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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.
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Animals , Female , Humans , Mice , Apoptosis , Cell Line, Tumor , Cisplatin/pharmacology , Cysteine Endopeptidases/metabolism , Endometrial Neoplasms/genetics , Side-Population Cells/pathology , SumoylationABSTRACT
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 (siRNA) eukaryotic expression plasmid targeting BCRP/ABCG2.
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Objective To isolate side population (SP) cells from an established ovarian cancer (OC)cell line,characterize these cells,and examine their drug resistance. Methods SP and non-SP (NSP) cells were isolated by fluorescence-activated cell sorting (FACS),and cultured in differential conditions,then detected their SP ratio to compare their capability of differentiation and self-renewal. Moreover,SP and NSP cell tumorigenesis was examined by subcutaneous and intraperitoneal injection of these cells to nonobes ediabetic(NOD)-severe combined immundeficient(SCID)mice. Drug resistance to cisplatin was examined by cell counting kit-8 (CCK-8).Results SP cells could be isolated stablly and insistently. There was(4.81 ± 0.43)%of SP cells in the established OC cell line and(4.89 ± 0.33)%of SP cells after cultured the isolated SP cells in differentiation condition,and there was no significant different between these two quantities (P>0.05). However,after cultured the NSP cells,there was only (0.10 ± 0.03)%of SP cells which was significantly lower than that contained in the OC cell line(P<0.01). In the tumorigenesis assay 1.0 × 103 SP cells were injected subcutaneously and formed the xenografted tumors in 6 weeks(3/3),and 1.0×104 NSP cells were injected subcutaneously and did not form xenografted tumors in 12 weeks(0). The tumorigenic capability of SP cells was higher than that of NSP cells(P<0.01). Both the original and the xenografted tumors were low differentiated serous cystadenocarcinomas and expressed the ovarian serous cystadenocarcinomas CA125 marker after stained by HE and immunohistochemistry. Simultaneously,the SP cells were also capable to form tumors as shown by intraperitoneal injection. In the drug resistance assay shown that the 50% inhibitory concentration (IC50) of the SP and NSP cells were respectively(2.33 ± 0.14)μg/ml and(1.60 ± 0.04)μg/ml(P<0.05). After treated the unsorted OC cells with cisplatin,the quantity of SP cells increased to(40.10 ± 4.22)%and there was significant difference,when compared to the untreated cells which was(4.81±0.43)%(P<0.01). The SP cells survival rate was(58.7± 3.3)%when treated with cisplatin at its IC50 dose,and the rate decreased to(7.2±1.3)%(P<0.01)when verapamil was present. Conclusions The SP cells could be isolated from the established OC cell line. They had the capacities of self-renewal,differentiation,and tumorigenesis,and the new tumor demonstrated the original tumor′s phenotype. The SP cells also had stem cells′ biological characteristics and is resistant to cisplatin.
