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BACKGROUND:There is a certain cel subset in esophageal cancer tissues, with certain invasive and metastatic properties, which is closely related to the clinical therapeutic effect on tumors. OBJECTIVE:To isolate tumor stem cel spheres in human esophageal carcinoma cel lines KYSE-150 and TE-1 and to analyze their proliferation and invasion ability. METHODS:KYSE-150 and TE-1 cels were cultured in serum-free medium to observe the formation of cel spheres. Cel proliferation and invasion were detected using MTT and Transwel chamber culture. Surface markers of cels were detected using flow cytometry. RESULTS AND CONCLUSION: Cel spheres that were stably subcultured were obtained from KYSE-150 and TE-1 cels cultured in serum-free medium. The proliferation and invasion abilities of cel spheres were significantly stronger than those of parent cels (P < 0.05). The number of CD44+, CD271+ and CD44+CD271+ cels in TE-1 and KYSE-150 cel spheres was significantly higher than that in the TE-1 and KYSE-150 parent cels (P < 0.05). These experimental results show that cel spheres isolated from human esophageal carcinoma cel lines TE-1 and KYSE-150 have tumor stem cel properties as wel as strong proliferation and invasion abilities. And moreover, CD44 and CD271 can be used as important surface markers of esophageal carcinoma stem cels. Cite this article:Wang YL, Wang ZM, Wang Y, Tao YP, Han GY.Culture, differentiation, proliferation and invasion of tumor stem cels in human esophageal carcinoma cel lines KYSE-150 and TE-1. Zhongguo Zuzhi Gongcheng Yanjiu. 2016;20(1):55-59.
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BACKGROUND:Effective sorting of prostate cancer stem cels is the basis of experimental studies in prostate cancer developing. The most common sorting method is magnetic-activated cel sorting. OBJECTIVE:To separate CD133+/CD44+ cels in prostate cancer tissues using self-designed magnetic beads folowed by culture, passage and immunological identification. METHODS:Self-designed magnetic microspheres were applied to establish immunomagnetic beads to sort CD133+/CD44+ cels in prostate cancer tissues. The sorted cels were cultured in serum-free medium. The sphere formation, cel morphology, and proliferation ability after cel passage were statisticaly compared between the sorted cels and the normal tumor cel lines. Immunofluorescence detection was performed to detect the expression of specific antibodies. RESULTS AND CONCLUSION:Self-designed immunomagnetic beads had smal diameter and a high-sorting effect. The sorted cels possessed a high capacity of microsphere formation. After cel culture and passage, the cels highly expressed CD133 and CD44 antigens. The sorted cels with no induction had varying shapes and grew vigorously. After induction with transforming growth factor-β, the cultured cels were noted to have a single shape and grow slowly. The cel proliferation ability of sorted cels in these two groups differed significantly from that of the normal cancer cel lines (bothP < 0.05). In conclusion, the CD133+/CD44+ cels sorted from prostate tumor cels possessed cel morphology and function characteristics of stem cels which can provide a basis for extraction and culture of prostate cancer stem cels. Cite this article:Gong R, Li SY, Huo ZX, Ding H, Sun EL. Extraction of prostate cancer stem cels using self-designed magnetic beads. Zhongguo Zuzhi Gongcheng Yanjiu. 2016;20(1):36-41.
