Influence of the interaction between tumor microenvironment and liver cancer stem cells on the development and progression of hepatocellular carcinoma / 临床肝胆病杂志

In recent years, liver cancer stem cells (LCSC) have been considered one of the main causes of treatment failure and recurrence of hepatocellular carcinoma (HCC). Many studies have shown that LCSC are a small fraction of cells with the abilities of self-renewal, differentiation, and tumorigenesis in HCC tumor, which can initiate the onset of HCC and affect its proliferation, invasion, metastasis, recurrence, and drug resistance. Therapies based on tumor microenvironment (TME) have been developed recently, and a number of studies have found that targeting the relevant elements of TME has a higher therapeutic value than targeting tumor cells themselves. TME is the microenvironment for the growth of LCSC and HCC cells, and it interacts with LCSC and has a synergistic effect, thereby playing a positive role in the development and progression of HCC. This article introduces how various cellular components and non-cellular components in TME interact with LCSC to regulate the development and progression of the HCC. In addition, this article also describes the molecular targets, therapies, and drugs associated with the main components of TME and LCSCs, in order to seek safer and more effective targeted therapies for HCC.

Effects of autophagy-mediated crizotinib resistance on cancer stem-like cell subsets in anaplastic lymphoma kinase-positive anaplastic large cell lymphoma and its significance / 白血病·淋巴瘤

Objective:To investigate the effects of autophagy-mediated crizotinib resistance on cancer stem-like cell subsets in anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALK + ALCL). Methods:The preliminary research of our group divided ALK + ALCL Karpas299 cell line into two subgroups: reporter unresponsive (RU) and reporter responsive (RR) cells through the implantation of Sox2 reporter genes, among which the RR cells had the characteristics of stem cells. Fluorescent labeled LC3 overexpressing RR and RU cells (RR-LC3 and RU-LC3) were constructed by lentiviral transfection technique, and the transfection efficiency was verified by using Western blotting and flow cytometry. RU-LC3 and RR-LC3 were treated with crizotinib at different concentrations (0, 250, 500, 1 000 nmol/L). The RED and GEN signals were detected by using double-signal flow cytometry to observe autophagy flux (RED represents the red signal B695 of the next generation of far-red fluorescent protein TagFP635 mKate; GEN represents the green signal from pH-sensitive GFP variant pHluorin B530), and the RED to GEV ratio represents autophagy flux. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect autophagy related genes ULK1, WIPI1 and LC3B mRNA expression levels in cells. The effects of different concentrations of crizotinib (250, 500, 1 000 nmol/L) combined with chloroquine (5, 10 μmol/L) on the cell survival were detected by using MTS assay. Results:RU-LC3 and RR-LC3 cells with overexpression of LC3 were successfully constructed. After induction of 250, 500 and 1 000 nmol/L crizotinib, the RED to GEN ratio in RU-LC3 cells was 1.135±0.017, 1.453±0.017 and 1.755±0.021, respectively; the RED to GEN ratio in RR-LC3 cells was 1.193±0.018, 2.116±0.013 and 3.307±0.189, respectively; the RED to GEN ratio in RU-LC3 cells and RR-LC3 cells showed a dose-dependent manner. The RED to GEN ratio in RR-LC3 cells was higher than that in RU-LC3 cells when treated with same concentrations of crizotinib, and the differences were statistically significant (all P < 0.01). The autophagy flux of RR-LC3 cells was larger than that of RU-LC3 cells. When treated without crizotinib, mRNA relative expression levels of ULK1, WIPI1 and LC3B in RR cells were higher than those in RU cells (1.69±0.05 vs.1.01±0.02, t = -1.62, P < 0.01; 1.24±0.04 vs. 1.03±0.05, t = -2.11, P < 0.01; 1.70±0.22 vs. 1.02±0.05, t = -1.74, P = 0.033). In the absence of chloroquine, the half-inhibitory concentration ( IC50) of crizotinib in RR cells was higher than that of RU cells (950 nmol/L vs. 709 nmol/L). After treated with chloroquine, IC50 of RU cells did not change, while IC50 of RR cells was decreased with the increase of chloroquine concentration. Conclusions:Compared with RU cells, autophagy reaction of cancer stem-like RR cells is more rapid and intense, which is considered to be one of the important reasons for their resistance to crizotinib.

