Objective: To investigate the expression of programmed death protein-ligand 1 (PD-L1) in liver cancer stem-like cells (LCSLC) and its effect on the characteristics of tumor stem cells and tumor biological function, to explore the upstream signaling pathway regulating PD-L1 expression in LCSLC and the downstream molecular mechanism of PD-L1 regulating stem cell characteristics, also tumor biological functions. Methods: HepG2 was cultured by sphere-formating method to obtain LCSLC. The expressions of CD133 and other stemness markers were detected by flow cytometry, western blot and real-time quantitative polymerase chain reaction (RT-qPCR) were used to detect the expressions of stemness markers and PD-L1. The biological functions of the LCSLC were tested by cell function assays, to confirm that the LCSLC has the characteristics of tumor stem cells. LCSLC was treated with cell signaling pathway inhibitors to identify relevant upstream signaling pathways mediating PD-L1 expression changes. The expression of PD-L1 in LCSLC was down regulated by small interfering RNA (siRNA), the expression of stem cell markers, tumor biological functions of LCSLC, and the changes of cell signaling pathways were detected. Results: Compared with HepG2 cells, the expression rate of CD133 in LCSLC was upregulated [(92.78±6.91)% and (1.40±1.77)%, P<0.001], the expressions of CD133, Nanog, Oct4A and Snail in LCSLC were also higher than those in HepG2 cells (P<0.05), the number of sphere-formating cells increased on day 7 [(395.30±54.05) and (124.70±19.30), P=0.001], cell migration rate increased [(35.41±6.78)% and (10.89±4.34)%, P=0.006], the number of transmembrane cells increased [(75.77±10.85) and (20.00±7.94), P=0.002], the number of cloned cells increased [(120.00±29.51) and (62.67±16.77), P=0.043]. Cell cycle experiments showed that LCSLC had significantly more cells in the G(0)/G(1) phase than those in HepG2 [(54.89±3.27) and (32.36±1.50), P<0.001]. The tumor formation experiment of mice showed that the weight of transplanted tumor in LCSLC group was (1.32±0.17)g, the volume is (1 779.0±200.2) mm(3), were higher than those of HepG2 cell [(0.31±0.06)g and (645.6±154.9)mm(3), P<0.001]. The expression level of PD-L1 protein in LCSLC was 1.88±0.52 and mRNA expression level was 2.53±0.62, both of which were higher than those of HepG2 cells (P<0.05). The expression levels of phosphorylation signal transduction and transcription activation factor 3 (p-STAT3) and p-Akt in LCSLC were higher than those in HepG2 cells (P<0.05). After the expression of p-STAT3 and p-Akt was down-regulated by inhibitor treatment, the expression of PD-L1 was also down-regulated (P<0.05). In contrast, the expression level of phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) in LCSLC was lower than that in HepG2 cells (P<0.01), there was no significant change in PD-L1 expression after down-regulated by inhibitor treatment (P>0.05). After the expression of PD-L1 was knockdown by siRNA, the expressions of CD133, Nanog, Oct4A and Snail in LCSLC were decreased compared with those of siRNA-negative control (NC) group (P<0.05). The number of sphere-formating cells decreased [(45.33±12.01) and (282.00±29.21), P<0.001], the cell migration rate was lower than that in siRNA-NC group [(20.86±2.74)% and (46.73±15.43)%, P=0.046], the number of transmembrane cells decreased [(39.67±1.53) and (102.70±11.59), P=0.001], the number of cloned cells decreased [(57.67±14.57) and (120.70±15.04), P=0.007], the number of cells in G(0)/G(1) phase decreased [(37.68±2.51) and (57.27±0.92), P<0.001], the number of cells in S phase was more than that in siRNA-NC group [(30.78±0.52) and (15.52±0.83), P<0.001]. Tumor formation in mice showed that the tumor weight of shRNA-PD-L1 group was (0.47±0.12)g, the volume is (761.3±221.4)mm(3), were lower than those of shRNA-NC group [(1.57±0.45)g and (1 829.0±218.3)mm(3), P<0.001]. Meanwhile, the expression levels of p-STAT3 and p-Akt in siRNA-PD-L1 group were decreased (P<0.05), while the expression levels of p-ERK1/2 and β-catenin did not change significantly (P>0.05). Conclusion: Elevated PD-L1 expression in CD133(+) LCSLC is crucial to maintain stemness and promotes the tumor biological function of LCSLC.
Humans ; Animals ; Mice ; Proto-Oncogene Proteins c-akt/metabolism* ; B7-H1 Antigen/metabolism* ; Ligands ; Liver Neoplasms/pathology* ; RNA, Small Interfering/metabolism* ; Neoplastic Stem Cells/physiology* ; Cell Line, Tumor ; Cell Proliferation

