, lenvatinib-resistant cell lines increased Notch signaling pathway, whereas anlotinib inhibited Notch signaling pathway. Conclusions The antitumor effect of anlotinib on HCC and lenvatinib-resistant HCC cells may occur through inhibition of the Notch signaling pathway. Anlotinib may be the drug of choice for sequential therapy in lenvatinib-resistant liver cancer.

A positive feedback between PDIA3P1 and OCT4 promotes the cancer stem cell properties of esophageal squamous cell carcinoma

BackgroundIncreasing evidence has indicated that long non-coding RNAs (lncRNAs) have been proven to regulate esophageal cancer progression. The lncRNA protein disulfide isomerase family A member 3 pseudogene 1 (PDIA3P1) has been shown to promote cancer stem cell properties; however, its mechanism of action remains unclear. In this study, we investigated the regulation of esophageal cancer stem cell properties by the interaction of PDIA3P1 with proteins.MethodsThe GEPIA2 and Gene Expression Omnibus databases were used to analyze gene expression. PDIA3P1 expression in human esophageal squamous cell carcinoma (ESCC) tissues and cell lines was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Loss-of-function experiments were performed to determine the effects of PDIA3P1 on ESCC cell proliferation, migration, and invasion. The sphere formation assay, number of side population cells, and CD271 + /CD44 + cells were detected by flow cytometry to identify the cancer stem cell properties. RNA immunoprecipitation (RIP), RNA pull-down, co-immunoprecipitation (co-IP), dual luciferase reporter, and cleavage under targets and tagmentation (CUT&Tag) assays were performed to elucidate the underlying molecular mechanisms.ResultsPDIA3P1 expression was upregulated in ESCC cell lines and tissues. Functionally, higher PDIA3P1 expression promoted cell proliferation, invasion, and metastasis and inhibited apoptosis in esophageal cancer. Importantly, PDIA3P1 promoted cancer stem cell properties in ESCC. Mechanistically, PDIA3P1 interacted with and stabilized octamer-binding transcription factor 4 (OCT4) by eliminating its ubiquitination by the ubiquitinating enzyme WW domain-containing protein 2 (WWP2). Moreover, as a transcription factor, OCT4 bound to the PDIA3P1 promoter and promoted its transcription.ConclusionsOur research revealed a novel mechanism by which a positive feedback loop exists between PDIA3P1 and OCT4. It also demonstrated that the PDIA3P1-WWP2-OCT4 loop is beneficial for promoting the cancer stem cell properties of ESCC. Owing to this regulatory relationship, the PDIA3P1-WWP2-OCT4-positive feedback loop might be used in the diagnosis and prognosis, as well as in the development of novel therapeutics for esophageal cancer.

Trk Inhibition Reduces Tumorsphere Formation and Changes Expression of Stemness Markers in SK-ES-1 Ewing Sarcoma Cells

Introduction: Ewing sarcoma (ES) is a highly aggressive type of childhood cancer characterized by a chromosomal translocation resulting in fusions between the gene encoding EWS RNA Binding Protein 1 (EWSR1) and one gene of the ETS family, most frequently FLI-1, resulting in the EWS-FLI1 aberrant transcription factor. ES tumors can contain a subpopulation of cells showing cancer stem cell (CSC) features, which express stemness markers including CD133, OCT4 (Octamer-binding transcription factor 4), and NANOG, and display capacity to form tumorspheres likely enriched in CSCs. Neurotrophin (NT) receptors of the tropomyosin receptor kinase (Trk) family (TrkA, TrkB, and TrkC) may play a role in stimulating ES progression, but their possible role in CSCs remains unknown. Objective: To verify the effect of Trks inhibition on the formation of tumorspheres as well as the gene expression of stem markers. Method: The cells were dissociated and the formation of spheres was induced with supplemented culture medium and the K252a treatment was performed. After RNA extraction, mRNA expression levels of target genes Prom1 (CD133), OCT4 (POU5F1), SOX2, and Musashi-1 (MSI1) were analyzed by qPCR. Results: The pan-Trk inhibitor K252a (100 or 500 mM) hindered tumorsphere formation in human SK-ES-1 ES cell cultures. K252a also reduced mRNA expression of Prom1 (CD133-coding gene) while enhancing expression of OCT4. No changes in mRNA levels of SOX2 or Musashi-1 were observed. Conclusion: These findings provide the first evidence suggesting that Trk activity can influence stemness in ES cells.