Enrichment and isolation of breast cancer stem cells from breast cancer transplantation tumors in nude mice / 肿瘤研究与临床

Objective:Enriching and isolating breast cancer stem cells from breast cancer transplantation tumors in nude mice.Methods:Human breast cancer MDA-MB-231 cells were injected into the right axilla subcutaneous of 20 nude mice, and the tumor growth was observed .After 30 days, tumors were isolated and stained with hematoxylin and eosin, and then tumor cells from tissues were isolated. DMEM medium containing serum was used to cultivate isolated transplantation tumor cells, cell morphology and growth were also observed. Flow cytometry was used to detect the proportion of stem cells (CD44 +/CD24 -/low cells) in transplantation tumor cells. Serum-free DMEM medium containing epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and B27 cell supplement were used to cultivate transplantation tumor cells and to obtain cell microspheres. The proportion of stem cells on the 10th day in cell microspheres was detected by using flow cell sorter and stem cells were isolated according to the markers of cell surface. Results:After subcutaneously injecting MDA-MB-231 cells into 20 nude mice for 9 days, 17 nude mice had subcutaneous tumors with more parenchymal cells, little interstitial cells, arranged cords tumor cells, large volume of the cell and abundant cytoplasm, the nuclei in different sizes and hyperchromatic state, mitotic more common, the nucleoli clear and obvious pleomorphy. After cultivating transplantation tumor cells with DMEM medium containing serum, the cells began to grow adherent after 24 h, and the adherent proportion rose to 60% after 3 days; after 7 days, the cell proliferation was accelerated; and the cell morphology was more consistent, most of which were spindle shaped and were not significantly different from MDA-MB-231 cells; the proportion of stem cells in transplantation tumor cells was (0.10±0.02)%. After cultivating transplantation tumor cells with serum-free DMEM medium containing cell cultured supplement, the cells grow in spherical patterns, the proportion of stem cells in cell microspheres got up to (70.47±2.03)% on the 10th day.Conclusions:Subcutaneously injecting MDA-MB-231 cells in nude mice can build breast cancer nude mice ectopic transplantation tumor model. Breast cancer stem cells in the transplantation tumors can be enriched from isolated transplantation tumor cells through serum-free medium, and more stem cells can be isolated to provide the research basis for the biological characteristics of breast cancer stem cells.

Relationship between CDCA8 and tumor progression as well as that between CDCA8 and stemness maintenance of stem cells / 国际肿瘤学杂志

Cell division cycle associated 8 (CDCA8) is involved in the accurate regulation of cell division cycle. The expression change of CDCA8 can affect the progression of many kinds of tumors as well as the prognosis of patients. In recent years, studies have suggested that CDCA8 may play an important role in maintaining the stemness of cancer stem cells. Exploring the mechanism of CDCA8 affecting tumor is of great significance for the diagnosis and treatment of tumors.

Immunohistochemical expression of OCT4 and CD44 in major and minor salivary gland neoplasms