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Objective To detect and separate the side population cells(SP) from multiple myeloma (MM) cell lines PRIM8226,and to study their biological characteristics.Methods Fluorescence activated cell sorter (FACS) and Hoechst33342/PI dye were used to sort SP cells of PRIM8226.The multiplication capacity was tested by the growth curve and MTT test,SP cells proportion and the cell cycle were analyzed by flow cytometry,the colony-formtion ability was compared in terms of colony forming experiment,the expression of c-myc,KIF4,SOX2,OCT4 was tested by RT-PCR.The oncogenicity of the cells was analyzed by nude mouse transplantation tumor experiment in vivo.Results The ratio of SP cells in PRIM8226 was (1.78±0.89) ％.More SP cells in the G0 / G1 period,(44.34±3.09) ％ vs (28.49±1.97) ％,P ＜ 0.05,and fewer cells in S phase than MP cells,(38.83±3.69) ％ vs (51.49±4.62) ％,P ＜ 0.05.There were no difference in the expression of CD38 and CD138 between SP cells and MP cells,respectively,(78.5±8.5) ％ vs (82.0±4.0) ％ and (72.3±15.7) ％ vs (84.3±11.9) ％,P ＞ 0.05.Colony formation assay showed that the colony forming efficiency of the SP cells was higher than the MP cells,single cell clone diameter,the number of clone forming,the clone formtion rate were significantly higher than MP cells,0.280±0.016 vs 0.118±0.019,1 722±127 vs 358±14,(86.1±3.46) ％ vs (17.9±1.88) ％,P ＜ 0.05.The mRNA expression levels of c-myc,KIF4,SOX2,OCT4 in SP cells were higher than those in MP cells,c-myc (29.90±3.73) ％ vs (16.84±2.35) ％,KIF4 (29.97±2.89) ％ vs (19.06±1.23) ％,SOX2 (40.00±4.58) ％ vs (16.62±2.09) ％,OCT4 (32.96±0.92) ％ vs (23.27±0.92) ％,all P ＜ 0.05.Nude mouse transplantation tumor experiment in vivo showed the oncogenicity of the SP cells was more stronger than MP cells (5×103 vs 5×l05).Conclusion There are notably difference in the cell cycle,colony formation assay,the mRNA expression levels and oncogenicity,but no difference in the expression of CD38 and CD138,the proliferation ability between SP cells and MP cells.
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Side population cells (SPs) are selected by the efflux features of fluorescent dye Hoechst33342.SPs have the characteristics of cancer stem cells,and play an extremely important role in the occurrence and development of tumors.At present,the SPs in common types of malignant tumor have been studied extensively at home and abroad.The study of SPs may shed some light on the diagnosis and treatment of cancer.
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AIM:To investigate the role of side population ( SP) cells in multidrug resistance of colon cancer cells and microRNA biomarkers of SP cells in colon cancer cells .METHODS:SP cells in colon cancer cells were sorted by flow cytometry .The cell viability was measured by MTT method .MicroRNA expression profiles were detected by mi-croRNA chip.MicroRNA expression was verified by real-time PCR.RESULTS:The ratios of SP cells in HCT-15, HT-29 and LoVo colon cancer cell lines were 16.75%, 13.02%and 9.52%, respectively.In all 3 colon cancer cell lines, IC50 of the antitumor drugs including 5-fluorouracil , oxaliplatin and adriamycin for the SP cells were significantly higher than those for non-SP cells (P<0.05).MicroRNA profiling showed that miR-5000-3p, miR-5009-3p and miR-552 were all up-regulated in the SP cells of all 3 colon cancer cell lines .This result was verified by real-time PCR.CONCLUSION:miR-5000-3p, miR-5009-3p and miR-552 are all up-regulated in the SP cells of colon cancer cell lines , and may be the poten-tial microRNA biomarkers of SP cells in colon cancer .
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Objective To assay and determine whether the human acute monocytic leukemia cell line THP-1 contains side populations (SP) cells,and to increase the proportion of SP cells using cytarabine (Ara-C).Methods Fluorescent microscope and flow cytometry (FCM) were employed for detecting the percentage of SP cells in THP-1 cells.Then,SP and non-SP (NSP) subpopulations were collected and identified.Finally,THP-1 cells were incubated with different concentrations of Ara-C for 24 hours and detected the proportion of SP cells,respectively.Results The results demonstrated that the percentage of SP cells was (1.81 ± 0.99) ％ in THP-1 cells.A majority of the SP cells remained in the G0/G1 phase,and the expressions of CD34 + and CD34 + CD38-and the proliferative ability of the SP cells were higher than those of NSP cells (P ＜ 0.05).The mRNA expression of multidrug resistance genes (ABCG2,ABCB1),apoptosis regulation genes (Bcl-2) and the Bcl-2/Bax ratio of SP cells were higher than those of NSP cells.SP cells have been shown to be more tumorigenic than NSP cells.After co-culture with Ara-C,the proportion of SP cells increased significantly and presented in a concentration-dependent manor.Conclusions All of these findings suggest that the THP-1 cell line contains SP cells and the SP cells possess some intrinsic stem cell properties.The proportion of SP cells can be increased when co-cultured with Ara C,and this technique is a useful and important application for the study of LSCs.