OBJECTIVE: To investigate the effect of Piwil2-induced cancer stem-like cell (Piwil2-iCSC)-derived exosomes on the proliferation,migration and invasion of human umbilical cord blood-derived mesenchymal stem cells (hucMSCs). METHODS: Piwil2-iCSC-derived exosomes were isolated by ultracentrifugation and identified using transmission electron microscopy,nanoparticle tracking analysis and Western blotting.Exosome uptake assay was used to identify the pathway that Piwil2-iCSCderived exosomes utilized.HucMSCs were divided into control group,PBS intervention group and exosome intervention group,and CCK-8 assay,wound healing assay,Transwell assay,Western blotting and cell karyotype analysis were used to observe the proliferation,migration,invasion,expression levels of MMP2 and MMP9 proteins,and chromosome structure of hucMSCs. RESULTS: The diameter of Piwil2-iCSC-derived exosomes ranged from 50 nm to 100 nm,and most of them were oval or spherical capsules rich in CD9,CD63 and Piwil2 proteins.Exosomal uptake assay showed that the exosomes executed theirs functions after entering the cells.Compared with the control cells and PBS-treated cells,hucMSCs treated with the exosomes showed significantly increased number of proliferating cells (<0.05) with accelerated healing rate (<0.05 at 24 h;<0.01 at 48 h),increased invasive cells (<0.01),enhanced protein expressions of MMP2(<0.05 PBS group;<0.01 control group) and MMP9(<0.05),but their karyotype still remained 46XY without any abnormalities. CONCLUSIONS Piwil2-iCSC-derived exosomes can promote the proliferation,migration and invasion but does not cause cancer-like heterogeneity changes in hucMSCs.
Argonaute Proteins ; Cell Movement ; physiology ; Cell Proliferation ; physiology ; Exosomes ; physiology ; Fetal Blood ; cytology ; Humans ; Karyotyping ; Mesenchymal Stem Cells ; pathology ; Neoplasm Invasiveness ; Neoplastic Stem Cells ; Umbilical Cord ; Wound Healing

To investigate the effect of apigenin on self-renewal for sphere-forming cells in human small cell lung cancer cell line NCI-H446 and the underlying mechanisms.
 Methods: Sphere-forming cells from NCI-H446 cell line were cultured in stem cell-conditioned culture medium with ultra-low attachment surface plates. The rate of sphere-forming cells in the second passage sphere-forming cells was used to evaluate the inhibitory effects of apigenin on the self-renewal for sphere-forming cells. The protein level of urokinase-type plasminogen activator receptor (uPAR) in spheroids was analyzed by Western blot.
 Results: Apigenin signifcantly inhibited the self-renewal of the second passage sphere-forming cells [0, 5.0, 10.0, 20.0 μmol/L apigenin: (18.2±1.9)%, (13.6±1.7)%, (10.6±1.6)%, (6.9±1.3)%, respectively] and down-regulated uPAR expression in a concentration-dependent manner (P<0.05).
 Conclusion: Apigenin inhibits the self-renewal capacity of sphere-forming cells in NCI-H446 cells, which may be associated with down-regulation of uPAR expression.
Apigenin ; pharmacology ; Cell Line, Tumor ; Down-Regulation ; drug effects ; genetics ; Humans ; Lung Neoplasms ; Neoplastic Stem Cells ; drug effects ; pathology ; physiology ; Receptors, Cell Surface ; Receptors, Urokinase Plasminogen Activator ; drug effects ; genetics ; metabolism ; Signal Transduction ; Small Cell Lung Carcinoma ; drug therapy ; pathology ; Spheroids, Cellular ; drug effects ; physiology ; Stem Cells

Epithelial mesenchymal transition (EMT) is a process of normal cell physiological development, in which epithelial cells transform into mesenchyme cells through a specific program. EMT plays a key role in inflammatory reaction, cell development, tumor invasion, and metastasis and has an interrelation with prostate cancer stem cells. Recent researches show the involvement of EMT in the development and metastasis of prostate cancer. This article reviews the specific roles and action mechanisms of EMT in the progression of prostate cancer.
Biomedical Research ; Cell Differentiation ; Disease Progression ; Epithelial Cells ; physiology ; Epithelial-Mesenchymal Transition ; physiology ; Humans ; Male ; Mesenchymal Stromal Cells ; Neoplastic Stem Cells ; physiology ; Prostatic Neoplasms ; pathology

