Objective: To inhibit the stemness maintenance potential of endometrial cancer and increase the sensitivity of endometrial cancer side population cells to chemotherapy drugs by inducing extensive deSUMOylation modification of proteins. Methods: Flow cytometry was used to sort and culture CD133(+) CD44(+) KLE endometrial cancer cell clone spheres. Protein expression level of small ubiquitin-related modifier 1 (SUMO1) and two stemness maintenance genes of tumor side population cells, octamer binding transcription factor-4 (Oct4) and sex determining region Y-box2 (Sox2), were detected by western blotting method. Lentivirus-mediated Sentrin/SUMO-specific proteases 1 (SENP1) gene was stably transfected into KLE side population cells. Western blotting was used to detect the protein expressions of SENP1, SUMO1, Oct4 and Sox2. The clone formation rate was compared between KLE side population cells with or without SENP1 overexpression. Flow cytometry was applied to detect cell cycle changes. 3-(4, 5-Dimethylthiazole-2)-2, 5-diphenyl-tetrazolium bromide (MTT) experiment and flow cytometry apoptosis method were used to detect the chemosensitivity of the side population of endometrial cancer cells to cisplatin. Tumor-bearing mouse models of endometrial cancer were established to detect the effect of SENP1 overexpression on the chemotherapy sensitivity of cisplatin. Results: Compared with CD133(-)CD44(-) KLE cells, CD133(+) CD44(+) KLE side population cells could form clonal spheres and express higher levels of SUMO1, Oct4 and Sox2 proteins (P<0.05). Compared with KLE side population cells that were not transfected with SENP1 gene, the expression level of SENP1 protein in KLE side population cells overexpressing SUMO1、Oct4 and Sox2 were lower. The clonal sphere formation rate was reduced from (25.67±5.44)% to (7.46±1.42)%, and cell cycle shifted from G(0)/G(1) phase to G(2) phase. IC(50) of cisplatin decreased from (55.46±6.14) μg/ml to (11.55±3.12) μg/ml, and cell apoptosis rate increased from (9.76±2.09)% to (16.79±3.44)%. Overexpression of SENP1 could reduce the tumorigenesis rate of KLE side population cells in vivo and increase their chemotherapy sensitivity to cisplatin (P<0.05). Conclusion: Overexpression of SENP1 can induce protein deSUMOylation modification, inhibit the stemness maintenance potential of endometrial cancer side population cells, and enhance their chemotherapy sensitivity, which provides a new reference for gene therapy of endometrial cancer.
Animals ; Female ; Humans ; Mice ; Apoptosis ; Cell Line, Tumor ; Cisplatin/pharmacology* ; Cysteine Endopeptidases/metabolism* ; Endometrial Neoplasms/genetics* ; Side-Population Cells/pathology* ; Sumoylation

OBJECTIVE: To investigate the biological characteristics of ABCG2 and its effect on side population cells (SP cells) of multiple myeloma (MM) so as to find the way for reversing drug resistance. METHODS: The silence of ABCG2 expression was performed throngh interfering the MM cells by using siRNA. then the ratio of SP cell of MM, speed of cell proliferation and sensitivity of cells to chermotherapentic drugs before and after interference were compared, the biological functions of ABCG2 and its role in regulation of SP cells and PTEN/PI3K/AKT signaling pathway was clarified. RESULTS: siRNA interference could down regulate the expression of ABCG2 at both mRNA and protein level. After siRNA interference, the proliferation of multiple myeloma cells was decreased slightly, the expression of PTEN was increased, the activity of PI3K/AKT pathway was inhibited, and the ratio of SP cells was decreased. CONCLUSION In multiple myeloma, down-regulation of expression ABCG2 can negatively regulate the expression of PTEN, the PI3K/AKT signaling pathway is inhibited,the ratio of SP decreased,and the response to drug is increased.
ATP Binding Cassette Transporter, Subfamily G, Member 2 ; Cell Line, Tumor ; Humans ; Multiple Myeloma ; Neoplasm Proteins ; PTEN Phosphohydrolase ; Phosphatidylinositol 3-Kinases ; Proto-Oncogene Proteins c-akt ; Side-Population Cells ; Signal Transduction