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Abstract BACKGROUND:Previous studies have found that cryptotanshinone represses multiple tumors, but little is reported on its effect on renal carcinoma. OBJECTIVE:To explore the effect of cryptotanshinone on the proliferation and apoptosis of the renal carcinoma stem cels. METHODS:CD133+ renal carcinoma stem cels were separated from OS-RC-2 cels by immunomagnetic bead separation. Effects of 0, 0.2, 1, 5 mg/L cryptotanshinone on the proliferation and apoptosis of CD133+ renal carcinoma stem cels were detected by MTT and flow cytometry, respectively. Expression levels of Ki67, Bcl-2, Caspase-3 and p-Caspase-3 protein were detected by western blot assay. RESULTS AND CONCLUSION:After magnetic cel sorting, the percentage of CD133+ cels was increased from 6.32% to 82.73%, and there was a significant difference before and after cel sorting (P < 0.001). Cryptotanshinone could repress the proliferation of CD133+ renal carcinoma stem cels and promote cel apoptosis in a dose-dependent manner. The protein expression levels of Ki67 and Bcl-2 in the 5 mg/L cryptotanshinone group were significantly decreased compared with the control group, while the protein expression level of p-Caspase-3 protein was significantly increased. In addition, there was no difference in the protein expression of Caspase-3 between cryptotanshinone and control group. These findings indicate that cryptotanshinone may be a potent anticancer drug for the treatment of renal carcinoma by inhibiting expression of Ki67 and Bcl-2 and promoting protein expression of p-Caspase-3. Cite this article:Feng M, Jia MH. Cryptotanshinone effects on the proliferation and apoptosis of renal carcinoma stem cels. Zhongguo Zuzhi Gongcheng Yanjiu. 2016;20(1):49-54.
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BACKGROUND:Retinoic acid is the most promising inducer for neuroblastoma minimal residual lesion, and it can induce cel differentiationin vivo, accompanied by reducing tumor cel proliferation. OBJECTIVE:To study the effect of nanoparticle labeling on biological characteristics of neuroblastoma stem cels, and the role of 13-cis retinoic acid to induce differentiation of neuroblastoma stem cels. METHODS:Neuroblastoma stem cels were isolated and culturedin vitro using serum-free suspension culture method, labeled with polylysine-modified γ-Fe2O3 nanoparticles and induced in culture medium containing 13-cis retinoic acid. RT-PCR was used to detect the expression of Oct-4 before and after labeling as wel as before and after induction. Immunofluorescence method was used to detect the expression of nestin before and after labeling as wel as before and after induction. RESULTS AND CONCLUSION:Neuroblastoma stem cels were successfuly cultured in the bone marrow samples from 5 of 20 cases. Polylysine-modified γ-Fe2O3 nanoparticle labeling did no influence the viability and proliferation ability of neuroblastoma stem cels, and also had no effect on Oct-4 mRNA and nestin expression. After cultured in the culture medium containing 13-cis retinoic acid, the cel shape changed and the growth rate slowed down. Moreover, the expression of Oct-4 mRNA and nestin was gradualy reduced. These findings indicate that polylysine-modified gamma-Fe2O3 nanoparticles can be used to label neuroblastoma stem cels, and 13-cis retinoic acid can induce the differentiation of neuroblastoma stem cels.
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BACKGROUND:Thinking from ovarian cancer stem cel theory shows that: in the tumor cels, there are a fraction of stem cels with self-renewing ability and multipotent differentiation, which are the root causes of ovarian cancer recurrence and drug resistance. Studies have shown that CD90 can be used as a surface marker of mesenchymal stem cels and stem cels of other cancers. OBJECTIVE:To explore the biological features of CD90+ tumor cels from ovarian cancer tissues. METHODS: Primary ovarian cancer cels were isolated from the abdominal dropsy of ovarian cancer patients to sort CD90+ and CD90- cels using flow cytometry. RT-PCR was used to detect expressions of stem cel-related genes and epithelial to mesenchymal transition-related genes. Cel invasion was observed by Transwel invasion assay, cel proliferation and differentiation observed by clone formation assay, stem cel potential observed by suspension sphere-forming assay, and tumor formation rate observed byin vivo tumorigenicity experiment. RESULTS AND CONCLUSION:Compared with the CD90- cels, the expressions of CD44, CD133, ALDH1, N-cad and Vimentine were significantly higher in the CD90+cels (P < 0.05), but the expression of E-cad was significantly decreased in the CD90+ cels (P < 0.05). Tumor formation rates of CD90- and CD90+ cels were increased significantly with the increase of seeded cel number, which was more obvious in CD90+ cels. The number of transmembrane cels, the number of cel clones and the number of suspended spheres were significantly higher in the CD90+ cels than the CD90- cels (P < 0.05). Experimental findings from this study show that CD90+ cels highly express epithelial to mesenchymal transition-related genes and stem cel-related genes, with higher invasion, proliferation and differentiation, in vivo tumorigenicity and potential of stem cels. CD90+ cel separation may be a new method to separate ovarian cancer stem cels.