Abstract The aim of this study was to identify tumor parenchyma cells exhibiting immunohistochemical profile of stem cells by evaluating the immunoreactivity of OCT4 and CD44 in a number of cases of salivary gland neoplasms. The sample consisted of 20 pleomorphic adenomas, 20 mucoepidermoid carcinomas, and 20 adenoid cystic carcinomas located in major and minor salivary glands. The expression of OCT4 and CD44 was evaluated by the percentage of positive cells and the intensity of expression. All studied cases showed positive expression of OCT4 and CD44 and higher values than the control groups. For OCT4, luminal and non-luminal cells were immunostained in the case of pleomorphic adenomas and adenoid cystic carcinomas. Moreover, the immunoreactivity of CD44 was particularly evident in the non-luminal cells of these lesions. In mucoepidermoid carcinomas, there was immunoreactivity for both markers in squamous and intermediate cells and absence of staining in mucous cells. For both markers, a significantly higher immunostaining was verified in neoplasms located in the major salivary glands compared with lesions in minor salivary glands (p<0.001). In the total sample and in minor salivary glands, malignant neoplasms exhibited higher immunoreactivity for OCT4 than pleomorphic adenoma. A significant moderate positive correlation (r = 0.444 and p ≤ 0.001) was found between OCT4 and CD44 immunoexpression in the total sample. The high expression of OCT4 and CD44 may indicate that these proteins play an important role in identifying tumor stem cells.

Effects of Arf6 downregulation on biological characteristics of human prostate cancer cells

ABSTRACT Objective To evaluate the effects of Arf6 downregulation on human prostate cancer cells. Materials and Methods The effects of Arf6 downregulation on cell proliferation, migration, invasion and apoptosis were assessed by MTT, BrdU, scratch, Transwell assays and flow cytometry respectively. AKT, p-AKT, ERK1/2, p-ERK1/2 and Rac1 protein expressions were detected by Western blot. Results Downregulating Arf6 by siRNA interference suppressed the mRNA and protein expressions of Arf6. The proliferation capacities of siRNA group at 48h, 72h, and 96h were significantly lower than those of control group (P <0.05). The migration distance of siRNA group at 18h was significantly shorter than that of control group (P <0.01). The number of cells penetrating Transwell chamber membrane significantly decreased in siRNA group compared with that of control group (P <0.01). After 24h, negative control and normal control groups had similar apoptotic rates (P >0.05) which were both significantly lower than that of siRNA group (P <0.01). After Arf6 expression was downregulated, p-ERK1/2 and Rac1 protein expressions were significantly lower than those of control group (P <0.05). Conclusion Downregulating Arf6 expression can inhibit the proliferation, migration and invasion of prostate cancer cells in vitro, which may be related to ERK1/2 phosphorylation and Rac1 downregulation.

miR-125a-5p inhibits cancer stem cells phenotype and epithelial to mesenchymal transition in glioblastoma

SUMMARY OBJECTIVE Glioblastoma (GBM) is a common type of cancer with high mortality. Epithelial to mesenchymal transition (EMT) plays a vital role in the development of glioblastoma. The aim of this study is to evaluate the role of miR-125a-5p in glioblastoma and in the tumorigenesis of chemotherapeutic drug-resistant cancer stem-like cells in brain glioma. METHODS The role of miR-125a-5p in the regulation of CSCs, EMT, migration, and invasion in glioblastoma was measured in this study. RESULTS We showed the roles of miR-125a-5p in the regulation of CSCs, EMT, migration, and invasion in glioblastoma. miR-125a-5p can inhibit the CSCs phenotype and EMT in glioblastoma cells. In addition, its over-expression can significantly regulate CSCs-associated genes and EMT-associated gene expression in glioblastoma cells. CONCLUSIONS We concluded that miR-125a-5p is one of the key microRNAs regulating CSCs and EMT programs in glioblastoma. The results suggested that miR-125a-5p might be a novel therapy target for glioblastoma.