Severe graft-versus-host disease (GVHD) is an often lethal complication of allogeneic hematopoietic stem cell transplantation (HSCT). The safety of clinical-grade mesenchymal stem cells (MSCs) has been validated, but mixed results have been obtained due to heterogeneity of the MSCs. In this phase I study, the safety of bone marrow-derived homogeneous clonal MSCs (cMSCs) isolated by a new subfractionation culturing method was evaluated. cMSCs were produced in a GMP facility and intravenously administered to patients who had refractory GVHD to standard treatment resulting after allogeneic HSCT for hematologic malignancies. After administration of a single dose (1x10(6) cells/kg), 11 patients were evaluated for cMSC treatment safety and efficacy. During the trial, nine patients had 85 total adverse events and the rate of serious adverse events was 27.3% (3/11 patients). The only one adverse drug reaction related to cMSC administration was grade 2 myalgia in one patient. Treatment response was observed in four patients: one with acute GVHD (partial response) and three with chronic GVHD. The other chronic patients maintained stable disease during the observation period. This study demonstrates single cMSC infusion to have an acceptable safety profile and promising efficacy, suggesting that we can proceed with the next stage of the clinical trial.
Bone Marrow ; Drug-Related Side Effects and Adverse Reactions ; Graft vs Host Disease* ; Hematologic Neoplasms ; Hematopoietic Stem Cell Transplantation ; Humans ; Mesenchymal Stromal Cells* ; Myalgia ; Population Characteristics

OBJECTIVETo observe the proliferation changes of the side population of gastric cancer cell line SGC-7901 cells (SP), the non-side population (NSP) cells, and unsorted cells (Total) after intervened by Sijunzi Decoction (SD) containing serum.

METHODSSixteen pure bred New Zealand rabbits were equally divided into the normal control group, the low dose SD group (at the daily dose of 7 mL/kg), the middle dose SD group (at the daily dose of 14 mL/kg), and the high dose SD group (at the daily dose of 28 mL/kg) according to the random digit table. Rabbits' serum was extracted after equal volume of corresponding medication was given by gastrogavage twice daily for 2 consecutive weeks. The drug serum was identified using high performance liquid chromatography. SP cells of SGC-7901 were detected using flow cytometry, SP and NSP cells were screened. The proliferation curve of SP, NSP, and Total cells were detected with CCK-8 assay. Changes of their proliferation were also observed.

RESULTSGinsenoside Rg1, an effective ingredient in SD was detected in prepared drug serum. The proliferation of SGC-7901 SP cells was significantly higher than that of NSP cells and Total cells (P < 0.05). Drug serum on gastric cancer cell line SGC-7901 SP, NSP, and Total cells could inhibit their proliferation, but its inhibition on SP cells' proliferation was significantly lower than on NSP and Total cells (P < 0.05).

CONCLUSIONSSD could significantly inhibit the proliferation of gastric cancer cell line SGC-7901 SP, NSP, and Total cells. But there exist obvious difference in the inhibition among the three groups.

Animals ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Rabbits ; Side-Population Cells ; drug effects ; pathology ; Stomach Neoplasms ; pathology

This study was aimed to sort the side population (SP) cells from human multiple myeloma cell lines, then detect the biological characteristics of those SP cells. After Hoechst33342 staining, intracellular Hoechst33342 fluorescence staining differences of myeloma cell lines observed by the fluorescence microscopy. The fluorescence-activated cell sorting (FACS) technology was used to isolate SP cells and main population (MP) cells; proliferative capacity in vitro was determined by cell growth curve; the cell colony forming ability was compared by colony forming test. The CD138 expression was detected by flow cytometry. The expression of ABCG2 mRNA was detected by reverse transcription PCR; CCK-8 assay and colony forming test were used to evaluate the effect of bortezomib on the cell proliferation, vitality and colony forming ability of the two populations. The results showed that the myeloma cell lines had a small proportion of SP cells, especially, RPMI 8226 cells accounted for the highest proportion of SP cells (7.10 ± 2.69)%, which have also been confirmed under the fluorescence microscope; the proliferative activity and cell colony forming ability of SP cells were significantly higher than those of MP cells (P < 0.05). The expression levels of CD138 in SP and MP cells were not significantly different (P > 0.05). RT-PCR results showed that SP cells expressed the drug-resistance gene ABCG2, but MP cells hardly express these genes. The inhibition rate of bortezomib on SP cells was significantly lower than that on MP cells (P < 0.05), however, the difference was not significant (P > 0.05) at bortezomib 40 nmol/L. Bortezomib could reduce colony formation in the both two cell populations, but more severe reduction appeared in the MP cells. It is concluded that the myeloma cell line contain a small amount of SP cells with the cancer stem cell characteristics.
Cell Line, Tumor ; Cytological Techniques ; methods ; Humans ; Multiple Myeloma ; Neoplastic Stem Cells ; cytology ; Side-Population Cells ; cytology