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BACKGROUND:Recent studies have shown that cancer stem cels play a key role in the development of tumors, therefore, the research about cancer stem cels’ markers can deepen the understanding of the development and clinical diagnosis of the tumor. OBJECTIVE:To review the research progression of stem cel marker LGR5. METHODS: The first author retrieved PubMed database and Wanfang database for papers regarding LGR5 and stem cel/cancer stem cel published between January 1998 and December 2014 using the key Words “LGR5, stem cel, cancer stem cel” in English and Chinese, respectively. A total of 178 papers were initialy retrieved. After 123 papers with independent objective and out-of-date contents were excluded, 55 papers were suitable for final analysis. RESULTS AND CONCLUSION: LGR5 is the surface marker of intestinal, stomach, hair folicle stem cels, which has a great relationship with occurrence, development and prognosis of colorectal cancer, stomach cancer, lung cancer, ovarian cancer, liver cancer, basal cel carcinoma. As a candidate marker of cancer stem cels, LGR5 can be the new treatment target. Studies have shown that R-spondins (RSPOs) is a high affinity ligand of LGR5. They participate in the Wnt signaling pathway, regulating cel proliferation and differentiation.
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BACKGROUND:Breast cancer stem cels are relatively special cels in the body, which have the self-renewal and multi-differentiation ability to promote tumor formation and development, and maintain tumor growth for a long-term. Therefore, it is of great significance to analyze the expression of resistance proteins of breast cancer stem cels. OBJECTIVE:To isolate breast cancer stem cels from human breast cancer tissues, to observe their differentiation and morphology characteristics and to analyze their resistance proteins. METHODS:Thirty tumor samples of breast invasive ductal carcinoma were selected to separate single cel suspension using mechanical separation method, and breast cancer stem cels and differentiated cels were sorted with two-step immunomagnetic bead method. Two-step immunocytochemistry method was used to detect the expression of resistance proteins in breast cancer stem cels. RESULTS AND CONCLUSION:Percentage of breast cancer stem cels had no significant correlations with age, long diameter of the tumor, lymph node metastasis and histological grading (P > 0.05). P-gp and GST-π positive rates in the breast cancer stem cels were significantly higher than those in the differentiated cels (P < 0.05); while TopoII and LRP positive rates in the breast cancer stem cels were significantly lower than those in the differentiated cels (P < 0.05). To conclude, breast cancer stem cels show stronger drug resistance than the differentiated cels by highly expressing P-gp and GST-π and lowly expressing TopoII and LRP, which may be the key reason for chemotherapy resistance in breast cancer.
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BACKGROUND:Neuroblastoma is the most common solid tumor in infants and children. Targeting cancer stem cel therapy can be expected to cure cancer radicaly, but because of smal number, anti-apoptotic and anti-chemotherapy capacity, cancer stem cels are not sensitive to the radiotherapy and chemotherapy. OBJECTIVE:To explore the expression of stem cel marker CD133 in neuroblastoma and its clinical significance. METHODS:Fifty-eight children with neuroblastoma admitted at Department of Pediatric Surgery, the First Affiliated Hospital of Xinjiang Medical University, China from June 2004 to February 2014 were enroled, including 46 cases of neuroblastoma and 12 cases of ganglion neuroblastoma. Then, the expression level of CD133 was analyzed by immunohistochemistry method, and the relationship between pathological types, International Neuroblastoma Staging System stage, survival time after surgery and the expression level of CD133 was explored. RESULTS AND CONCLUSION:There were 22 cases (48%) of neuroblastoma positive for CD133, and 4 cases (33%) of ganglion neuroblastoma positive for CD133. CD133 mainly expressed in the tumor cel cytoplasm. The expression rates of CD133 in different clinical stages were significantly different (P=0.011), which were 21% for stages 1 and 2, 64% for stages 3 and 4, 36% for stage 4S. And the positive rates of CD133 between patients with good prognosis and patients with poor prognosis were significantly different (P=0.031), which were 29% and 57%, respectively. The life cycle of CD133-negative patients was significantly longer than that of CD133-positive infants (P < 0.05). There were tightly connections between CD133 and the occurrence, development, and prognosis of neuroblastoma, and thus, CD133 is of great significance to assess the survival time after surgery and improve of the diagnosis and treatment of neuroblastoma.