RESUMO OBJETIVO O glioblastoma (GBM) é um câncer comum e de alta mortalidade. A transição epitélio-mesênquima (EMT) desempenha um papel vital no desenvolvimento do glioblastoma. O objetivo deste estudo é avaliar o papel do miR-125a-5p no glioblastoma e a tumorigênese de células-troco cancerígenas resistentes a medicamentos quimioterápicos em gliomas cerebrais. METODOLOGIA Os papéis do miR-125a-5p na regulação de células-tronco cancerígenas, EMT, migração e invasão do glioblastoma foram medidos neste estudo. RESULTADOS Mostramos a função do miR-125a-5p na regulação das células-tronco cancerígenas, EMT, migração e invasão do glioblastoma. O miR-125a-5p pode inibir o fenótipo e a EMT de células-tronco cancerígenas em células de glioblastoma. Além disso, a sua superexpressão pode regular de forma significante genes associados às células-tronco cancerígenas e a expressão de genes associados à EMT em células de glioblastoma. CONCLUSÕES Concluímos que o miR-125a-5p é um dos principais microRNAs na regulação de células-tronco cancerígenas e programas de EMT em glioblastomas, e os resultados sugerem que o miR-125a-5p pode ser um novo alvo terapêutico em casos de glioblastoma.

Activation of wnt/β-catenin signaling enhanced stemness of prostate cancer cells / 肿瘤

Objective: To explore the relationship between the activity of Wnt/β-catenin signaling pathway and prostate cancer stem cells by utilizing a Wnt/β-catenin signaling reporter system. Methods: Immunofluorescence staining was used to explore the activation of Wnt/β-catenin signaling pathway in prostate cancer. Wnt/β-catenin signaling reporter plasmid 7TGP carrying T cell specific transcription factor (TCF) binding site and enhanced green fluorescent protein (GFP) was transfected into prostate cancer PC3 and DU145 cells by liposomes, respectively. Furthermore, the top 5% (GFP+) and bottom 5% (GFP-) groups of PC3 and DU145 cells according to the fluorescent intensity of GFP were collected via flow cytometry. Then Western blotting assay was used to compare the expression level of activated β-catenin protein in the nucleus of GFP+ and GFP- cell groups. The real-time fluorescent quantitative PCR was used to detect the expressions of Wnt/β-catenin signaling downstream target genes in GFP+ and GFPcell groups. Sphere formation assay and real-time fluorescent quantitative PCR were used to compare the stemness of GFP+ and GFP- cells. Results: The activity of canonical Wnt signaling pathway was low in most of prostate cancer cells, in which β-catenin was not activated and translocated to the nucleus. While β-catenin was increasingly translocated to the nucleus in a small percentage of prostate cancer cells resulting in high activation of Wnt signaling pathway. PC3-GFP+ and DU145-GFP+ cells had more expression of nuclear β-catenin comparing with the corresponding GFP- cells (both P < 0.05), and the expression level of Wnt signaling pathway down-stream target gene Axis inhibition protein 2 (AXIN2) was up-regulated (both P < 0.05). In addition, the expression levels of cancer stem cell markers including B-cell-specific moloney leukemia virusinsert site 1 (BMI1) and aldehyde dehydrogenase family 1 member A1 (ALDH1A1) were up-regulated in PC3-GFP+ and DU145-GFP+ cells comparing with the corresponding GFP- cells (all P < 0.05). The sphere formation capacity was remarkedly up-regulated in PC3-GFP+ and DU145-GFP+ cells comparing with the corresponding GFP- cells (both P < 0.05). Conclusion: Prostate cancer cells with higher activity of Wnt/β-catenin signaling show the enhanced stemness.

Advances in targeted mitochondria inhibiting cancer stem cells / 肿瘤

Cancer stem cells (CSCs) have long been considered as the origin of tumorigenesis, recurrence and metastasis. The changes of mitochondrial structure and morphology, abnormal activation of signaling pathways, mitochondrial dysfunction, reactive oxygen species (ROS) and mitochondrial autophagy in CSCs are the key factors to maintain the stem cell nature of CSCs and to regulate the proliferation and apoptosis of CSCs, and are also the major cause of anticancer treatment failure. This review particularly elucidates the mechanisms of mitochondria in CSCs affecting apoptosis, signaling pathway, energy metabolism, ROS and mitochondrial autophagy, in order to provide a powerful basis for the potential anti-tumor therapy targeting mitochondria in CSCs.