Polyploid giant cancer cells induced by Docetaxel exhibit a senescence phenotype with the expression of stem cell markers in ovarian cancer cells.

Docetaxel (Doc) plays a crucial role in clinical antineoplastic practice. However, it is continuously documented that tumors frequently develop chemoresistance and relapse, which may be related to polyploid giant cancer cells (PGCCs). The aim of this study was investigate the formation mechanism and biological behavior of PGCCs induced by Doc. Ovarian cancer cells were treated with Doc, and then the effect of Doc on cellular viability was evaluated by MTT assay and microscopic imaging analysis. The biological properties of PGCCs were further evaluated by Hoechst 33342 staining, cell cycle and DNA content assay, DNA damage response (DDR) signaling detection, β-galactosidase staining, mitochondrial membrane potential detection, and reverse transcription-quantitative polymerase chain reaction. The results indicated that Doc reduced cellular viability; however, many cells were still alive, and were giant and polyploid. Doc increased the proportion of cells stayed in the G2/M phase and reduced the number of cells. In addition, the expression of γ-H2A.X was constantly increased after Doc treatment. PGCCs showed senescence-associated β-galactosidase activity and an increase in the monomeric form of JC-1. The mRNA level of octamer-binding transcription factor 4 (OCT4) and krüppel-like factor 4 (KLF4) was significantly increased in PGCCs. Taken together, our results suggest that Doc induces G2/M cell cycle arrest, inhibits the proliferation and activates persistent DDR signaling to promote the formation of PGCCs. Importantly, PGCCs exhibit a senescence phenotype and express stem cell markers.

Acoustic vibration promotes in vitro expansion of human embryonic stem cells.

This study aimed to investigate the effect of acoustic vibration on the pluripotency of human embryonic stem cells (hESCs) and evaluate cell proliferation and self-renewal ability post-treatment. The human ES cell line H1 was used for the experiments. hESCs were treated with an acoustic vibration device. Their proliferative ability was subsequently detected using a colony formation assay, while the expression of pluripotency-related markers was detected via immunofluorescence staining. Finally, changes in gene expression levels were examined using quantitative polymerase chain reaction (qPCR) in the presence of appropriate primers. Compared with normal cells in the control group, the morphology of experimental cells subjected to acoustic vibration did not significantly change. Contrastingly, the colony-forming efficiency of the experimental cells significantly increased. Immunofluorescence staining results showed the cells in experimental group were positive for the pluripotency markers NANOG, octamer-binding transcription factor 4 gene (OCT4), and SRY (sex determining region Y)-box 2 (SOX2). In addition, the expression levels of pluripotency genes NANOG, OCT4, SOX2, and Yes-associated protein (YAP)-related genes were up-regulated following acoustic vibration. Our results revealed that acoustic vibration enhanced the proliferative ability of hESCs and increased the expression levels of NANOG, OCT4, SOX2, and YAP-related genes, indicating that acoustic vibration can optimize the self-renewal ability of hESCs and that the YAP signaling pathway may play a critical role in the functional process of acoustic vibration.

Angiotensin-(1-7) Improves Islet β-cell Dedifferentiation by Activating PI3K/Akt/FoxO1 Pathway.