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BACKGROUND:Studies have shown that microRNAs (miRNAs) have moderating effect on the renewal and differentiation of cancer stem cels. However, there is no complete understanding on the effect of microRNA-17-92 gene on gastric cancer stem cel renewal and proliferation. OBJECTIVE:To explore the effect of miRNA-17-92 in promoting self-renewal and proliferation of gastric cancer stem cels. METHODS:(1) The gradualy reduced miRNAs during gastric cancer stem cel self-renewal were investigated using miRNA array based on RNAs from differentiated and adherent cels. (2) The miRNA-17-92 was constructed and transfected to gastric cancer stem cels. (3) The effects of miRNA-17-92 on the self-renewal of gastric cancer stem cels were studied by tumor sphere assayin vitro. (4) The effects of miRNA-17-92 on the proliferation of gastric cancer stem cels were investigated by MTT assay and colony formation assay. RESULTS AND CONCLUSION:(1) miR-19b/20a/92a expression gradualy reduced in the adhesion and differentiation of gastric cancer stem cels. (2) The expression of lentivirus carrying miRNA-17-19 gene in MKN28 cels and CD44-/EpCAM- cels were significantly increased; transient transfection of pre-miR-19b/20a/92a increased the expression of CD44-/EpCAM- and MKN28 miRNA, transient transfection of pre-miR-19b/20a/92a antagonists reduced the expression of SGC7901 and CD44+/EpCAM+ miRNA; overexpression of lenti-miR-19b/20a/92a significantly increased the ability of gastric cels to form tumor spheres; chemotherapy drugs prolonged the survival time of lenti-miR-19b/20a/92a-infected cels; transient transfection of pre-miR-19b/20a/92a significantly increased the number of CD44+/EpCAM+ cels, but transfection of pre-miR-19b/20a/92a antagonist reduced the number of CD44+/EpCAM+ cels. (3) MTT proliferation assay showed that gastric cancer cel proliferation rate in miR-19b/20a/92a stably expressing group was faster than that in the control group. Transient transfection of miR-19b/20a/92a precursor accelerated the growth rate of gastric cancer cels, and transient transfection of its antagonist slowed down the growth rate of gastric cancer cels. Colony formation assay showed that transient transfection of miR-19b/20a/92a precursor significantly increased the colony formation number as compared with the control group; transient transfection of miR-19b/20a/92a antagonist reduced the colony formation as compared with the control group. These findings indicate that miR-19b/20a/92a gene presents with continuous deletion in gastric cancer stem cel differentiation process, and miRNA-17-92 gene can promote the renewal and proliferation of gastric cancer stem cels.