Signal transduction mechanism and targeted therapy of melanoma stem cells / 肿瘤

Melanoma is the most serious type of skin cancer with high metastasis potential and very low survival rate. Although the molecular targeted therapy and immunotherapy have prolonged the survival time of some patients with melanoma in the past few years, a considerable number of patients still can not obtain a longer stable remission period. According to the recent study, melanoma stem cells (MSCs) are one of potential causes of tumor invasion, drug resistance, distant metastasis and recurrence. MSCs can express a variety of cellular markers, and participate into the regulation of tumor-related signal transduction. At the same time, these signaling pathways can retroregulate the expression of cellular markers, and promote the development of melanoma. Therefore, targeting MSCs markers and blocking the related signal transduction pathway can inhibit the growth of melanoma. In this review, the cellular markers, signal transduction mechanism and targeted therapy of MSCs have been discussed in order to provide new ideas for the treatment of melanoma.

Effect of Yanghe decoction serum on the proliferation of breast cancer stem cells / 国际中医中药杂志

Objective To investigate the effect of Yanghe decoction serum on the proliferation of breast cancer stem cells HMLER90hi and its mechanism. Methods Twenty female Sprague-Dawley rats were randomly divided into low,medium and high dose groups containing drug serum and control group, in order to prepare the Yanghe decoction serum and blank serum. After 24 hours of drug intervention,the effects of each group on the proliferation of HMLER90hi cells at 24 h,48 h,and 72 h were detected by MTT assay. The expression of EphA4 and p50 mRNA in each group were detected by real-time fluorescence quantitative PCR. Results Compared with the blank control group,the cell proliferation activity of each dose group of Yanghe decoction significantly decreased at 24 h (0.818 ± 0.061, 0.706 ± 0.073, 0.587 ± 0.052 vs. 0.928 ± 0.075), 48h (0.760 ± 0.047, 0.638 ± 0.056, 0.510 ± 0.059 vs. 0.973 ± 0.095), and 72 h (0.672 ± 0.102, 0.508 ± 0.092, 0.448 ± 0.048vs.1.023 ± 0.099) (P<0.05 orP<0.01), respectively. After 24 h of drug intervention, compared with the control group, the expression of EphA4 mRNA (0.54 ± 0.07, 0.54 ± 0.07, 0.33 ± 0.04 vs.0.68 ± 0.09) significantly decreased, and p50 mRNA (0.69 ± 0.10, 0.54 ± 0.08, 0.41 ± 0.06vs. 0.85 ± 0.13) significantly decreased in each dose group of Yanghe decoction (P<0.05 orP<0.01).ConclusionsTheYanghe decoction can inhibit the proliferation of breast cancer stem cell HMLER90hi,and its mechanism may be related to its inhibition of the conduction of the juxtacrine pathway of monocyte macrophage.

CD47 expression is decreased in hematopoietic progenitor cells in patients with myelofibrosis

Myelofibrosis (MF) is characterized by increased circulating hematopoietic progenitor cells (HPCs), abnormal cytokine levels, and the survival advantage of neoplastic progenitors over their normal counterparts, which leads to progressive disappearance of polyclonal hematopoiesis. CD47 is a surface glycoprotein with many functions, such as acting as a phagocytosis inhibitor of the expressing cell, that is increased in normal hematopoietic stem and progenitor cells mobilized into the blood and several human cancer-initiating cells, such as in acute myeloid leukemia. We compared CD47 expression in hematopoietic stem and progenitor cells of patients with MF and controls and found it to be decreased in progenitors of MF. Exposure of control HPCs to the cytokines transforming growth factor β and stromal-derived factor 1, which are important regulators of hematopoietic stem cell cycling and are overexpressed in patients with MF, did not modulate CD47 expression.

Research progress of cancer stem cells in triple-negative breast cancer / 国际肿瘤学杂志

There are abundant cancer stem cells (CSCs) in triple-negative breast cancer TNBC).CSCs can maintain their phenotype by unique molecular mechanism and tumor microenvironment,and can promote the chemotherapy resistance and recurrence of TNBC.Blocking these key molecules or altering tumor microenvironment can reduce CSCs,and then inhibited cancer growth and reverse chemotherapy resistance of TNBC.