Hepatocellular carcinoma (HCC) is one of leading causes of death in the world. Although various treatments have been developed, the therapeutic side effects are far from desirable. Chinese medicines (CMs, including plants, animal parts and minerals) have drawn a great deal of attention in recent years for their potential in the treatment of HCC. Most studies have shown that CMs may be able to retard HCC progression with multiple actions, either alone or in combination with other conventional therapies to improve quality of life in HCC patients. Additionally, CMs are used for preventing HCC occurrence. The aim of this study is to review the potential prophylactic and curative effects of CMs on human HCC and the possible mechanisms that underlie these pharmacological actions. Publications were collected and reviewed from PubMed and China National Knowledge Infrastructure from 2000 to 2014. Keywords for literature searches include "Chinese medicine", "Chinese herb", "traditional Chinese Medicine", "hepatocellular carcinoma" and "liver cancer". CMs in forms of pure compounds, isolated fractions, and composite formulas are included. Combination therapies are also considered. Both in vitro and in vivo efficacies of CMs are being discussed and the translational potential to bedside is to be discussed with clinical cases, which show the actions of CMs on HCC may include tumor growth inhibition, antimetastatic activities, anti-inflammation, anti-liver cancer stem cells, reversal on multi-drug resistance and induction/reduction of oxidative stress. Multiple types of molecules are found to contribute in the above actions. The review paper indicated that CMs might have potential to both prevent HCC occurrence and retard HCC progression with several molecular targets involved.
Carcinoma, Hepatocellular ; drug therapy ; prevention & control ; Drug Resistance, Multiple ; Humans ; Liver Neoplasms ; drug therapy ; prevention & control ; Medicine, Chinese Traditional ; NF-E2-Related Factor 2 ; physiology ; Neoplastic Stem Cells ; drug effects ; Reactive Oxygen Species ; metabolism

Stem cells are known to maintain stemness at least in part through secreted factors that promote stem-like phenotypes in resident cells. Accumulating evidence has clarified that stem cells release nano-vesicles, known as exosomes, which may serve as mediators of cell-to-cell communication and may potentially transmit stem cell phenotypes to recipient cells, facilitating stem cell maintenance, differentiation, self-renewal, and repair. It has become apparent that stem cell-derived exosomes mediate interactions among stromal elements, promote genetic instability in recipient cells, and induce malignant transformation. This review will therefore discuss the potential of stem cell-derived exosomes in the context of stromal remodeling and their ability to generate cancer-initiating cells in a tumor niche by inducing morphologic and functional differentiation of fibroblasts into tumor-initiating fibroblasts. In addition, the immunosuppressive potential of stem cell-derived exosomes in cancer immunotherapy and their prospective applications in cell-free therapies in future translational medicine is discussed.
Apoptosis ; Cell Communication ; Cell Transformation, Neoplastic ; Disease Progression ; Exosomes ; physiology ; Humans ; Immunotherapy ; methods ; Mesenchymal Stromal Cells ; physiology ; Neoplasms ; blood supply ; pathology ; therapy ; Neoplastic Stem Cells ; ultrastructure ; Neovascularization, Pathologic ; pathology ; Organelle Biogenesis ; Tumor Microenvironment

OBJECTIVE: To investigate the self-renewal mechanism of CD133+ cancer stem cells from Hep-2 cell line. METHOD: The CD133+ cells were sorted by flow cytometry from Hep-2 cell line. Then the sorted CD133+ cells were cultured in RPMI1640. The ability of self-renewal of CD133+ cells were tested by MTT assay. mRNA and protein expression of self-renewal related genes were detected by western blot and RT- PCR. RESULT: (3.10 ± 0.21)% of Hep-2 cells expressed the membrane antigen CD133. CD133+ fraction was raised to (90.20 ± 5.51)% by flow cytometry. In vitro culture and growth curve showed CD133+ cells had more active proliferation ability than CD133- cells, which showed statistically significant difference between these two group (P < 0.01). RT- PCR and western blot results showed upregulated mRNA and protein expression of Fas, c-myc, survivin in CD133+ group (P < 0.01). In the same time, the ratio of Bcl-2/Bax gene expression was obviously increased in CD133+ group. Self-renewal related gene such as β-catenin, SHH, SMOH and Bmi-1,Gli-1 were all up-regulated in CD133+ group both in mRNA and protein. On the contrary, PTCH gene was down-regulated. CONCLUSION CD133 positive cells are a small proportion of a Hep-2 cell line. The results of this experiment verified that CD133 positive cells owned the properties of cancer stem cells. Upregulated anti-apoptotic gene is the foundatiom of self-renewal mechanism of CD133+ cells. Cancer stem cells related signal pathways such as Hedgehog, Wnt and Bmi-1 pathway are in state of activation. The identification of self-renewal mechanism about cancer stem cell provides a powerful tool to investigate the tumorigenic process in the larynx and to develop therapies targeting to these signal pathways.
AC133 Antigen ; Antigens, CD ; Apoptosis ; Cell Physiological Phenomena ; physiology ; Down-Regulation ; Flow Cytometry ; Glycoproteins ; Humans ; Laryngeal Neoplasms ; Neoplastic Stem Cells ; physiology ; Patched Receptors ; Patched-1 Receptor ; Peptides ; Receptors, Cell Surface ; genetics ; metabolism ; Signal Transduction ; beta Catenin ; genetics