Islet β-cell dedifferentiation may be the main cause of reduced insulin secretion. Angiotensin-(1-7) [Ang-(1-7)] can attenuate high glucose-induced apoptosis and dedifferentiation of pancreatic β-cell, but the specific signal transduction pathway and mechanism are not yet clear. This study aimed to investigate the effects of Ang-(1-7) on high glucose-induced islet β-cell dedifferentiation by activating the phosphatidylinositol-3-kinase/Protein kinase B/ Forkhead box transcription factor O1 (PI3K/Akt/FoxO1) signaling pathway. The mouse islet β-cell line MIN6 cells were passaged and cultured and randomly divided into five groups: control (Con) group, high glucose (HG) group, HG with Ang-(1-7) group, HG with Ang-(1-7) and specific MasR antagonist A-779 group, and HG with Ang-(1-7) and PI3K inhibitor LY294002 group. After 48 hours, glucose-stimulated insulin secretion (GSIS) was detected by Enzyme-Linked Immunosorbent Assay (ELISA). The mRNA and protein expression levels of β-cell-specific factors (Pancreatic duodenal homeobox-1 (Pdx1), v-maf musculoaponeurotic fibrosarcoma oncogene homolog A(MafA)) and endocrine progenitor cell-specific factors (Octamer binding transcription factor 4(Oct4), Nanog) were measured by Real Time-PCR and Western blot. The factors of protein expression levels of PI3K/Akt/FoxO1 signaling pathway (Akt, p-Akt, Fox- O1, p-FoxO1) were determined by Western blot. We observed for the first time that high glucotoxicity can induce dedifferentiation of pancreatic islet β-cell, causing a decrease in insulin secretion levels and expression of Pdx1, MafA, p-- FoxO1, and p-Akt and an increase in expression of Oct4 and Nanog. After Ang-(1-7) intervention, insulin secretion levels and expression of Pdx1, MafA, p-FoxO1 and p-Akt were increased, and the levels of Oct4 and Nanog were reduced. However, A-779 and LY294002 could reverse this effect. During these processes, the total Akt and total FoxO1 expression did not change significantly. Ang-(1-7) may prevent high glucose-induced pathological dedifferentiation of pancreatic β-cell by activating the PI3K/Akt/FoxO1 signaling pathway.

A paradigm for post-embryonic Oct4 re-expression: E7-induced hydroxymethylation regulates Oct4 expression in cervical cancer.

The Octamer-binding transcription factor-4 (Oct4) is upregulated in different malignancies, yet a paradigm for mechanisms of Oct4 post-embryonic re-expression is inadequately understood. In cervical cancer, Oct4 expression is higher in human papillomavirus(HPV)-related than HPV-unrelated cervical cancers and this upregulation correlates with the expression of the E7 oncogene. We have reported that E7 affects the Oct4-transcriptional output and Oct4-related phenotypes in cervical cancer, however, the underlying mechanism remains elusive. Here, we characterize the Oct4-protein interactions in cervical cancer cells via computational analyses and Mass Spectrometry and reveal that Methyl-binding proteins (MBD2 and MBD3), are determinants of Oct4-driven transcription. E7 triggers MBD2 downregulation and TET1 upregulation, thereby disrupting the methylation status of the Oct4 gene. This coincides with an increase in the total DNA hydroxymethylation leading to the re-expression of Oct4 in cervical cancer and likely affecting broader transcriptional patterns. Our findings reveal a previously unreported mechanism by which the E7 oncogene can regulate Oct4 re-expression and global transcriptional patterns by increasing DNA hydroxymethylation and lowering the barrier to cellular plasticity during carcinogenesis.

Crosslinked modified decellularized rabbit conjunctival stroma for reconstruction of tissue-engineered conjunctiva in vitro

