Introduction:Sulfatase 2(SULF2),an endogenous extracellular sulfatase,can remove 6-O-sulfate groups of glucosamine residues from heparan sulfate(HS)chains to modulate the Wnt/β-catenin signaling pathway,which plays a...Introduction:Sulfatase 2(SULF2),an endogenous extracellular sulfatase,can remove 6-O-sulfate groups of glucosamine residues from heparan sulfate(HS)chains to modulate the Wnt/β-catenin signaling pathway,which plays an important role in both liver carcinogenesis and embryogenesis.Side population(SP)cells are widely identified as stem-like cancer cells and are closely related to carcinoma metastasis,recurrence,and poor patient prognosis.However,the roles of SULF2 in SP cells of hepatomas are unclear,and the underlying mechanism is undefined.Objectives:This study aimed to compare the heterogeneity between SP cells and non-side population(NSP)cells derived from three different liver cancer cell lines and to elucidate the involvement of the SULF2-Wnt/β-catenin axis in liver cancer stem cells(CSCs)and its impact on the processes of carcinogenesis and invasiveness.Methods:In this work,three different liver cancer SP cells(HepG2,Huh7,and PRC/PRL/5)were sorted by flow cytometry.We also examined the migration and invasion behaviors of SP and NSP cells.To determine if this high tumorigenic potential of SP cells is correlated to SULF2,qPCR,western blotting,and immunofluorescence analysis were conducted.We also performed nude mouse xenograft experiments for in vivo analysis.Results:The results from the in vitro colony formation assay showed that SP cells exhibited a 2-fold higher colony formation efficiency compared to their NSP counterparts.The SP cells exhibited significantly higher potentials in terms of their migratory capacity and invasive ability compared to NSP cells.We found that higher expression of SULF2 in SP cells was associated with greater capabilities for clonogenicity,migration,and invasion.It was also linked to higher activation of the Wnt/β-catenin signaling pathway via stimulation of key downstream factors,particularlyβ-catenin,c-Myc,and cyclin D1.Further,a positive correlation between the upregulated SULF2 expression and tumorigenesis in the in vivo nude mouse xenograft models was demonstrated,highlighting that the potential underlying mechanism was Wnt/β-catenin signaling pathway activation.Conclusion:Our findings show that variable SULF2 expression was associated with differential activation of the Wnt/β-catenin signaling pathway,which could lead to behavioral differences between SP and NSP cells and also among the SP cells of the three liver cancer cell lines assessed.It was reasonably concluded that the SULF2-Wnt/β-catenin axis could play an important role in the tumorigenicity of liver cancer stem cells.展开更多
AIM： To understand the role and significance of side population （SP） cells from hepatocellular carcinoma （HCC） in hepatocarcinogenesis, development, relapse and metastasis, we simulated the denutrition conditions...AIM： To understand the role and significance of side population （SP） cells from hepatocellular carcinoma （HCC） in hepatocarcinogenesis, development, relapse and metastasis, we simulated the denutrition conditions that cancer cells experience in clinical therapy, observed the different anti-apoptosis ability of SP cells and non-SP cells under such conditions, and established the possible effects of P53, Bcl-2 and Bax on survival of SP cells. METHODS： We used flow cytometry to analyze and sort the SP and non-SP cells in established HCC lines MHCC97 and hHCC. We evaluated cell proliferation by methyl thiazolyl tetrazolium （MTT） assay and investigated the expression of p53, bcl-2 and bax genes during denutrition, by RT-PCR and immunofluorescence staining. RESULTS： The percentage of SP cells in the two established HCC lines was 0.25% and 0.5%, respectively. SP cells had greater anti-apoptosis and proliferation ability than non-SP cells. Expression of Bcl-2 and Bax in SP and non-SP cells differed during denutrition. The former was up-regulated in SP cells, and the latter was up-regulated in non-SP cells. CONCLUSION： It may be that different upstream molecules acted and led to different expression levels of Bcl-2 and Bax in these two cell lines. There was a direct relationship between up-regulation of Bcl-2 and down-regulation of Bax and higher anti-apoptosis ability in SP cells. It may be that the existence and activity of SP cells are partly responsible for some of the clinical phenomena which are seen in HCC, such as relapse or metastasis. Further research on SP cells may have potential applications in the field of anticancer therapy.展开更多
AIM: To investigate the persistence of side population (SP) cells in pancreatic cancer and their role and mechanism in the drug resistance. METHODS: The presentation of side population cells in pancreatic cancer cell ...AIM: To investigate the persistence of side population (SP) cells in pancreatic cancer and their role and mechanism in the drug resistance. METHODS: The presentation of side population cells in pancreatic cancer cell line PANC-1 and its proportion change when cultured with Gemcitabine, was detected by Hoechst 33342 staining and FACS analysis. The expression of ABCB1 and ABCG2 was detected by real- time PCR in either SP cells or non-SP cells. RESULTS: SP cells do exist in PANC-1, with a median of 3.3% and a range of 2.1-8.7%. After cultured with Gemcitabine for 3 d, the proportion of SP cells increased significantly (3.8% ± 1.9%, 10.7% ± 3.7%, t = 4.616, P = 0.001 < 0.05). ABCB1 and ABCG2 expressed at higher concentrations in SP as compared with non-SP cells (ABCB1: 1.15 ± 0.72, 5.82 ± 1.16, t = 10.839, P = 0.000 < 0.05; ABCG2: 1.16 ± 0.75, 5.48 ± 0.94, t = 11.305, P = 0.000 < 0.05), which may contribute to the efflux of fluorescent staining and drug resistance. CONCLUSION: SP cells with inherently high resistance to chemotherapeutic agents do exist in pancreatic cancers, which may be candidate cancer stem cells contributing to the relapse of the tumor.展开更多
Mounting evidence has shown that side population （SP） cells are enriched for cancer stem cells （CSCs） responsible for cancer malignancy. In this study, SP technology was used to isolate a small subpopulation of SP...Mounting evidence has shown that side population （SP） cells are enriched for cancer stem cells （CSCs） responsible for cancer malignancy. In this study, SP technology was used to isolate a small subpopulation of SP cells in human gallbladder cancer cell line GBC-SD, and SP cells which had superior potential for proliferation in vitro and tumorigenesis in vivo were identified. Importantly, the abundance of GBC-SD SP cells was increased by a transforming growth factor-β （TGF-β）-induced epithelial-mesenchymal transition （EMT）, and this effect was accompanied with a strong up-regulation of ABCG2 mRNA expression, and a decreased sensitivity to mitoxantrone. SP cells were restored upon the removal of TGF-β and the reversion of the cells to an epithelial phenotype, and smad3-specific siRNA reduced SP abundance in response to TGF-β. In conclusion, TGF-β-induced EMT by smad-dependent signaling pathway promotes cancer development and anti-cancer drug resistant phenotype by augmenting the abundance of GBC-SD SP cells, and a better understanding of mechanisms involved in TGF-β-induced EMT may provide a novel strategy for preventing cancer progression.展开更多
AIM: To investigate whether the side population (SP) cells possess cancer stem cell-like characteristics in vitro and the role of SP cells in tumorigenic process in gastric cancer. METHODS: We analyzed the presence of...AIM: To investigate whether the side population (SP) cells possess cancer stem cell-like characteristics in vitro and the role of SP cells in tumorigenic process in gastric cancer. METHODS: We analyzed the presence of SP cells indifferent human gastric carcinoma cell lines, and then isolated and identified the SP cells from the KATO Ⅲ human gastric cancer cell line by flow cytometry. The clonogenic ability and self-renewal were evaluated by clone and sphere formation assays. The related genes were determined by reverse transcription polymerase chain reaction. To compare tumorigenic ability, SP and non-side population (NSP) cells from the KATO Ⅲ human gastric cancer cell line were subcutaneously injected into nude mice. RESULTS: SP cells from the total population accounted for 0.57% in KATO Ⅲ, 1.04% in Hs-746T, and 0.02% in AGS (CRL-1739). SP cells could grow clonally and have self-renewal capability in conditioned media. The expression of ABCG2, MDRI, Bmi-1 and Oct-4 was different between SP and NSP cells. However, there was no apparent difference between SP and NSP cells when they were injected into nude mice. CONCLUSION: SP cells have some cancer stem celllike characteristics in vitro and can be used for studying the tumorigenic process in gastric cancer.展开更多