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BACKGROUND:Lung adenocarcinoma stem cels are a very important marker for diagnosis of lung adenocarcinoma. OBJECTIVE:To investigate the relationship between human lung adenocarcinoma stem cel phenotypes and patient’s prognosis. METHODS:From February 2010 to January 2013, 48 patients with lung adenocarcinoma admitted in the First Affiliated Hospital of Xinjiang Medical University were enroled. Lung adenocarcinoma cancer stem cel phenotypes were detected with immunofluorescence method, and the relationship of different phenotypes and clinical characteristics with patient’s prognosis was compared. RESULTS AND CONCLUSION: SP-C and CCSP expression was observed in the lung adenocarcinoma tissues of 48 cases, possessing the phenotypic characteristics of bronchioloalveolar stem cels. Of the 48 patients, OCT4 was positive in 34 cases (OCT4+ group) and negative in 14 cases (OCT4- group). There was no difference between the two groups in terms of patient’s age, gender, smoking history and tumor stage (P > 0.05). There were 23 cases (68%) in the OCT4+ group with cancer cel metastasis, which was significantly higher than that in OCT4- group (21%;P > 0.05). In the aspects of patient’s age, gender, smoking history, staging and cancer metastasis and other clinical pathological stratification, the 2-year survival rate in the OCT4- group were generaly higher than that in the OCT4+ group, and there was a significant difference in the survival curves of two groups (P < 0.05). Human lung adenocarcinoma stem cels have the phenotypic characteristics of bronchioloalveolar stem cels, and meanwhile, if the cels are positive for OCT4, patient’s prognosis is poor and associated with lung adenocarcinoma metastasis.
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BACKGROUND:Tumor cels are resistant to chemotherapeutic drugs, and drug resistance is closely correlated with tumor stem cels. Therefore, how to kil tumor stem cels wil become the key to the treatment of oral squamous cel carcinoma. OBJECTIVE:To study the effect of 5-fluorouracil on biological characteristics of tongue squamous cel carcinoma Tca8113 cels. METHODS:Viability of Tca8113 cels treated with different concentrations of 5-fluorouracil was determined by cel counting kit-8, and the best drug concentration and time were screened for subsequent experiments. Tca8113 cels without 5-fluorouracil acted as control group. Then the cel cycle and percentage of the side population cels in Tca8113 cels were determined by flow cytometry. Scratch test was used to determine the migration ability of Tca8113 cels. RESULTS AND CONCLUSION:Results from cel counting kit-8 showed that 5-fluorouracil inhibited the viability of Tca8113 cels positively in a time- and dose-dependent manner. Tca8113 cels under intervention with 50 mg/L 5-fluorouracil for 48 hours showed lowest cel viability. Flow cytometry results showed that in the experimental group, G0/G1 phase cels increased significantly compared with the control group (P=0.01), S phase cels decreased significantly compared with the control group (P=0.244), and G2/M phase cels disappeared completely. After treatment with 5-fluorouracil, the percentage of side population cels was increased significantly (P=0.00). The scratch test showed that in the experimental group, the cels had better ability of wound healing than those in the control group. In conclusion, 5-fluorouracil can enrich the cancer stem cel population in Tca8113 cels.
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BACKGROUND:EpCAMhighCD44+ colon cancer stem cels have been isolated in recent years, and most of them are related to the study of isolating tumor stem cels in colorectal cancer cel lines. There are less reports on the function of colon cancer stem cels. OBJECTIVE:To isolate, screen and culture colon cancer stem cels from colorectal carcinoma tissues and to explore the protein expression and biological characteristics of colon cancer stem cels. METHODS: The primary cels were isolated from the colon cancer tissues. EpCAMhighCD44+ cels were detected and sorted by flow cytometry. Then, these cels were cultured in serum-free medium. After the cels were replanted subcutaneously into the mice, we observed the daily performance and tumor growth in the mice. When the tumor diameter was above 1 cm, tumor tissues were taken for immunohistochemical testing. The growth of EpCAMhighCD44+ cels cultured in the serum-free medium was observed; expressions of neutral epithelial mucin and CK-20 in EpCAMhighCD44+ cels were detected. RESULTS AND CONCLUSION: EpCAMhighCD44+ colon cancer stem cels were detected in the colon cancer tissues, and the proportion of EpCAMhighCD44+ cels in primary colon cancer cels was 1.7%-38%. After 24 hours of serum-free culture, there were a few of smal suspended cel spheres which were incompact. After 21 days, dense cel spheres with uniform size were found. The formation of transplanted tumor was found in the nude mice. Hematoxylin-eosin staining showed the transplanted tumor had the typical characteristics of colon cancer cels. The expression of neutral epithelial mucin and CK-20 was observed in al the transplanted tumors. Additionaly, the proliferation of EpCAMhighCD44+ cels could be promoted by 5-fluorouracil (10, 1, and 0.1 PPC) with time. These findings indicate that EpCAMhighCD44+ colon cancer stem cels in the colon cancer have a certain ability of proliferation, regeneration, differentiation, tumor formation and chemotherapy resistance.