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Based on the theory of cancer stem cells (CSCs),people have been searching for the treatments of malignant cancers.Gastric cancer side population cells (SP) have the characteristics of CSCs.Searching for the molecular targeted therapy strategy of gastric cancer which embarks from the gastric cancer SP and is based on the theory of CSCs provides a new direction for the treatment,early diagnosis,therapeutic effect and prognosis of gastric cancer.
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ObjectiveTo identify the presence of side population (SP) cells in human ovarian cancer cell line OVCAR-3 and to investigate whether SP cells have the characteristics of cancer stem cells.MethodsSP and non-SP (NSP) cells from OVCAR-3 were isolated by fluorescence-activated cell sorting after being stained by DNA-binding dye Hoechst 33342.Limiting dilution transplantation assay,realtime PCR,and drug sensitivity assay were performed to compare the tumorigenic ability,differentiation ability in vivo,the mRNA expressiou of stemness marker (Oct-4,Klf4,and Nanog) and ATP-binding cassette (ABC) transporter (ABCG2,ABCB1,and ABCC2),and response to multiple drugs (cisplatin,paclitaxel,doxorubicin,and mitoxantrone )between SP and NSP cells.ResultsA few of SP cells [ ( 1.13 ±0.39) ％ ] which were sensitive to reserpine were identified in OVCAR-3 cells.The injection of as few as 102 SP cells initiated tumors in two of five mice.Tumor latency was 52 -61 days.However,the NSP cells did not generate any tumors in mice until 104 NSP cells were injected (two of five mice).Tumor latency was 64 - 98 days.Tumorigenicity of SP cells was enhanced by at least 100-fold than that of NSP cells.The SP cells regenerated both SP [ ( 2.09 ± 0.73 ) ％ ] and NSP populations in vivo with a fraction size that was comparable to the original population.The mRNA expression ofstemness genes Oct-4,Klf4 and ABC transporters ABCG2,ABCC2 genes were elevated in SP cells compared to NSP cells,the fold changes were 1.95±0.41 (P＜0.05),4.26 ±0.63 (P＜0.01),3.22±0.36 (P＜0.01),and 1.76±0.26 (P＜0.01 ),respectively.The relative activity of SP and NSP cells were 0.757 ± 0.105 versus 0.474 ± 0.035 (P＜0.01),0.521 ±0.092 versus 0.384 ±0.073 (P＜0.05),0.742 ±0.051 versus 0.526 ±0.088 (P ＜0.01 ),and 0.690 ± 0.096 versus 0.466 ± 0.112 ( P ＜ 0.01 ) when they exposed to 0.25 μg/ml cisplatin, 0.01μmol/Lpaclitaxel, 0.25μmol/Ldoxorubicin, and0.05μg/mlmitoxantrone,respectively.ConclusionsSP cells from OVCAR-3 have enhanced self-renewal,differentiation,and tumorinitiating capacity compared to NSP cells.The mRNA expression of stemness genes and ABC transporters are markedly elevated in SP cells,which showed resistance to multiple chemotherapeutic drugs and have characteristics of cancer stem-like cells.Therefore,SP phenotype could be used as a marker to isolate the cancer stem-like cells in ovarian cancer.