It has been widely verified by various sorting methods that cancer stem cells (CSCs) exist in different types of tumor cells or tissues.However,due to lack of specific stem cell surface markers,CSCs are very difficult...It has been widely verified by various sorting methods that cancer stem cells (CSCs) exist in different types of tumor cells or tissues.However,due to lack of specific stem cell surface markers,CSCs are very difficult to be separated from some cancer cells,which becomes the key barrier of functional studies of CSCs.The sorting method by side population cells (SP) lays a solid foundation for in-depth and comprehensive study of CSCs.To identify the existence of SP in prostate cancer cell lines,we applied flow cytometry sorting by SP to cultures of prostate cancer cell lines (TSU,LnCap,and PC-3),and the cancer stem-like characteristics of SP were verified through experiments in vitro and in vivo.The proportion of SP in TSU cells was calculated to be 1.60%±0.40% (±s),and that in PC-3 and LnCap cells was calculated to be 0.80%±0.05% and 0.60%±0.20%,respectively.The colony formation assay demonstrated that the colony formation rate of SP to non-SP sorted from TSU via flow cytometry was 0.495±0.038 to 0.177±0.029 in 500 cells,0.505±0.026 to 0.169±0.024 in 250 cells,and 0.088±0.016 to 0.043±0.012 in 125 cells respectively.In the in vivo experiments,tumors were observed in all the mice on the 10th day after injecting 50 000 cells subcutaneously in SP group,whereas when 5×106 cells were injected in non-SP group,tumors were developed in only 4 out of 8 mice until the 3rd week before the end of the experiment.Our results revealed that prostate cancer cells contain a small subset of cells,called SP,possessing much greater capacity of colony formation and tumorigenic potential than non-SP.These suggest that SP in prostate cancer cells may play a key role in the self-renewal and proliferation,and have the characteristics of cancer stem-like cells.Dissecting these features will provide a new understanding of the function of prostate CSCs in tumorigenicity and transformation.展开更多
Objective：Side population cells （SP cells） are a new type of stem cells. They mainly express ABCG2/BCRP1 and have the ability to eliminate DNA dye Hoechst33342. Many studies showed that side population cells were a...Objective：Side population cells （SP cells） are a new type of stem cells. They mainly express ABCG2/BCRP1 and have the ability to eliminate DNA dye Hoechst33342. Many studies showed that side population cells were able of self-renewal, differentiation and carcinogenesis in cancers. Our investigation aimed at isolation of side population cells and ABCG2 positive subpopulation.from colon cancer cell line SW480 and identification of their characteristics of cancer stem cells. Methods： side population cells and non-side population cells of colon cancer cell line SW480 were isolated with DNA dye Hoechst33342 and their cell cycles were measured by flow cytometry. Expression of ABCG2 of SW480 was measured by immunohistochemistry and immunofluorescence, and its proportion was measured by flow cytometry. Results： SW480 contained 2.29% side population ceils. The fraction of side population ceils decreased greatly to 0.40% by treatment with verapamil. The fraction of side population cells in S-G2M cell cycle was 16.14%, which was much lower than the fraction （34.05%） of non-side population cells in S-G2M. In SW480, ABCG2 positive cells, which proportion was 9.66%, were small, circular or oval, lack of psuedopods, similar to poor differentiation. On the contrary, the ABCG2 negative cells were large, polygonal, with many psuedopods, similar to high differentiation. ConclusJon： our assay identified that side population cells did exist in SW480 and had a quiescence characteristic of stem cells. ABCG2 positive subpopulation occupied about 9.66% of SW480 and may have the ability to promote cell self-renewal and inhibit cell differentiation. Therefore, to isolate ABCG2 positive subpopulation from side population cells may be an alternative to study colorectal cancer stem cells.展开更多
Objective：There has been an increasing interest in recent years in the role of stem cells.With an extensive understanding of their biology,a major role for stem cells in the malignant process has been proposed and th...Objective：There has been an increasing interest in recent years in the role of stem cells.With an extensive understanding of their biology,a major role for stem cells in the malignant process has been proposed and the existence of cancer stem cells（CSCs） has been confirmed in hematopoietic malignancies and solid organ malignancies including brain cancer,breast,prostate,colon,and pancreatic cancer.Lung cancer is the leading cause of cancer mortality in most large cities of China.It is possible that lung cancer contains cancer stem cells responsible for its malignancy.The aim of this study is to identify,characterize and enrich the CSC population that drives and maintains lung adenocarcinoma growth and metastasis.Methods：Side population（SP） cell analysis and sorting were applied on human lung adenocarcinoma cell line and an attempt to further enrich them by preliminary serum-free culture before fluorescence activated cell sorting（FACS） was done.Stem cell properties of SP cells were evaluated by their proliferative index,colony-forming efficiency,tumorigenic potential,bi-differentiation capacity and the expression of common stem cell surface markers.Results：Lung cancer cells could grow in a serum-free Medium（SFM） as non-adherent spheres similar to neurospheres or mammospheres.The proportion of SP cells in cell spheres was significantly higher than that in cells grown as monolayers.SP cells had a greater proliferative index,a higher colony-forming efficiency and a greater ability to form tumor in vivo.SP cells were both CCA positive and SP-C positive while non-SP cells were only SP-C positive.Flow cytometric analysis of cell phenotype showed that SP cells expressed CD133 and CD44,the common cell surface markers of cancer stem cells,while non-SP cells only expressed CD44.Conclusion：SP cells existed in human lung adenocarcinoma cell lines and they could be further enriched by preliminary serum-free culture before FACS sorting.SP cells possessed the properties of cancer stem cells.展开更多
Relapse and metastasis are frequent in colon cancer and may be linked to stem cell characteristics.This study isolated side population（SP） cells from a colon cancer cell line（Colo-320） and examined their self-rene...Relapse and metastasis are frequent in colon cancer and may be linked to stem cell characteristics.This study isolated side population（SP） cells from a colon cancer cell line（Colo-320） and examined their self-renewal and differentiation abilities.Compared to non-SP（NSP） cells,SP colon cancer cells were more tumorigenic in vivo and exhibited more invasive characteristics and a greater ability to form colonies.Additionally,more cells were in G0/G1 phase and more highly expressed the multidrug resistance protein BCRP/ABCG2.We achieved enhanced chemotherapy sensitivity by transfecting SP cells with a hairpin-like,small interfering RNA（si RNA） eukaryotic expression plasmid targeting BCRP/ABCG2.展开更多
Objective: To investigate whether the side population cells (SP cells) exist in human gallbladder carcinoma cell line and the differences of drug resistance gene ABCG2 expression in SP cells, non-SP cells and GBC-SD c...Objective: To investigate whether the side population cells (SP cells) exist in human gallbladder carcinoma cell line and the differences of drug resistance gene ABCG2 expression in SP cells, non-SP cells and GBC-SD cell lines. Methods: Fluorescence activated cell sorter (FACS) was used to sort the SP and non-SP cells from GBC-SD cell line of gallbladder carcinoma of human being. Then, the sorting cells were cultured and detected the expression of ABCG2 gene among the SP cells, non-SP cells and GBC-SD cell lines by using reverse transcription polymerase chain reaction (RT-PCR), immunofluo-rescence chemistry, western blot and flow cytometry techniques. Results: A very small fraction cells (0.64 ± 0.08%) were iso-lated through FACS analysis which had the potency of stem cells and highly expressed ABCG2 gene (89.56 ± 3.86%). On the contrary, there were nearly no expression in non-SP cells (1.32 ± 0.49%) and lower expression in GBC-SD cell line (12.37 ± 1.61%). Conclusion: The side population cells that had the potency of stem cells existed in human gallbladder carcinoma cell line and over-expressed the drug resistance gene ABCG2. They may be play an important role in drug resistance of tumor.展开更多