BACKGROUNDInfantile hemangioma (IH) is the most common benign tumor in children with prevalence in the face and neck. Various treatment options including oral propranolol have been described for IH, but the mechanism of drugs remains enigmatic. The aim of this study was to investigate the pathogenesis and establish a reliable in vivo model of IH which can provide platform for drug exploration.

METHODSStem cells from the proliferating hemangiomas (HemSCs) were isolated by CD133-tagged immunomagnetic beads. Their phenotype and angiogenic property were investigated by flow cytometry, culturing on Matrigel, real-time polymerase chain reaction (PCR), immunofluorescent staining and injection into BALB/c-nu mice.

RESULTSHemSCs had robust ability of proliferating and cloning. The time of cells doubling in proliferative phase was 16 hours. Flow cytometry showed that HemSCs expressed mesenchymal markers CD29, CD44, but not endothelial/hematopoietic marker of CD34 and hematopoietic marker CD45. The expression of CD105 was much lower than that of the reported hemangioma derived or normal mesenchymal stem cell (MSC). Real-time PCR showed that the mRNA levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and matrix metalloproteinase-1 (MMP-1) of HemSCs were higher than that of neonatal human dermal fibroblasts (NHDFs) and human umbilical vein endothelial cells (HUVECs). After HemSCs were cultured on Matrigel in vitro, they formed tube-like structure in a short time (16 hours) and differentiated into endothelial cells in 7 days. After 1 - 2 weeks of implantation into immunodeficient mice, HemSCs generated glucose transporter 1 positive blood vessels. When co-injected with HUVECs, the vascularization of HemSCs was greatly enhanced. However, the single implantation of HUVECs hardly formed blood vessels in BALB/c-nu mice (P < 0.05).

CONCLUSIONSHemSCs may be some kinds of primitive mesoderm derived stem cells with powerful angiogenic ability, which can recapitulate human hemangioma by co-injecting into immunodeficient mice with HUVECs.

AC133 Antigen ; Animals ; Antigens, CD ; analysis ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Collagen ; Disease Models, Animal ; Drug Combinations ; Glycoproteins ; analysis ; Hemangioma ; pathology ; Humans ; Laminin ; Male ; Mice ; Mice, Inbred BALB C ; Neoplastic Stem Cells ; chemistry ; pathology ; Peptides ; analysis ; Proteoglycans ; Vascular Endothelial Growth Factor A ; physiology

Cancer stem cells (CSCs) and angiogenesis play important roles in generation and development of malignant tumours. The number of researches concerned both of them is increasing rapidly and many impressive conclusions have been achieved based on recent studies. It is indicated that the CSCs have complicated interaction with the adjacent vascular microenvironment and they act on the disease progression together. CSCs may enhance angiogenesis while the vascular microenvironment has effects on maintenance and even induction of stemness, and new illustrations of mechanisms are constantly obtained. In this review, we summarize the current research status of mutual actions between CSCs and the vascular microenvironment, and also overview the latest progresses about relevant targeted therapies, to provide advisable information for future preclinical and clinical explorations.
Humans ; Neoplasms ; blood supply ; pathology ; Neoplastic Stem Cells ; pathology ; physiology ; Neovascularization, Pathologic ; pathology ; physiopathology ; Tumor Microenvironment

The identification and characterization of stem cells is a major focus of developmental biology and regenerative medicine. The advent of genetic inducible fate mapping techniques has made it possible to precisely label specific cell populations and to follow their progeny over time. When combined with advanced mathematical and statistical methods, stem cell division dynamics can be studied in new and exciting ways. Despite advances in a number of tissues, relatively little attention has been paid to stem cells in the oral epithelium. This review will focus on current knowledge about adult oral epithelial stem cells, paradigms in other epithelial stem cell systems that could facilitate new discoveries in this area and the potential roles of epithelial stem cells in oral disease.
Adult Stem Cells ; cytology ; physiology ; Animals ; Asymmetric Cell Division ; Biomarkers ; Cell Proliferation ; Clone Cells ; Epithelial Cells ; cytology ; Genetic Drift ; Humans ; Mouth Mucosa ; cytology ; Mouth Neoplasms ; pathology ; Neoplastic Stem Cells