Conjunctival reconstruction is an essential part of ocular surface restoration, especially in severe conjunctival disorders. Decellularized conjunctival tissues have been used in tissue engineering. In this study, we investigated the feasibility of constructing tissue-engineered conjunctiva using stem cell (human amniotic epithelial cells, hAECs), and cross-linked modified decellularized rabbit conjunctival stroma (DRCS-Asp-hEGF), and decellularized rabbit conjunctiva stroma (DRCS). With phospholipase A2 and sodium dodecyl, DRCS were nearly DNA-free, structurally intact and showed no cytotoxic effects in vitro, as confirmed by DNA quantification, histology, and immunofluorescence. The results of Fourier transform infrared, Alcian blue staining and human epidermal growth factor (hEGF) release assays showed that DRCS-Asp-hEGF was successfully prepared via crosslinking with aspartic acid (Asp) and modified by hEGF at pH 7.7. The hAECs were positive for octamer-binding transcription factor-4 and ABCG2 cell markers. The hAECs were directly placed on the DRCS and DRCS-Asp-hEGF for five days respectively. Tissue-engineered conjunctiva was constructed in vitro for five days, and the fluorescence staining results showed that hAECs grew in monolayers on DRCS-Asp-hEGF and DRCS. Flow cytometry results showed that compared with DRCS, the number of apoptotic cells stained in DRCS-Asp-hEGF was small, 86.70 ± 0.79% of the cells survived, and 87.59 ± 1.43% of the cells were in the G1 phase of DNA synthesis. Electron microscopy results showed that desmosome junction structures, which were similar to the native conjunctival tissue, were formed between cells and the matrix in the DRCS-Asp-hEGF.

Angiotensin (1-7) reverses glucose-induced islet β cell dedifferentiation by Wnt/β-catenin/FoxO1 signalling pathway.

Recent studies have shown that a decline in isletβ cells quality is due to β-cell dedifferentiation, not only β-cell apoptosis. Angiotensin (1-7) [Ang(1-7)] could attenuate high glucose-induced apoptosis and dedifferentiation of pancreaticβ cells by combining with MAS receptors. However, the mechanism of such action has not been elucidated. Recent studies have revealed that Wnt/β-catenin and forkhead box transcription factor O1 (FoxO1) are associated with β-cell dedifferentiation. Our study aims to explore whether the effects of Ang(1-7)on islet b cell dedifferentiation are mediated through the Wnt/β-catenin/FoxO1 pathway. Isletβ cells were divided into 6 groups: a control group, a high-glucose group, high glucose with Ang(1-7) group, high-glucose with Ang(1-7) and A779 group, high-glucose with angiotensin(1-7) and CHIR99021 group, and high-glucose with CHIR99021 group. A779 is a kind of MAS receptor antagonist that blocks the action of Ang(1-7), and CHIR99021 is a Wnt pathway activator. The morphology of pancreaticβ cells was observed in each group after 48 hours of intervention. β-cell insulin secretory function and expressions of relevant factors were measured. Compared with the control group, the cell morphology became degraded in the high-glucose group and the capability of insulin secretion was reduced. Meanwhile, the expressions of matureβ cells markers [pancreatic and duodenal homeobox 1 (Pdx1) and MAF BZIP transcription factor A (MafA)] were reduced, while the expressions of endocrine progenitor cells makers [octamer-binding transcription factor 4 (Oct4) and Nanog] were increased. The addition of CHIR99021 resulted in profound deep destruction ofβ cells compared with the high-glucose group. However, such changes were dramatically reversed following the treatment of Ang(1-7). The addition of A779 significantly inhibited the improvement caused by Ang(1-7). Ang(1-7) can effectively reverseβ cell dedifferentiation through Wnt/β-catenin/FoxO1 pathway. It might be a new strategy for preventing and treating diabetes.

Seminoma and dysgerminoma: evidence for alignment of clinical trials and de-escalation of systemic chemotherapy.

Malignant germ cell tumours are a group of rare cancers whose incidence peaks in late adolescence and early adulthood. Dysgerminomas of the ovary and seminomas of the testis are analogous diseases, but seminomas have a 10-fold higher incidence. The two tumours are morphologically identical and are only differentiated by surrounding organ-specific tissue or testicular germ cell neoplasia in situ. They share genetic features including KIT and RAS mutations, amplification of chromosome 12p, and expression of pluripotency markers (NANOG (Nanog homeobox), OCT3/4 (Octamer-binding transcription factor 3/4), and SAL4 (Spalt-like trascription factor 4)). Both histologies are exquisitely sensitive to platinum chemotherapy, and the combination of bleomycin, etoposide, and cisplatin (BEP) yields survival rates greater than 90%. However, BEP causes significant, lifelong toxicity (cardiovascular, renal, respiratory, and neurological) in these young patients with an expectation of cure. Here, we comprehensively review the biological features of dysgerminoma and seminoma to demonstrate that they are biologically analogous diseases. We present available clinical trial data supporting de-escalation of chemotherapy treatment. Finally, we propose that future trials should enrol men, women, and children to benefit all patients regardless of age or sex.