AIM: To ascertain whether side population (SP) cells in HXO-Rb44 retinoblastoma cell line have cancer stem cell-like property in vitro and in vivo. METHODS: We analyzed and sorted SP from HXO-Rb44 retinoblastoma cell ...AIM: To ascertain whether side population (SP) cells in HXO-Rb44 retinoblastoma cell line have cancer stem cell-like property in vitro and in vivo. METHODS: We analyzed and sorted SP from HXO-Rb44 retinoblastoma cell line by Hoechst 33342 staining on flow cytometry. SP and NSP cells were determined their ability of proliferation and self-renewal by SP reanalysis, soft agar assay and tumor sphere assay in vitro. Clone formation was detected by seeding HXO-Rb44 and HXO-Rb44 -RFP cells into soft agar. The expression of ABCGZ MDRI Bmi-1 and Oct-4 was determined by RT-PCR between SP and non-SP (NSP) cells. Moreover, they were injected into nude mice to determine their tumorigency in vivo. RESULTS: SP from HXO-Rb44 retinoblastoma cell line could grow clonally in soft agar assays and form tumor spheres from single cells in conditioned media. The expressions of ABCG2, MDRI Bmi-1 and Oct-4 were significantly higher in SP than NSP cells. As few as SP cells resulted in tumor formation in 6 of 12 injected sites, however, the injection of NSP cells failed to form new tumor. CONCLUSION: SP cells isolated by Hoechst 33342 from the HXO-Rb44 retinoblastoma cell line had property of high tumorigency in vivo and in vitro Therefore, SP might be a target while developing retinoblastoma therapies.展开更多
The stemness of different side population(SP)cell subtypes in ovarian cancer cells was studied,and the heterogeneity of ovarian cancer stem cells was analyzed.The cisplatin-resistant human serous ovarian cancer cell l...The stemness of different side population(SP)cell subtypes in ovarian cancer cells was studied,and the heterogeneity of ovarian cancer stem cells was analyzed.The cisplatin-resistant human serous ovarian cancer cell line C13 was stained with the bisbenzimide Hoechst 33342.A flow cytometry-based fluorescence-activated sorting method was used to obtain lower-SP(LSP)cells,upper-SP(USP)cells,and non-SP cells(NSP)based on their sensitivity to the staining time and Hoechst dye concentration.The sphere-forming capability,expression levels of stem cell markers,resistance to high concentrations of cisplatin,and subcutaneous tumorigenicity in NOD/SCID mice of the different cell subtypes were evaluated.The C13 cells contained SP cells with stemness characteristics,and the LSP cell subtype expressed higher levels of stem cell markers,had higher in vitro sphere-forming capability,higher cisplatin resistance and higher in vivo subcutaneous tumorigenesis than USP cells(P<0.05).NSP cells had no stemness.In conclusion,different subtypes of ovarian cancer SP cells have different stemness levels,and ovarian cancer stem cells may be heterogeneous.展开更多
Objective:Side population(SP) cells may play a crucial role in tumorigenesis and the recurrence of cancer.Many kinds of cell lines and tissues have demonstrated the presence of SP cells,including several gastric cance...Objective:Side population(SP) cells may play a crucial role in tumorigenesis and the recurrence of cancer.Many kinds of cell lines and tissues have demonstrated the presence of SP cells,including several gastric cancer cell lines.This study is aimed to identify the cancer stem-like cells in the SP of gastric cancer cell line MKN-45.Methods:We used fluorescence activated cell sorting(FACS) to sort SP cells in the human gastric carcinoma cell line MKN-45(cells labeled with Hoechst 33342) and then characterized the cancer stem-like properties of SP cells.Results:This study found that the SP cells had higher clone formation efficiency than major population(MP) cells.Five stemness-related gene expression profiles,including OCT-4,SOX-2,NANOG,CD44,and adenosine triphosphate(ATP)-binding cassette transporters gene ABCG2,were tested in SP and MP cells using quantitative real-time reverse transcription polymerase chain reaction(RT-PCR).Western blot was used to show the difference of protein expression between SP and MP cells.Both results show that there was significantly higher protein expression in SP cells than in MP cells.When inoculated into non-obese diabetic/severe combined immunodeficiency(NOD/SCID) mice,SP cells show higher tumorigenesis tendency than MP cells.Conclusions:These results indicate that SP cells possess cancer stem cell properties and prove that SP cells from MKN-45 are gastric cancer stem-like cells.展开更多
Side population （SP） cells are a small subset of cells isolated from a cultured cancer cell line that exhibit characteristics similar to those of cancer stem cells （CSCs）, such as high metastatic and tumorigenic p...Side population （SP） cells are a small subset of cells isolated from a cultured cancer cell line that exhibit characteristics similar to those of cancer stem cells （CSCs）, such as high metastatic and tumorigenic potential. The molecular mechanisms that give rise to the malignant properties of SP cells are not clear. We isolated SP cells from the MCF-7 breast cancer cell line and profiled microRNA （miRNA） expression patterns between SP cell-derived spheroids and non-SP cells. SP spheroids were found to possess 42 up-regulated miRNAs and 27 down-regulated ones （above 5-fold changes）. One of the up-regulated miRNAs, miR-888 computationally predicted to participate in the adherens junction （AJ） pathway, was investigated. Over-expression of miR-888 in MCF-7 cells reduced the mRNA levels of all four AJ pathway genes （E-cadherin, ACTG1, PTPRTand CDC42） that were selected for testing, whereas knocking down miR-888 reversed the trends. Western blot and flow cytometric quantitation of the membrane E-cadherin levels showed the same trend of change under these treatments. Luciferase reporter assay showed E-cadherin is a direct target of miR-888. As a potential role in intercellular adhesiveness and maintenance of malignant tissue architecture, the results indicate that miR-888 is a repressor of the AJ pathway in MCF-7 cells and that up-regulation of miR-888 contributes to aggressiveness in MCF-7 SP cells.展开更多
Human amnion mesenchymal cells （AMCs） contain muhipotent cells. To enrich such muhipotent stem cells, we applied to AMCs the new method for the isolation of side population （SP） cells used for the enrichment of mu...Human amnion mesenchymal cells （AMCs） contain muhipotent cells. To enrich such muhipotent stem cells, we applied to AMCs the new method for the isolation of side population （SP） cells used for the enrichment of muhipotent stem cells from many tissues. We succeeded in obtaining SP cells from AMCs （AMC-SP cells）. AMC-SP cells were found in 0.2 % of AMCs, irrespective of the length of pregnant period, ranging from 37 to 40 weeks. Cell cycle analyses suggested that AMC-SP cells belonged to a cell population that proliferated very slowly and/or was in a quiescent state in the amniotic membrane. Upon culturing, they proliferated with 40 to 80 cell doublings. However, they did not form colonies in a soft agarose culture, whereas HepG2 cells, representative human hepatoma cells formed many large colonies. These results suggest that AMC-SP cells that have considerable value for the use of regenerative medicine can be managed safely in vitro.展开更多
BACKGROUND： Overwhelming evidence suggests that tumor bulks are comprised of differentiated tumor cells and cancer stem cells （CSCs）. The stem cell-like side-population （SP） cells account for a minor fraction of ...BACKGROUND： Overwhelming evidence suggests that tumor bulks are comprised of differentiated tumor cells and cancer stem cells （CSCs）. The stem cell-like side-population （SP） cells account for a minor fraction of the total tumor cells, yet are apparently the cells capable of tumor initiation, growth, maintenance, and recurrence. OBJECTIVE： To identify potential stem cell-like cancer cells in a U87 human brain glioma cell line on the basis of dye efflux, clone formation, and multi-drug resistance capacity. DESIGN, TIME AND SETTING： The cellular and molecular biology experiment was performed at the Laboratory of Shanghai Institute of Hematology and Laboratory of Shanghai Institute of Endocrinology in Ruijin Hospital; in vivo contrast observational animal trial was performed at Experimental Animal Center, School of Medicine, Shanghai Jiao Tong University from June 2007 to May 2008. MATERIALS： The U87 cell line was provided by the Shanghai Institute of Cancer Research, Chinese Academy of Science; DMEM/F12 （1 ： 1） and fetal bovine serum were purchased from Gibco Invitrogen, USA; human recombinant basic fibroblast growth factors were purchased from BD Bioscience, USA; Hoechst 33342, Verapamil, and methyl thiazolyl tetrazolium were purchased from Sigma, USA; phycoerythrin-labeled anti-human-CD133 was purchased from Milteny Biotec, Germany; SYBR PrimeScriptTM RT-PCR kit was