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BACKGROUND:5-Fluorouracil is a common chemotherapy drug for gastric cancer, but it is more likely to develop drug resistance in clinical treatment. Studies have shown that tumor stem cels are lowly sensitive to chemotherapeutic drugs, which may be an important cause of chemotherapy resistance. OBJECTIVE:To analyze the sensitivity of gastric cancer stem cels to 5-fluorouracil in vitro, and to understand the mechanism of drug resistance associated with gastric cancer. METHODS:Based on the sorting strategy, human gastric cancer cel clones were isolated from AGS cel lines. CD44 and thymidylate synthetase expression in different clones was detected using immunocytochemistry analysis. Clone formation assay was used to evaluate self-renewal capacity of different clones. Cel counting kit-8 was used to determine the clonal growth inhibition rate of AGS under treatment with different concentrations of 5-fluorouracil. RESULTS AND CONCLUSION:The AGS cels that were inoculated with low density and cultured could differentiate into 32 forms, including the ful clone (16%, 5/32), the second clone (66%, 21/32) and the accessory clone (19%, 6/32). Among them, the ful clones highly expressed CD44 and thymidylate synthetase, and could generate a great amount of passage 2 clones after inoculation; the second clones showed a weak expression of CD44 and thymidylate synthetase, and could generate a smal number of passage 2 clones after inoculation; the accessory clones showed a weak or no expression of CD44 and thymidylate synthetase, and there was no passage 2 clone after inoculation. Under the effect of different concentrations of 5-fluorouracil, the growth inhibition rates of the secondary clones and AGS cels were both higher than that of the ful clones (bothP < 0.05). These findings indicate that gastric cancer stem cels have a relatively lower sensitivity to 5-fluorouracilin vitro, which is speculated to be an important mechanism of drug resistance.
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BACKGROUND:DCLK1 is a transmembrane microtubule-associated kinase in neurons after mitotic division, which may be the intestinal cancer stem cel marker. OBJECTIVE:To observe the expression and pathological significance of DCLK1 and Ki67 in colorectal cancer. METHODS: Expression of Ki67 and DCLK1 in 150 cases of colorectal cancer tissues was detected by immunohistochemical method in contrast to normal colorectal mucosa, para-carcinoma tissue, and adenoma tissue. RESULTS AND CONCLUSION:The expression rates of DCLK1 and Ki67 were 36.7% and 34.7% in cancer tissues, respectively, both of which were significantly higher than those in normal colorectal mucosa and adenoma. The expression of DCLK1 was associated with the location, depth of invasion, lymph node metastasis (P < 0.05), while the expression of Ki67 was just associated with the depth of invasion (P < 0.05). There was a negative correlation between the expression of DCLK1 and Ki67 (r=-0.460,P=0.000). The count of DCLK1+/Ki67-cels was about 2.01% in colorectal cancer tissues, and these cels mainly distributed at the bottom of intestinal mucosa base and common duct wal. DCLK1+/Ki67- cels were oval, the nuclei were large and deep-stained with prominent nucleolus, and there was rare nuclear fission and less cytoplasm. From the aspects of cel number, location, and cel morphology, DCLK1+/Ki67- cels are in line with the characteristics of cancer stem cels; therefore, DCLK1+/Ki67-can be used as a cancer stem cel marker of colorectal cancer.