In recent years, cancer stem cells have become a hotspot for global researchers. Cancer stem cell theory deems that cells with self-renewal and differentiation potential play a key role in tumor resistance and relapse. These cells are named cancer stem cells. At present, the sorting methods include the side population cell sorting technique, screening techniques based on cell surface special markers, tumor sphere cultures, label retaining cell, ALDEFLUOR assays and so on. Side population cells are a small part of cells with the capacity of efflux DNA fluorescent dye Hoechst 33342 and present a low staining intensity in flow cytometry plot. Side population cells are rich in cancer stem cells, and its sorting method has been considered simple and effective in cancer stem cell research.
Flow Cytometry ; Humans ; Neoplasms ; Neoplastic Stem Cells ; Side-Population Cells

OBJECTIVE: To investigate the optimizing conditions in isolation of the side population in laryngeal carcinoma cell line Hep-2. METHOD: Single-cell suspension cells were detached from the culture flask with trypsin EDTA, at a concentration of 1 x 10(6) cells/ml. (1) The trail Samples were incubated with Hoechst33342 at a concentration of 5 microg/ml, 9 microg/ml, 10 microg/ml, 11 microg/ml for 90 minutes. (2) They were incubated with Hoechst for 50, 70, 90, 110, 130 min in water bath individually. (3) The single-cell suspension were incubated Hoechst in water bath and in thermostat each. (4) The two different density of cells were harvested, which were 100% and 70%, and then di gest into single-cell suspension. Once incubation finished, suspended in phosphate buffered saline (PBS), then test SP% by flow cytometry. Among all groups,Verapamil hydrochloride was added to the control samples, incubated at 37 degrees C for 30 minutes, the other condition were keep the same with their trial groups. RESULT: (1) The percentage of Hoechst-negative cells in trial group was (39.96 +/- 0.24)%, (26.23 +/- 0.39)%. (18.79 +/- 0.02)%, (19.01 +/- 0.14)% at the concentration of 5 microg/ml, 9 microg/ml, 10 microg/ml, 11 microg/ml respectively, when the PI-positive cells were (30.45 +/- 0.63)%, (49.9 +/- 0.42)%, (50.12 +/- 0.68)%, (64.16 +/- 0.39)% separately. (2) Varying the duration of staining incubation showed that there was a typical FACS pattern and SP% was constant when the incubation was at least 90 min. (3) Compare to water bath, SP% was more than in thermostat, the SP% was (18.67 +/- 0.45)%, (22.6 +/- 0.50)% respectively; (4) Cell density is also responsible for SP%. The low density the cell is, the less in SP%. SPSS13.0 was used in statistical analysis, the groups were compared using t-Test. P < 0.05 was considered statistically significant. CONCLUSION The optimum concentration and duration of incubation of Hoechst33342 in isolation of the side population cells in laryngeal carcinoma cell line Hep-2 is 10 microg/ml and 90 min. Incubated in water bath is better than in thermostat. The best staining cell density is around 80%-90%.
Cell Count ; Cell Line, Tumor ; Flow Cytometry ; Humans ; Laryngeal Neoplasms ; pathology ; Neoplastic Stem Cells ; cytology ; Side-Population Cells ; cytology

OBJECTIVETo isolate the side population (SP) and non-side population (NSP) cells from A431 cells and compare their difference in tumorigenicity in mice and the expression profiles of epithelial-mesenchymal transition (EMT) markers.