purchased from TaKaRa Biotechnology, Dalian, China. METHODS： Monolayer cultured cells were harvested by 0.25% Trypsin-EDTA and suspended at a 1 ×10^6/mL dilution in PBS containing 2% FBS, and were stained with Hoechst 33342 dye, either alone or in combination with Verapamil. Following fluorescence-activated cell sorting, SP and non-SP subsets were cultivated with serum-containing （DMEM plus 10% fetal bovine serum） or serum-free culture medium [DMEM/F12 （1： 1） ＋ 1× B27 supplement ＋ 10 ng/mL basic fibroblast growth factors ＋ 1× L-glutamine] to determine growth characteristics in vitro. Finally, single free U87 cells and subsets （SP or non-SP cells） were subcutaneously injected into the backs of 5-week-old nude mice for in vivo tumorigenicity. MAIN OUTCOME MEASURES： Cell morphology and clonogenicity were observed under inverted microscope; SP phenotype and fluorescent antibody labeling were analyzed by MoFIoTM flow cytometry; ABC transporter mRNA expression was evaluated by semi-quantitative real-time RT-PCR; efflux capacity for anti-neoplastic drugs from the U87 cell line and subsets was measured with the MTT assay, then detected by enzyme-linked immunosorbent assay at a wavelength of 490 nm; in vivo tumorigenicity in immunodeficient nude mice was evaluated by diameter size. RESULTS： During in vitro passages, human U87 cells maintained a stable SP fraction profile and exhibited the ability to form neurosphere-like clones. SP cell proliferation decreased compared with non-treated U87 cells. CD133 expression was reduced in the SP and non-SP cells. Freshly sorted SP fractions expressed higher levels of ABC drug transporter genes, and exhibited increased potential for cytotoxic drug resistance. The in vivo malignancy of U87 cells was largely dependent on non-SP cells in nude mice, and tumors that formed from the non-SP fraction developed faster and larger compared with tumors from the SP fraction. CONCLUSION： The SP cell component was a key factor that influenced mRNA expression and cytotoxic drug resistance. In particular, cancer stem cells or tumor-initiating cells were not exclusively enriched in the SP subset of the U87 cell line, and non-SP cells were even more tumorigenic.展开更多
Gastric cancer stem-like cells（GCSCs） have been identified to possess the ability of self-renewal and tumor initi-ation.However,the mechanisms involved remain largely unknown.Here,we isolated and characterized the G...Gastric cancer stem-like cells（GCSCs） have been identified to possess the ability of self-renewal and tumor initi-ation.However,the mechanisms involved remain largely unknown.Here,we isolated and characterized the GCSCs by side population（SP） sorting procedure and cultured sphere cells（SC） from human gastric cancer cell lines SGC-7901,BGC-823,MGC-803,HGC-27 and MKN-28.The sorting and culture assay revealed that SP cells proliferated in an asymmetric division manner.In addition,SP cells exhibited a higher potential of spheroid colony formation and greater drug resistance than non-SP cells（NSP）.Moreover,the SC were found with enhanced capabilities of drug resistance in vitro and tumorigenicity in vivo.Sox2 mRNA and protein was highly and significantly overex-pressed in the SP cells and SC.Importantly,downregulation of Sox2 with siRNA obviously reduced spheroid colony formation and doxorubicin efflux,as well as increased apoptosis rate in sphere cells in vitro and suppressed tumori-genicity in vivo.These results suggest that both SP cells and cultured SC enrich with GCSCs and that Sox2 plays a pivotal role in sustaining stem cell properties and might be a potential target for gastric cancer therapy.展开更多
Objective:The aim of this study was to investigate the expression of ABCG2 in human gastric carcinoma and its clinical significance.Methods:Expression of ABCG2 was examined with immunohistochemical technique in the sp...Objective:The aim of this study was to investigate the expression of ABCG2 in human gastric carcinoma and its clinical significance.Methods:Expression of ABCG2 was examined with immunohistochemical technique in the specimens from 45 gastric carcinoma tissues and 30 surrounding normal tissues.The mRNA expression of ABCG2 was measured by RT-PCR and real-time quantitative PCR in 30 cases of gastric carcinoma and normal gastric mucosa, respectively.Results:ABCG2 expression was observed in 28 of 45(62.2%) cases by immunohistochemical analysis.In ABCG2-positive tumors, adjacent non-neoplastic tissue was similarly analyzed, revealed that ABCG2 was up-regulated in gastric carcinoma.ABCG2 expression in poorly differentiated/undifferentiated carcinoma was significantly higher than that in well/moderately-differentiated carcinoma(P < 0.05).The mRNA expression of ABCG2 was significantly higher than that in normal gastric mucosa(P < 0.05).Conclusion:ABCG2 plays an important role in the multi-drug resistance of gastric carcinoma.ABCG2 might be an important factor in the research of gastric cancer stem cell.展开更多
Objective： To characterize a novel chronic myeloid leukemia （CML） cell line and to further elucidate the mechanisms of resistance to STI571. Methods： A novel K562 cell line （K562NP16） was achieved after exposure...Objective： To characterize a novel chronic myeloid leukemia （CML） cell line and to further elucidate the mechanisms of resistance to STI571. Methods： A novel K562 cell line （K562NP16） was achieved after exposure of the K562 cells to VP16. A small subpopulation （K562NP16 SP） that was capable of excluding Hoechst 33342 in the K562NP16 cell line was isolated by fiow cytometry sorting. The rest of the K562NP16 cells were classified as non-SP K562NP16. The mechanisms involved in K562NP16 SP cells which became resistant to STI571 were studied. Results： The levels of Bcr-Abl and Abl proteins were similar in the K562 cell line and in non-SP K562NP16 and K562NP16 SP cells. The multidrug-resistant gene 1 （MDR1） expression of the 170 kDa P-glycoprotein （P-gp） was detected in K562NP16 non-SP and K562NP16 SP cells but not in K562 cells. The expression levels of P-gp in the two K562NP16 cell lines were similar. Compared with non-SP K562/ VP16, the K562NP16 SP cells were more resistant to STI571. This resistance could hardly be reversed by many multidrug resistance inhibitors. In addition, in vivo study showed that the K562NP16 SP cells induced tumorigenesis in mice, while the K562NP16 non-SP cells failed to do so. Conclusion： A novel K562 cell line, K562NP16, was generated. A small side population K562NP16 SP cells, had high resistance to STI571 treatment and more tumorigenic than the K562 cells. It may represent the cancer stem cells of the K562NP16 cell line.展开更多
The endometrium is the inner mucosal lining of the uterus that undergoes extensive cyclic growth, regeneration, differentiation, and shedding throughout the menstrual cycle in response to steroid hormones. It repeated...The endometrium is the inner mucosal lining of the uterus that undergoes extensive cyclic growth, regeneration, differentiation, and shedding throughout the menstrual cycle in response to steroid hormones. It repeatedly undergoes approximately 450 cycles of degeneration and regeneration in a woman's lifetime. Endometrial abnormalities can be associated with repeated embryo implantation failure, recurrent spontaneous abortion, and other physiological features responsible for female infertility. This significant regenerative capacity may occur as a result of tissue-resident stem cell populations within the endometrium. Indeed, the existence of endometrial stem cells was only observed in humans and rodents through several isolation and characterization methods in the last few years. Although endometrial stem cells share various biological characteristics with other types of mesenchymal stem cells, they also show some differences in phenotype, self-renewal, and multilineage differentiation potential. Extensive studies over many years on endometrial stem cells will provide new insights into the physiology and mechanisms underlying various gynaecological diseases related to endometrial abnormalities such as female infertility, endometriosis, and endometrial cancer. Here we summarized recent studies about cellular origins and biological characteristics of endometrial stem cells. We also reviewed various recent studies to improve our understanding of their physiological roles. Many preclinical studies on their potential therapeutic applications to various endometrial diseases that could lead to reproductive dysfunction were also reviewed.展开更多
基金supported by the Shenzhen Science and Technology Innovation Commission Fundamental Research Key Projects(Nos.JCYJ20180508153013853,JCYJ20180508152130899)the Shenzhen Science and Technology Innovation Fund(No.JCYJ20150331142757381)the National Natural Science Foundation of China(Nos.81641051,81872368).