Micro RNA-10b regulates aldehyde dehydrogenase 1 mRNA and protein expression in breast cancer MCF-7 cell line / 中国组织工程研究

BACKGROUND: MicroR-10 b can regulate the characteristics of breast cancer stem cells, and acetaldehyde dehydrogenase 1 (ALDH1) is one of the most important markers of breast cancer stem cells. The interaction between them in breast cancer cells needs further explorations. OBJECTIVE: To investigate whether over-expression of microRNA-10 b affects ALDH1 mRNA and protein levels in human breast cancer MCF-7 cells. METHODS: hsa-miR-10 b mimics or its negative control was transfected into breast cancer MCF-7 cell line. At 48 hours after transfection, the mRNA and protein expression of ALDH1 in the cells was detected using real-time RT-PCR and western blot assays, respectively. RESULTS AND CONCLUSION: Over-expression of microR-10 b was found in the MCF-7 cell line transfected with hsa-miR-10 b mimics, which was significantly higher than that in the control group (P=0.003 47). Both of ALDH1 mRNA and protein levels were up-regulated in the MCF-7 cell line overexpressing microR-10 b, as compared with the control group (P=0.009 54 and P=0.003 11, respectively). To conclude, over-expression of microR-10 b induces the ALDH1 mRNA and protein expression in the breast cancer MCF-7 cell line, providing new evidence that microR-10 b regulates the invasion and metastasis of breast cancer cells.

RNA interferes with Id2 gene expression to inhibit proliferation and invasion of PC-3 prostate cancer stem cells / 中国组织工程研究

BACKGROUND: The Id2 gene is an endogenous negative regulator of basic helix-loop-helix factor, which is involved in the cell proliferation, differentiation and existence. Id2 also shows functional diversity in the progression and infiltration of different types of tumors OBJECTIVE: To observe the changes of proliferation and invasiveness of PC-3 human prostate cancer stem cells after shRNA-Id2 transfection. METHODS: PC-3 human prostate cancer stem cells in logarithmic growth phase were harvested to isolate tumor stem cell spheres by serum-free suspension culture. The expression of CD44+CD24-on the surface of tumor stem cells was detected by flow cytometry. The shRNA-Id2 expression vector was constructed and transfected into PC-3 human prostate cancer stem cells. Untransfected PC-3 human prostate cancer stem cells were used as control. At 48 hours after transfection, the expression of Id2 gene and protein in shRNA-Id2 transfected prostate cancer stem cells, NC-shRNA empty vector transfected prostate cancer stem cells and untransfected prostate cancer stem cells were detected by RT-PCR and western blot, respectively. The proliferation and invasion of shRNA-Id2 transfected prostate cancer stem cells and untransfected prostate cancer stem cells were detected by MTT assay and Transwell chamber, respectively. The expressions of E-cadherin, vimentin and Twist were detected by western blot and RT-PCR. RESULTS AND CONCLUSION: Tumor stem cell spheres were successfully isolated by the serum-free suspension culture. The expression rate of CD44+CD24-on the surface of the third-generation PC-3 human prostate cancer stem cells was (85.69±8.96) ％, indicating that the cultured tumor stem cell spheres overexpressed the phenotype of tumor stem cells. At 48 hours after transfection, the expression of Id2 gene and protein was significantly lower in the shRNA-Id2 transfection group than the non-transfection group (P < 0.05) , indicating that the expression of Id2 was successfully interfered with the expression of PC-3 prostate cancer stem cells. The invasive ability of the cells in the shRNA-Id2 transfection group was significantly lower than that in the non-transfection group (P < 0.05). Western blot and RT-PCR detection showed that the expression of E-cadherin, an epithelial marker of PC-3 prostate cancer stem cells, in the shRNA-Id2 transfection group was significantly higher than that in non-transfection group (P < 0.05) , while the expression of vimentin, a marker of mesenchymal stem cells, and Twist, a transcription factor regulating cell-mesenchymal transformation, in the shRNA-Id2 transfection group was significantly lower than that in the non-transfection group (P < 0.05). These findings indicate that RNA interference with Id2 gene can inhibit the proliferation and invasion of PC-3 prostate cancer stem cells by regulating the expression of E-cadherin, vimentin and Twist.