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ObjectiveTo investigate the expression of stem cell marker adenosine triphosphate (ATP)-binding cassette transporter 2 (ABCG2) in the tissue and side population (SP) of a cell line A431 of human cutaneous squamous cell carcinoma(SCC).MethodsSP cells were separated by flow cytometry from cultured A431 cells.Methyl thiazolyl tetrazolium(MTT) assay was used to evaluate the proliferative ability of,reverse transcription-PCR to determine the expression of ABCG2 in,SP and non-SP cells.Immunohistochemistry (MaxVision method) was carried out to detect the expression of ABCG2 protein in tissue specimens from 10 patients with SCC.ResultsSP cells existed in cultured A431 cells,and accounted for about 1.1％ of A431 cells.The SP cells had a stronger growth and colony-forming ability than non-SP cells did.The number of cell clones formed by SP cells and non-SP cells was 114.8 ± 4.95 and 44.5 ± 3.67,respectively,per well in a 24-well plate( t =27.92,P ＜ 0.01 ).The expression level of ABCG2 mRNA was significantly higher in SP cells than in non-SP cells(t =5.22,P＜ 0.01).There existed a small number of ABCG2 positive cells in SCC tissue,and ABCG2 was mainly expressed in the cytoplasm and membrane of tumor cells.ConclusionsSP cells exist in A431 cells,which have characteristics of stem cells and highly express ABCG2.ABCG2 may be a potential stem cell surface marker of SCC.
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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.
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In the occurrence and development of tumors, side population cells play an extremely important role. They have the characteristics of cancer stem cells, especially their potential of tumor originating, and stronger drug-resistance. The study of biological characteristics, sorting and training methods of side population cells and the relationship between side population cells and tumor drug-resistance may shed some light on the diagnosis and treatment of cancer.
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Objective To investigate the drug resistance of side population cells in human gallbladder cancer cell line GBC-SD and explore its mechanism. Methods Drug sensitivity assays of 5chemotherapeutic agents were performed on side population cells (SP) and non-SP cells of GBC-SD.GBC-SD was cultured and then treated with the chemotherapeutic agent gemcitabine. The frequency of SP by FACS was measured. RT-PCR and Western blotting were used to detect the expression of AB-CG2 in both the SP and the corresponding non-SP subsets. Results After 1 d treatment with 4 chemotherapeutic agents (gemcitabine, cisplatin, 5-fluorouracil and mitoxantrone) in IC50 concentration to GBC-SD cell line, the reproductive ability of SP was higher than that of non-SP (P0.05). The percentage of SP in GBC-SD treated with chemotherapeutic agent gemcitabine after 3 weeks was sharply elevated by FACS (8.02% ±0.13% vs 0.62% ±0.08%, P<0.05), and the expression of ABCG2mRNA and protein were increased in SP as compared with non-SP. Conclusion SP from human gallbladder cancer cell line GBC-SD, like stem cell, showed a heighten resistance to drugs. Increased expression of ABCG2 was largely responsible for the multi-drug resistance.
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Objective The fluorescence-activated cell sorting (FACS) technique is a method for the identification and isolation of different cell populations.At present,the special surface marker for limbal stem cells has been not found yet.This study aimed to investigate the application of FACS technique in the research of rabbit limbal stem cells.MethodsCorneal limbal tissue was obtained from New Zealand white rabbits and cultured using the explant culture method in SHEM.Side population cells (SP cells) and non-SP cells were sorted from cultured rabbit limbal epithelium cells by FACS at a excitation wavelength 350 nm,and acquistion length 450 nm (blue light) and 675 nm (red light).The SP cells and non-SP cells were identified by detecting the expression of ABCG2 and K3/K12.The colony-forming efficiency of SP cells and non-SP cells were evaluated by the observation of cellular vitality with trypan blue staining.The percentage of colony formation was calculated as the colony number in various group/200×100%.ResultsIn 48-72 hours after primary culture,limbal epithelial cells migrated from the cultured tissue mass to form the mambrane-like structure and achieved 70%-80% confluence.The cells showed round,polygon and flattened shape.The proportion of SP cells in cultured limbal epithelial cells was 0.22%±0.09% with a colony-forming efficiency of 5.52±0.45% in SP cells and 0.78%±0.73% in non-SP cells,with a statistically significant difference between the two populations (t＝2.17,P＜0.01).After verapamil,an inhibitor of the expression of the ABCG2 protein,was added into the medium,the proportion of SP cells in the cultured limbal epithelial cells declined to 0.04%±0.006%.The SP cells presented a positive immunoresponse for ABCG2 and absence of immunoresponse for K3/K12,but a contradictory staining result was found in non-SP cells.ConclusionFACS can be applied in the research of limbal stem cells.