文摘Introduction:Sulfatase 2(SULF2),an endogenous extracellular sulfatase,can remove 6-O-sulfate groups of glucosamine residues from heparan sulfate(HS)chains to modulate the Wnt/β-catenin signaling pathway,which plays an important role in both liver carcinogenesis and embryogenesis.Side population(SP)cells are widely identified as stem-like cancer cells and are closely related to carcinoma metastasis,recurrence,and poor patient prognosis.However,the roles of SULF2 in SP cells of hepatomas are unclear,and the underlying mechanism is undefined.Objectives:This study aimed to compare the heterogeneity between SP cells and non-side population(NSP)cells derived from three different liver cancer cell lines and to elucidate the involvement of the SULF2-Wnt/β-catenin axis in liver cancer stem cells(CSCs)and its impact on the processes of carcinogenesis and invasiveness.Methods:In this work,three different liver cancer SP cells(HepG2,Huh7,and PRC/PRL/5)were sorted by flow cytometry.We also examined the migration and invasion behaviors of SP and NSP cells.To determine if this high tumorigenic potential of SP cells is correlated to SULF2,qPCR,western blotting,and immunofluorescence analysis were conducted.We also performed nude mouse xenograft experiments for in vivo analysis.Results:The results from the in vitro colony formation assay showed that SP cells exhibited a 2-fold higher colony formation efficiency compared to their NSP counterparts.The SP cells exhibited significantly higher potentials in terms of their migratory capacity and invasive ability compared to NSP cells.We found that higher expression of SULF2 in SP cells was associated with greater capabilities for clonogenicity,migration,and invasion.It was also linked to higher activation of the Wnt/β-catenin signaling pathway via stimulation of key downstream factors,particularlyβ-catenin,c-Myc,and cyclin D1.Further,a positive correlation between the upregulated SULF2 expression and tumorigenesis in the in vivo nude mouse xenograft models was demonstrated,highlighting that the potential underlying mechanism was Wnt/β-catenin signaling pathway activation.Conclusion:Our findings show that variable SULF2 expression was associated with differential activation of the Wnt/β-catenin signaling pathway,which could lead to behavioral differences between SP and NSP cells and also among the SP cells of the three liver cancer cell lines assessed.It was reasonably concluded that the SULF2-Wnt/β-catenin axis could play an important role in the tumorigenicity of liver cancer stem cells.
文摘AIM： To understand the role and significance of side population （SP） cells from hepatocellular carcinoma （HCC） in hepatocarcinogenesis, development, relapse and metastasis, we simulated the denutrition conditions that cancer cells experience in clinical therapy, observed the different anti-apoptosis ability of SP cells and non-SP cells under such conditions, and established the possible effects of P53, Bcl-2 and Bax on survival of SP cells. METHODS： We used flow cytometry to analyze and sort the SP and non-SP cells in established HCC lines MHCC97 and hHCC. We evaluated cell proliferation by methyl thiazolyl tetrazolium （MTT） assay and investigated the expression of p53, bcl-2 and bax genes during denutrition, by RT-PCR and immunofluorescence staining. RESULTS： The percentage of SP cells in the two established HCC lines was 0.25% and 0.5%, respectively. SP cells had greater anti-apoptosis and proliferation ability than non-SP cells. Expression of Bcl-2 and Bax in SP and non-SP cells differed during denutrition. The former was up-regulated in SP cells, and the latter was up-regulated in non-SP cells. CONCLUSION： It may be that different upstream molecules acted and led to different expression levels of Bcl-2 and Bax in these two cell lines. There was a direct relationship between up-regulation of Bcl-2 and down-regulation of Bax and higher anti-apoptosis ability in SP cells. It may be that the existence and activity of SP cells are partly responsible for some of the clinical phenomena which are seen in HCC, such as relapse or metastasis. Further research on SP cells may have potential applications in the field of anticancer therapy.
基金The National Natural Science Foundation of China, No. 30571817
文摘AIM: To investigate the persistence of side population (SP) cells in pancreatic cancer and their role and mechanism in the drug resistance. METHODS: The presentation of side population cells in pancreatic cancer cell line PANC-1 and its proportion change when cultured with Gemcitabine, was detected by Hoechst 33342 staining and FACS analysis. The expression of ABCB1 and ABCG2 was detected by real- time PCR in either SP cells or non-SP cells. RESULTS: SP cells do exist in PANC-1, with a median of 3.3% and a range of 2.1-8.7%. After cultured with Gemcitabine for 3 d, the proportion of SP cells increased significantly (3.8% ± 1.9%, 10.7% ± 3.7%, t = 4.616, P = 0.001 < 0.05). ABCB1 and ABCG2 expressed at higher concentrations in SP as compared with non-SP cells (ABCB1: 1.15 ± 0.72, 5.82 ± 1.16, t = 10.839, P = 0.000 < 0.05; ABCG2: 1.16 ± 0.75, 5.48 ± 0.94, t = 11.305, P = 0.000 < 0.05), which may contribute to the efflux of fluorescent staining and drug resistance. CONCLUSION: SP cells with inherently high resistance to chemotherapeutic agents do exist in pancreatic cancers, which may be candidate cancer stem cells contributing to the relapse of the tumor.
基金supported by a grant from the National Natural Science Foundation of China(No.30772127)
文摘Mounting evidence has shown that side population （SP） cells are enriched for cancer stem cells （CSCs） responsible for cancer malignancy. In this study, SP technology was used to isolate a small subpopulation of SP cells in human gallbladder cancer cell line GBC-SD, and SP cells which had superior potential for proliferation in vitro and tumorigenesis in vivo were identified. Importantly, the abundance of GBC-SD SP cells was increased by a transforming growth factor-β （TGF-β）-induced epithelial-mesenchymal transition （EMT）, and this effect was accompanied with a strong up-regulation of ABCG2 mRNA expression, and a decreased sensitivity to mitoxantrone. SP cells were restored upon the removal of TGF-β and the reversion of the cells to an epithelial phenotype, and smad3-specific siRNA reduced SP abundance in response to TGF-β. In conclusion, TGF-β-induced EMT by smad-dependent signaling pathway promotes cancer development and anti-cancer drug resistant phenotype by augmenting the abundance of GBC-SD SP cells, and a better understanding of mechanisms involved in TGF-β-induced EMT may provide a novel strategy for preventing cancer progression.
文摘AIM: To investigate whether the side population (SP) cells possess cancer stem cell-like characteristics in vitro and the role of SP cells in tumorigenic process in gastric cancer. METHODS: We analyzed the presence of SP cells indifferent human gastric carcinoma cell lines, and then isolated and identified the SP cells from the KATO Ⅲ human gastric cancer cell line by flow cytometry. The clonogenic ability and self-renewal were evaluated by clone and sphere formation assays. The related genes were determined by reverse transcription polymerase chain reaction. To compare tumorigenic ability, SP and non-side population (NSP) cells from the KATO Ⅲ human gastric cancer cell line were subcutaneously injected into nude mice. RESULTS: SP cells from the total population accounted for 0.57% in KATO Ⅲ, 1.04% in Hs-746T, and 0.02% in AGS (CRL-1739). SP cells could grow clonally and have self-renewal capability in conditioned media. The expression of ABCG2, MDRI, Bmi-1 and Oct-4 was different between SP and NSP cells. However, there was no apparent difference between SP and NSP cells when they were injected into nude mice. CONCLUSION: SP cells have some cancer stem celllike characteristics in vitro and can be used for studying the tumorigenic process in gastric cancer.