Effect of targeted silencing Notch1 on proliferation and apoptosis of human non-small cell lung cancer stem cells / 国际肿瘤学杂志

Objective To investigate the effect of targeted silencing Notch1 on proliferation and apoptosis of human non-small cell lung cancer stem cells.Methods Lung cancer A549 cells and SPC-A-1 cells were selected and divided into control group,Nc-shRNA group and Notch1-shRNA group.The Nc-shRNA group was a negative control RNAi lentivirus group,and the Notch1-shRNA group was a Notch1 inhibitory RNAi lentivirus group.The lentiviral-mediated shRNA interference technology was used to target the silencing of Notch1.The silencing effect of Notch1 gene was verified by quantitative real time polymerase chain reaction (qRT-PCR) and Western blotting.Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) and sarcosphere formation assay.Apoptosis was detected by Annexin V/7-AAD double staining.Western blotting was used to detect the expression of proliferating cell nuclear antigen (PCNA),B-cell lymphoma-2 (Bcl-2) and Notch1 downstream gene Hes-1.Results The results of qRT-PCR showed that the relative expression levels of Notch1 in control group,Nc-shRNA group and Notch1-shRNA group in A549 cells and SPC-A-1 cells were 1.000 ± 0.000,0.937 ± 0.025,0.490 ± 0.036 and 1.000 ± 0.000,1.077 ± 0.070,0.373± 0.038,with statistically significant differences (F =359.707,P ＜0.001;F =210.455,P ＜0.001),further paired comparison,the relative expression of Notch1 in Notch1-shRNA group was significantly lower than that in Nc-shRNA group (all P ＜ 0.05).Western blotting showed that the expressions of Notch1 protein in A549 cells and SPC-A-1 cells were consistent with the mRNA results.MTT assay showed that the 24 h A values of A549 cells in control group,Nc-shRNA group and Notch1-shRNA group were 0.209 ± 0.005,0.219 ± 0.009,0.159 ±0.006,48 h A values were 0.293 ± 0.004,0.302 ± 0.004,0.205 ± 0.005,72 h A values were 0.450 ± 0.003,0.430 ± 0.012,0.348 ± 0.017,with statistically significant differences (F =79.487,P＜0.001;F =508.664,P ＜0.001;F =57.156,P ＜0.001),further paired comparison,the proliferation ability of Notch1-shRNA group was significantly lower than that of Nc-shRNA group at 24,48,72 h (all P ＜ 0.05).The 48 h A values of SPC-A-1 cells in control group,Nc-shRNA group and Notch1-shRNA group were 0.438 ±0.022,0.412 ± 0.015,0.364 ± 0.010,72h A values were 0.540 ± 0.016,0.519 ± 0.009,0.438 ± 0.019,with statistically significant differences (F =15.667,P =0.004;F =37.299,P ＜ 0.001),further paired comparison,the proliferation ability of Notch1-shRNA group was significantly lower than that of Nc-shRNA group at 48 h and 72 h (all P ＜ 0.05).The sphere sizes of control group,Nc-shRNA group and Notch1-shRNA group in A549 cells were (149.667 ± 6.506) μm,(136.667 ± 7.095) μm,(86.676 ± 7.638) μm,with statistically significant difference (F =65.940,P ＜ 0.001).The sphere sizes of the three groups in SPC-A-1 cells were (118.667 ± 6.658) μm,(128.000 ± 7.000) μm,(60.675 ± 4.509) μm,with statistically significant difference (F =105.372,P ＜0.001).Further paired comparison,the sphere size of Notch1shRNA group was significandy smaller than that of Nc-shRNA group in the two kinds of cells (all P ＜ 0.05).The apoptosis rates of control group,Nc-shRNA group and Notch1-shRNA group in A549 cells and SPC-A-1cells were (0.489 ± 0.014)％,(0.633 ± 0.021)％,(1.683 ± 0.221)％ and (1.323 ± 0.194)％,(1.690 ± 0.188) ％,(3.017 ± 0.356) ％,with statistically significant differences (F =77.660,P ＜ 0.001;F=32.200,P =0.001),further paired comparison,the apoptosis rate of Notch1-shRNA group was significantly higher than that of Nc-shRNA group in the two kinds of cells (all P ＜ 0.05).Western blotting showed that the expressions of PCNA,Bcl-2 and Hes-1 in control group,Nc-shRNA group and Notch1-shRNA group in A549 cells were statistically significant (F =155.343,P ＜ 0.001;F =22.576,P =0.002;F =70.108,P＜0.001),and the expressions of PCNA,Bcl-2 and Hes-1 in the three groups in SPC-A-1 cells were statistically significant (F =49.419,P ＜0.001;F =28.090,P =0.001;F =12.040,P =0.007).Further paired comparison,the expressions of PCNA,Bcl-2 and Hes-1 in Notch1-shRNA group were significantly lower than those in Nc-shRNA group in the two kinds of cells,and the differences were statistically significant (all P ＜0.05).Conclusion Targeted silencing of Notch1 can reduce the proliferation activity of lung cancer stem cells and promote apoptosis,which may be related to the down-regulation of its downstream gene Hes-1.