METHODSA431 cells stained with Hoechst 33342 were sorted with flow cytometry. The isolated SP cells and NSP cells were inoculated into NOD/SCID mice and the tumorigenicity of the cells was observed. EMT markers E-cadherin, β-catenin, vimentin, AXL, and Erbb3 in the tumor tissues were detected by immunohisto-staining.

RESULTSThe tumors generated by SP cells were larger than those by NSP cells in NOD/SCID mice. Compared with the tumors generated by NSP cells, the cells in the periphery of tumors generated by SP cells showed up-regulated expressions of AXL, vimentin and β-catenin and down-regulated ERBB3 and E-cadherin.

CONCLUSIONThe SP cells in A431 cells have a strong tumorigenicity and show more EMT phenotypes in tissues.

Animals ; Biomarkers, Tumor ; metabolism ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Line, Tumor ; Epithelial-Mesenchymal Transition ; Humans ; Male ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Neoplastic Stem Cells ; metabolism ; Side-Population Cells ; metabolism ; Skin Neoplasms ; metabolism ; pathology

Cancer stem cell (CSC) research has increased exponentially to gain further insight into the mechanisms underlying both carcinogenesis and chemotherapy resistance. The present study was performed to explore the potential value of a side population (SP) assay for identifying and characterizing putative CSCs among canine lymphoma cells. Canine lymphoma cells from cell lines and clinical samples were subjected to the SP assay consisting of Hoechst 33342 staining and subsequent flow cytometric analysis. The SP assay revealed various amounts of a SP fraction among the canine lymphoma cells. The percentages of SP were not affected by inhibitors of membrane transporters, verapamil hydrochloride, or fumitremorgin C. Most of the canine lymphoma cells expressed high levels of Bmi-1 and membrane transporter proteins such as ABCG2 and phosphorylated (p)-glycoprotein. This investigation lays the groundwork for further studies of the biological behaviors and molecular characteristics of CSCs in cases of canine lymphoma.
Animals ; Benzimidazoles/*metabolism ; Cell Line, Tumor ; Dog Diseases/*diagnosis/drug therapy/pathology ; Dogs ; Flow Cytometry/*methods/veterinary ; Fluorescent Dyes/*metabolism ; Gene Expression Regulation, Developmental ; Lymphoma/diagnosis/drug therapy/pathology/*veterinary ; Neoplastic Stem Cells/drug effects/*metabolism/pathology ; Side-Population Cells/drug effects/*metabolism/pathology

OBJECTIVEIsolate and characterize the side population (SP) cells with potency of stem cells from human gastric carcinoma cell line BGC-823.

METHODSSP and non-SP cells were sorted from BGC-823 cells by fluorescence-activated cell sorting (FACS) using Hoechst33342 staining. The tumorigenic ability of the SP cells was assessed by in vivo transplantation into non-obese diabetic/severe combined immunodeficiency mice.

RESULTSSP cells were isolated from BGC-823 cells in a proportion of 0.9% to 2.1% with respect to the whole cell population. The colony formation assay showed that the colony formation rate of the SP cells was significantly higher than that of the non-SP cells (72.56% vs. 49.00%, P < 0.01). The drug sensitivity test showed that the SP cells showed stronger drug resistance to 5-Fu than the non-SP cells. The in vivo transplantation of SP cells in mice showed that the tumor weight was (0.176 ± 0.034) g, significantly higher than that of non-SP cells (0.045 ± 0.046) g (P < 0.01).

CONCLUSIONSThe results of this study indicate the existence of cancer stem-like SP cells in the human gastric cancer line BGC-823 cells. Further characterization of this SP cell population may provide new insights for diagnosis and treatment of gastric cancer.

Animals ; Antimetabolites, Antineoplastic ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; Colony-Forming Units Assay ; Drug Resistance, Neoplasm ; Female ; Fluorouracil ; pharmacology ; Humans ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Side-Population Cells ; drug effects ; pathology ; transplantation ; Stomach Neoplasms ; pathology