基金supported by Beijing Natural Science Foundation (No.7102047)
文摘It has been widely verified by various sorting methods that cancer stem cells (CSCs) exist in different types of tumor cells or tissues.However,due to lack of specific stem cell surface markers,CSCs are very difficult to be separated from some cancer cells,which becomes the key barrier of functional studies of CSCs.The sorting method by side population cells (SP) lays a solid foundation for in-depth and comprehensive study of CSCs.To identify the existence of SP in prostate cancer cell lines,we applied flow cytometry sorting by SP to cultures of prostate cancer cell lines (TSU,LnCap,and PC-3),and the cancer stem-like characteristics of SP were verified through experiments in vitro and in vivo.The proportion of SP in TSU cells was calculated to be 1.60%±0.40% (±s),and that in PC-3 and LnCap cells was calculated to be 0.80%±0.05% and 0.60%±0.20%,respectively.The colony formation assay demonstrated that the colony formation rate of SP to non-SP sorted from TSU via flow cytometry was 0.495±0.038 to 0.177±0.029 in 500 cells,0.505±0.026 to 0.169±0.024 in 250 cells,and 0.088±0.016 to 0.043±0.012 in 125 cells respectively.In the in vivo experiments,tumors were observed in all the mice on the 10th day after injecting 50 000 cells subcutaneously in SP group,whereas when 5×106 cells were injected in non-SP group,tumors were developed in only 4 out of 8 mice until the 3rd week before the end of the experiment.Our results revealed that prostate cancer cells contain a small subset of cells,called SP,possessing much greater capacity of colony formation and tumorigenic potential than non-SP.These suggest that SP in prostate cancer cells may play a key role in the self-renewal and proliferation,and have the characteristics of cancer stem-like cells.Dissecting these features will provide a new understanding of the function of prostate CSCs in tumorigenicity and transformation.
文摘Objective：Side population cells （SP cells） are a new type of stem cells. They mainly express ABCG2/BCRP1 and have the ability to eliminate DNA dye Hoechst33342. Many studies showed that side population cells were able of self-renewal, differentiation and carcinogenesis in cancers. Our investigation aimed at isolation of side population cells and ABCG2 positive subpopulation.from colon cancer cell line SW480 and identification of their characteristics of cancer stem cells. Methods： side population cells and non-side population cells of colon cancer cell line SW480 were isolated with DNA dye Hoechst33342 and their cell cycles were measured by flow cytometry. Expression of ABCG2 of SW480 was measured by immunohistochemistry and immunofluorescence, and its proportion was measured by flow cytometry. Results： SW480 contained 2.29% side population ceils. The fraction of side population ceils decreased greatly to 0.40% by treatment with verapamil. The fraction of side population cells in S-G2M cell cycle was 16.14%, which was much lower than the fraction （34.05%） of non-side population cells in S-G2M. In SW480, ABCG2 positive cells, which proportion was 9.66%, were small, circular or oval, lack of psuedopods, similar to poor differentiation. On the contrary, the ABCG2 negative cells were large, polygonal, with many psuedopods, similar to high differentiation. ConclusJon： our assay identified that side population cells did exist in SW480 and had a quiescence characteristic of stem cells. ABCG2 positive subpopulation occupied about 9.66% of SW480 and may have the ability to promote cell self-renewal and inhibit cell differentiation. Therefore, to isolate ABCG2 positive subpopulation from side population cells may be an alternative to study colorectal cancer stem cells.
文摘Objective：There has been an increasing interest in recent years in the role of stem cells.With an extensive understanding of their biology,a major role for stem cells in the malignant process has been proposed and the existence of cancer stem cells（CSCs） has been confirmed in hematopoietic malignancies and solid organ malignancies including brain cancer,breast,prostate,colon,and pancreatic cancer.Lung cancer is the leading cause of cancer mortality in most large cities of China.It is possible that lung cancer contains cancer stem cells responsible for its malignancy.The aim of this study is to identify,characterize and enrich the CSC population that drives and maintains lung adenocarcinoma growth and metastasis.Methods：Side population（SP） cell analysis and sorting were applied on human lung adenocarcinoma cell line and an attempt to further enrich them by preliminary serum-free culture before fluorescence activated cell sorting（FACS） was done.Stem cell properties of SP cells were evaluated by their proliferative index,colony-forming efficiency,tumorigenic potential,bi-differentiation capacity and the expression of common stem cell surface markers.Results：Lung cancer cells could grow in a serum-free Medium（SFM） as non-adherent spheres similar to neurospheres or mammospheres.The proportion of SP cells in cell spheres was significantly higher than that in cells grown as monolayers.SP cells had a greater proliferative index,a higher colony-forming efficiency and a greater ability to form tumor in vivo.SP cells were both CCA positive and SP-C positive while non-SP cells were only SP-C positive.Flow cytometric analysis of cell phenotype showed that SP cells expressed CD133 and CD44,the common cell surface markers of cancer stem cells,while non-SP cells only expressed CD44.Conclusion：SP cells existed in human lung adenocarcinoma cell lines and they could be further enriched by preliminary serum-free culture before FACS sorting.SP cells possessed the properties of cancer stem cells.
基金supported by grants from the National Nature Science Foundation of China(No.81101870)the National Key Clinical Specialist Construction Programs of China(No.2013-544)the Key Programs of National Health and Family Planning Commission of Tianjin(No.16KG127)
文摘Relapse and metastasis are frequent in colon cancer and may be linked to stem cell characteristics.This study isolated side population（SP） cells from a colon cancer cell line（Colo-320） and examined their self-renewal and differentiation abilities.Compared to non-SP（NSP） cells,SP colon cancer cells were more tumorigenic in vivo and exhibited more invasive characteristics and a greater ability to form colonies.Additionally,more cells were in G0/G1 phase and more highly expressed the multidrug resistance protein BCRP/ABCG2.We achieved enhanced chemotherapy sensitivity by transfecting SP cells with a hairpin-like,small interfering RNA（si RNA） eukaryotic expression plasmid targeting BCRP/ABCG2.
基金Supported in part by the National Natural Science Foundation of China (No. 30471963).
文摘Objective: To investigate whether the side population cells (SP cells) exist in human gallbladder carcinoma cell line and the differences of drug resistance gene ABCG2 expression in SP cells, non-SP cells and GBC-SD cell lines. Methods: Fluorescence activated cell sorter (FACS) was used to sort the SP and non-SP cells from GBC-SD cell line of gallbladder carcinoma of human being. Then, the sorting cells were cultured and detected the expression of ABCG2 gene among the SP cells, non-SP cells and GBC-SD cell lines by using reverse transcription polymerase chain reaction (RT-PCR), immunofluo-rescence chemistry, western blot and flow cytometry techniques. Results: A very small fraction cells (0.64 ± 0.08%) were iso-lated through FACS analysis which had the potency of stem cells and highly expressed ABCG2 gene (89.56 ± 3.86%). On the contrary, there were nearly no expression in non-SP cells (1.32 ± 0.49%) and lower expression in GBC-SD cell line (12.37 ± 1.61%). Conclusion: The side population cells that had the potency of stem cells existed in human gallbladder carcinoma cell line and over-expressed the drug resistance gene ABCG2. They may be play an important role in drug resistance of tumor.