Rapid responses of human pluripotent stem cells to cyclic mechanical strains applied to integrin by acoustic tweezing cytometry

Acoustic tweezing cytometry (ATC) is an ultrasound-based biophysical technique that has shown the capability to promote differentiation of human pluripotent stem cells (hPSCs). This study systematically examined how hPSCs respond to cyclic mechanical strains applied by ATC via displacement of integrin-bound microbubbles (averaged diameter of 4.3 µm) using ultrasound pulses (acoustic pressure 0.034 MPa, center frequency 1.24 MHz and pulse repetition frequency 1 Hz). Our data show downregulation of pluripotency marker Octamer-binding transcription factor 4 (OCT4) by at least 10% and increased nuclear localization of Yes-associated protein (YAP) by almost 100% in hPSCs immediately after ATC application for as short as 1 min and 5 min respectively. Analysis of the movements of integrin-anchored microbubbles under ATC stimulations reveals different stages of viscoelastic characteristic behavior and increasing deformation of the integrin-cytoskeleton (CSK) linkage. The peak displacement of integrin-bound microbubbles increased from 1.45 ± 0.16 to 4.74 ± 0.67 μm as the duty cycle of ultrasound pulses increased from 5% to 50% or the duration of each ultrasound pulse increased from 0.05 to 0.5 s. Real-time tracking of integrin-bound microbubbles during ATC application detects high correlation of microbubble displacements with OCT4 downregulation in hPSCs. Together, our data showing fast downregulation of OCT4 in hPSCs in respond to ATC stimulations highlight the unique mechanosensitivity of hPSCs to integrin-targeted cyclic force/strain dependent on the pulse duration or duty cycle of ultrasound pulses, providing insights into the mechanism of ATC-induced accelerated differentiation of hPSCs.

Dauricine attenuates Oct4/sonic hedgehog co-activated stemness and induces reactive oxygen species-mediated mitochondrial apoptosis via AKT/β-catenin signaling in human neuroblastoma and glioblastoma stem-like cells.

Neuroblastoma and glioblastoma are primary malignant tumors of the nervous system, with frequent relapse and limited clinical therapeutic drugs. The failure of their treatment is due to the tumor cells exhibiting cancer stem-like cells (CSLCs) properties. Octamer binding transcription factor 4 (Oct4) is involved in mediating CSLCs, our previous work found that Oct4-driven reprogramming of astrocytes into induced neural stem cells was potentiated with continuous sonic hedgehog (Shh) stimulation. In this study, we aimed to study the importance of Oct4 and Shh combination in the stemness properties induction of neuroblastoma and glioblastoma cells, and evaluate the anti-stemness effect of dauricine (DAU), a natural product of bis-benzylisoquinoline alkaloid. The effect of Oct4 and Shh co-activation on cancer stemness was evaluated by tumor spheres formation model and flow cytometry analysis. Then the effects of DAU on SH-SY5Y and T98-G cells were assessed by the MTT, colony formation, and tumor spheres formation model. DAU acts on Oct4 were verified using the Western blotting, MTT, and so on. Mechanistic studies were explored by siRNA transfection assay, Western blotting, and flow cytometry analysis. We identified that Shh effectively improved Oct4-mediated generation of stemness in SH-SY5Y and T98-G cells, and Oct4 and Shh co-activation promoted cell growth, the resistance of apoptosis. In addition, DAU, a natural product, was found to be able to attenuate Oct4/Shh co-activated stemness and induce cell cycle arrest and apoptosis via blocking AKT/β-catenin signaling in neuroblastoma and glioblastoma, which contributed to the neuroblastoma and glioblastoma cells growth inhibition by DAU. In summary, our results indicated that the treatment of DAU may be served as a potential therapeutic method in neuroblastoma and glioblastoma.