基金Supported by National Natural Science Foundation of China(No.81072108No:31050110429)+4 种基金China Postdoctoral Science Foundation(No.20090450167No.201003676)PH.D Programs Foundation of Ministry of Education of China(No. 20090201120068)Scientific and Technological Cooperation Projects of Shaanxi Province,China(2010K14-02)The Fundamental Research Funds for the Central Universities
文摘AIM: To ascertain whether side population (SP) cells in HXO-Rb44 retinoblastoma cell line have cancer stem cell-like property in vitro and in vivo. METHODS: We analyzed and sorted SP from HXO-Rb44 retinoblastoma cell line by Hoechst 33342 staining on flow cytometry. SP and NSP cells were determined their ability of proliferation and self-renewal by SP reanalysis, soft agar assay and tumor sphere assay in vitro. Clone formation was detected by seeding HXO-Rb44 and HXO-Rb44 -RFP cells into soft agar. The expression of ABCGZ MDRI Bmi-1 and Oct-4 was determined by RT-PCR between SP and non-SP (NSP) cells. Moreover, they were injected into nude mice to determine their tumorigency in vivo. RESULTS: SP from HXO-Rb44 retinoblastoma cell line could grow clonally in soft agar assays and form tumor spheres from single cells in conditioned media. The expressions of ABCG2, MDRI Bmi-1 and Oct-4 were significantly higher in SP than NSP cells. As few as SP cells resulted in tumor formation in 6 of 12 injected sites, however, the injection of NSP cells failed to form new tumor. CONCLUSION: SP cells isolated by Hoechst 33342 from the HXO-Rb44 retinoblastoma cell line had property of high tumorigency in vivo and in vitro Therefore, SP might be a target while developing retinoblastoma therapies.
基金the Fundamental Research Funds for the Central Universities(HUST:No.2017KFYXJJ122)a grant from the National Natural Science Foundation of China(No.81672580).
文摘The stemness of different side population(SP)cell subtypes in ovarian cancer cells was studied,and the heterogeneity of ovarian cancer stem cells was analyzed.The cisplatin-resistant human serous ovarian cancer cell line C13 was stained with the bisbenzimide Hoechst 33342.A flow cytometry-based fluorescence-activated sorting method was used to obtain lower-SP(LSP)cells,upper-SP(USP)cells,and non-SP cells(NSP)based on their sensitivity to the staining time and Hoechst dye concentration.The sphere-forming capability,expression levels of stem cell markers,resistance to high concentrations of cisplatin,and subcutaneous tumorigenicity in NOD/SCID mice of the different cell subtypes were evaluated.The C13 cells contained SP cells with stemness characteristics,and the LSP cell subtype expressed higher levels of stem cell markers,had higher in vitro sphere-forming capability,higher cisplatin resistance and higher in vivo subcutaneous tumorigenesis than USP cells(P<0.05).NSP cells had no stemness.In conclusion,different subtypes of ovarian cancer SP cells have different stemness levels,and ovarian cancer stem cells may be heterogeneous.
基金Project (Nos.81000706/H1108 and 81172368) supported by the National Natural Science Foundation of China
文摘Objective:Side population(SP) cells may play a crucial role in tumorigenesis and the recurrence of cancer.Many kinds of cell lines and tissues have demonstrated the presence of SP cells,including several gastric cancer cell lines.This study is aimed to identify the cancer stem-like cells in the SP of gastric cancer cell line MKN-45.Methods:We used fluorescence activated cell sorting(FACS) to sort SP cells in the human gastric carcinoma cell line MKN-45(cells labeled with Hoechst 33342) and then characterized the cancer stem-like properties of SP cells.Results:This study found that the SP cells had higher clone formation efficiency than major population(MP) cells.Five stemness-related gene expression profiles,including OCT-4,SOX-2,NANOG,CD44,and adenosine triphosphate(ATP)-binding cassette transporters gene ABCG2,were tested in SP and MP cells using quantitative real-time reverse transcription polymerase chain reaction(RT-PCR).Western blot was used to show the difference of protein expression between SP and MP cells.Both results show that there was significantly higher protein expression in SP cells than in MP cells.When inoculated into non-obese diabetic/severe combined immunodeficiency(NOD/SCID) mice,SP cells show higher tumorigenesis tendency than MP cells.Conclusions:These results indicate that SP cells possess cancer stem cell properties and prove that SP cells from MKN-45 are gastric cancer stem-like cells.
基金supported by the grant from the Natural Science Foundation of China (No. 30971625) to Dr. Liangbiao Chen
文摘Side population （SP） cells are a small subset of cells isolated from a cultured cancer cell line that exhibit characteristics similar to those of cancer stem cells （CSCs）, such as high metastatic and tumorigenic potential. The molecular mechanisms that give rise to the malignant properties of SP cells are not clear. We isolated SP cells from the MCF-7 breast cancer cell line and profiled microRNA （miRNA） expression patterns between SP cell-derived spheroids and non-SP cells. SP spheroids were found to possess 42 up-regulated miRNAs and 27 down-regulated ones （above 5-fold changes）. One of the up-regulated miRNAs, miR-888 computationally predicted to participate in the adherens junction （AJ） pathway, was investigated. Over-expression of miR-888 in MCF-7 cells reduced the mRNA levels of all four AJ pathway genes （E-cadherin, ACTG1, PTPRTand CDC42） that were selected for testing, whereas knocking down miR-888 reversed the trends. Western blot and flow cytometric quantitation of the membrane E-cadherin levels showed the same trend of change under these treatments. Luciferase reporter assay showed E-cadherin is a direct target of miR-888. As a potential role in intercellular adhesiveness and maintenance of malignant tissue architecture, the results indicate that miR-888 is a repressor of the AJ pathway in MCF-7 cells and that up-regulation of miR-888 contributes to aggressiveness in MCF-7 SP cells.
基金Scientific Research Common Program of Beijing Municipal Commission of Educationgrant number:KM200810025009+1 种基金Beijing Special Funds to Aid Returned Studentsgrant number:20080015
文摘Human amnion mesenchymal cells （AMCs） contain muhipotent cells. To enrich such muhipotent stem cells, we applied to AMCs the new method for the isolation of side population （SP） cells used for the enrichment of muhipotent stem cells from many tissues. We succeeded in obtaining SP cells from AMCs （AMC-SP cells）. AMC-SP cells were found in 0.2 % of AMCs, irrespective of the length of pregnant period, ranging from 37 to 40 weeks. Cell cycle analyses suggested that AMC-SP cells belonged to a cell population that proliferated very slowly and/or was in a quiescent state in the amniotic membrane. Upon culturing, they proliferated with 40 to 80 cell doublings. However, they did not form colonies in a soft agarose culture, whereas HepG2 cells, representative human hepatoma cells formed many large colonies. These results suggest that AMC-SP cells that have considerable value for the use of regenerative medicine can be managed safely in vitro.
基金the Natural Science Foundation of Shanghai,No. 08ZR 1413800
文摘BACKGROUND： Overwhelming evidence suggests that tumor bulks are comprised of differentiated tumor cells and cancer stem cells （CSCs）. The stem cell-like side-population （SP） cells account for a minor fraction of the total tumor cells, yet are apparently the cells capable of tumor initiation, growth, maintenance, and recurrence. OBJECTIVE： To identify potential stem cell-like cancer cells in a U87 human brain glioma cell line on the basis of dye efflux, clone formation, and multi-drug resistance capacity. DESIGN, TIME AND SETTING： The cellular and molecular biology experiment was performed at the Laboratory of Shanghai Institute of Hematology and Laboratory of Shanghai Institute of Endocrinology in Ruijin Hospital; in vivo contrast observational animal trial was performed at Experimental Animal Center, School of Medicine, Shanghai Jiao Tong University from June 2007 to May 2008. MATERIALS： The U87 cell line was provided by the Shanghai Institute of Cancer Research, Chinese Academy of Science; DMEM/F12 （1 ： 1） and fetal bovine serum were purchased from Gibco Invitrogen, USA; human recombinant basic fibroblast growth factors were purchased from BD Bioscience, USA; Hoechst 33342, Verapamil, and methyl thiazolyl tetrazolium were purchased from Sigma, USA; phycoerythrin-labeled anti-human-CD133 was purchased from Milteny Biotec, Germany; SYBR PrimeScriptTM RT-PCR kit was purchased from TaKaRa Biotechnology, Dalian, China. METHODS： Monolayer cultured cells were harvested by 0.25% Trypsin-EDTA and suspended at a 1 ×10^6/mL dilution in PBS containing 2% FBS, and were stained with Hoechst 33342 dye, either alone or in combination with Verapamil. Following fluorescence-activated cell sorting, SP and non-SP subsets were cultivated with serum-containing （DMEM plus 10% fetal bovine serum） or serum-free culture medium [DMEM/F12 （1： 1） ＋ 1× B27 supplement ＋ 10 ng/mL basic fibroblast growth factors ＋ 1× L-glutamine] to determine growth characteristics in vitro. Finally, single free U87 cells and subsets （SP or non-SP cells） were subcutaneously injected into the backs of 5-week-old nude mice for in vivo tumorigenicity. MAIN OUTCOME MEASURES： Cell morphology and clonogenicity were observed under inverted microscope; SP phenotype and fluorescent antibody labeling were analyzed by MoFIoTM flow cytometry; ABC transporter mRNA expression was evaluated by semi-quantitative real-time RT-PCR; efflux capacity for anti-neoplastic drugs from the U87 cell line and subsets was measured with the MTT assay, then detected by enzyme-linked immunosorbent assay at a wavelength of 490 nm; in vivo tumorigenicity in immunodeficient nude mice was evaluated by diameter size. RESULTS： During in vitro passages, human U87 cells maintained a stable SP fraction profile and exhibited the ability to form neurosphere-like clones. SP cell proliferation decreased compared with non-treated U87 cells. CD133 expression was reduced in the SP and non-SP cells. Freshly sorted SP fractions expressed higher levels of ABC drug transporter genes, and exhibited increased potential for cytotoxic drug resistance. The in vivo malignancy of U87 cells was largely dependent on non-SP cells in nude mice, and tumors that formed from the non-SP fraction developed faster and larger compared with tumors from the SP fraction. CONCLUSION： The SP cell component was a key factor that influenced mRNA expression and cytotoxic drug resistance. In particular, cancer stem cells or tumor-initiating cells were not exclusively enriched in the SP subset of the U87 cell line, and non-SP cells were even more tumorigenic.
基金supported in part by the Foundation of State Key Laboratory of Reproductive Medicine,the project funded by the Priority Academic Program Development(PAPD) of Jiangsu Higher Education Institutionsthe National Natural Science Foundation of China(No. 30930080 and 81161120537)
文摘Gastric cancer stem-like cells（GCSCs） have been identified to possess the ability of self-renewal and tumor initi-ation.However,the mechanisms involved remain largely unknown.Here,we isolated and characterized the GCSCs by side population（SP） sorting procedure and cultured sphere cells（SC） from human gastric cancer cell lines SGC-7901,BGC-823,MGC-803,HGC-27 and MKN-28.The sorting and culture assay revealed that SP cells proliferated in an asymmetric division manner.In addition,SP cells exhibited a higher potential of spheroid colony formation and greater drug resistance than non-SP cells（NSP）.Moreover,the SC were found with enhanced capabilities of drug resistance in vitro and tumorigenicity in vivo.Sox2 mRNA and protein was highly and significantly overex-pressed in the SP cells and SC.Importantly,downregulation of Sox2 with siRNA obviously reduced spheroid colony formation and doxorubicin efflux,as well as increased apoptosis rate in sphere cells in vitro and suppressed tumori-genicity in vivo.These results suggest that both SP cells and cultured SC enrich with GCSCs and that Sox2 plays a pivotal role in sustaining stem cell properties and might be a potential target for gastric cancer therapy.
基金Supported by a grant of the National Natural Science Foundation of China (No. 30570522)
文摘Objective:The aim of this study was to investigate the expression of ABCG2 in human gastric carcinoma and its clinical significance.Methods:Expression of ABCG2 was examined with immunohistochemical technique in the specimens from 45 gastric carcinoma tissues and 30 surrounding normal tissues.The mRNA expression of ABCG2 was measured by RT-PCR and real-time quantitative PCR in 30 cases of gastric carcinoma and normal gastric mucosa, respectively.Results:ABCG2 expression was observed in 28 of 45(62.2%) cases by immunohistochemical analysis.In ABCG2-positive tumors, adjacent non-neoplastic tissue was similarly analyzed, revealed that ABCG2 was up-regulated in gastric carcinoma.ABCG2 expression in poorly differentiated/undifferentiated carcinoma was significantly higher than that in well/moderately-differentiated carcinoma(P < 0.05).The mRNA expression of ABCG2 was significantly higher than that in normal gastric mucosa(P < 0.05).Conclusion:ABCG2 plays an important role in the multi-drug resistance of gastric carcinoma.ABCG2 might be an important factor in the research of gastric cancer stem cell.
基金grants from National Development Plan of HighTechnology 863(No.2002AA205061)Henan Outstanding YouthFoundation(No.0612000900).
文摘Objective： To characterize a novel chronic myeloid leukemia （CML） cell line and to further elucidate the mechanisms of resistance to STI571. Methods： A novel K562 cell line （K562NP16） was achieved after exposure of the K562 cells to VP16. A small subpopulation （K562NP16 SP） that was capable of excluding Hoechst 33342 in the K562NP16 cell line was isolated by fiow cytometry sorting. The rest of the K562NP16 cells were classified as non-SP K562NP16. The mechanisms involved in K562NP16 SP cells which became resistant to STI571 were studied. Results： The levels of Bcr-Abl and Abl proteins were similar in the K562 cell line and in non-SP K562NP16 and K562NP16 SP cells. The multidrug-resistant gene 1 （MDR1） expression of the 170 kDa P-glycoprotein （P-gp） was detected in K562NP16 non-SP and K562NP16 SP cells but not in K562 cells. The expression levels of P-gp in the two K562NP16 cell lines were similar. Compared with non-SP K562/ VP16, the K562NP16 SP cells were more resistant to STI571. This resistance could hardly be reversed by many multidrug resistance inhibitors. In addition, in vivo study showed that the K562NP16 SP cells induced tumorigenesis in mice, while the K562NP16 non-SP cells failed to do so. Conclusion： A novel K562 cell line, K562NP16, was generated. A small side population K562NP16 SP cells, had high resistance to STI571 treatment and more tumorigenic than the K562 cells. It may represent the cancer stem cells of the K562NP16 cell line.
基金supported by the Basic Science Research Program through the National Research Foundation of Korea(NRF)funded by the Ministry of Science,ICT and Future Planning(No.NRF-2021R1A2C2008424 and NRF-2021M3E5E5094127)This work was supported by the National Research Foundation of Korea(NRF)grant funded by the Korea government(MSIT)(No.NRF-2021R1A5A2030333)+3 种基金This work was supported by Korea Environment Industry&Technology Institute(KEITI)through the Project(Technology Development Project for Safety Management of Household Chemical Products)funded by Korea Ministry of Environment(MOE)(No.1485017593)This research was also supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute(KHIDI)funded by the Ministry of Health&Welfare,Republic of Korea(No HI21C1847).
文摘The endometrium is the inner mucosal lining of the uterus that undergoes extensive cyclic growth, regeneration, differentiation, and shedding throughout the menstrual cycle in response to steroid hormones. It repeatedly undergoes approximately 450 cycles of degeneration and regeneration in a woman's lifetime. Endometrial abnormalities can be associated with repeated embryo implantation failure, recurrent spontaneous abortion, and other physiological features responsible for female infertility. This significant regenerative capacity may occur as a result of tissue-resident stem cell populations within the endometrium. Indeed, the existence of endometrial stem cells was only observed in humans and rodents through several isolation and characterization methods in the last few years. Although endometrial stem cells share various biological characteristics with other types of mesenchymal stem cells, they also show some differences in phenotype, self-renewal, and multilineage differentiation potential. Extensive studies over many years on endometrial stem cells will provide new insights into the physiology and mechanisms underlying various gynaecological diseases related to endometrial abnormalities such as female infertility, endometriosis, and endometrial cancer. Here we summarized recent studies about cellular origins and biological characteristics of endometrial stem cells. We also reviewed various recent studies to improve our understanding of their physiological roles. Many preclinical studies on their potential therapeutic applications to various endometrial diseases that could lead to reproductive dysfunction were also reviewed.