HER2-expressing breast tumors are associated with breast cancer stem-cell phenotype CD44+/CD24- / Tumores de mama que superexpressam HER2 estão associados ao fenótipo de células-tronco tumorais CD44+/CD24-

ABSTRACT INTRODUCTION: According to the cancer stem-cell theory, tumors originate from a small population of cancer stem cells, which lose the mechanism of self-regulation and begin to differentiate and proliferate indefinitely. The CD44+/CD24- phenotype may be considered a stem-cell marker in breast cancer. OBJECTIVE: To evaluate the correlation between CD44+/CD24- phenotype and different molecular subtypes of breast cancer in invasive ductal carcinoma samples. METHODS: The expression of CD44, CD44v6, and CD24 markers was investigated in 133 cases of invasive mammary carcinoma with immunohistochemistry. CD44+/CD24- phenotype was identified and correlated with the molecular subtypes and classical prognostic factors such as age, histological grade, tumor size, and lymph node status. RESULTS: Eighteen (14%) cases were positive for CD44+/CD24- (CD44+/CD24- or CD44v6+/CD24-) phenotype; among these, 11.1%, 27.8%, 38.9%, and 22.2% were luminal, luminal B-human epidermal growth factor receptor 2 (HER2), HER2, and triple-negative subtype, respectively. CD44+/ CD24- phenotype was more common in HER2 subgroup (p = 0.0197). CONCLUSION: CD44+/CD24- phenotype was correlated with molecular subtypes of breast cancer. The highest expression of CD44+/CD24- phenotype was reported in patients with HER2+ disease, a molecular subtype associated with more aggressive behavior and worse prognosis.

RESUMO INTRODUÇÃO: De acordo com a teoria das células-tronco tumorais, os tumores são originários de uma pequena população de células-tronco que perdem o mecanismo de autorregulação e começam a se diferenciar e proliferar indefinidamente. O fenótipo CD44+/CD24- pode ser considerado um marcador de células-tronco tumorais no câncer de mama. OBJETIVO: Avaliar a correlação entre o fenótipo CD44+/CD24- e os diferentes subtipos moleculares do câncer de mama em amostras de carcinoma ductal invasor. MÉTODOS: A expressão dos marcadores CD44, CD44v6 e CD24 foi investigada em 133 casos de carcinoma mamário invasor por meio de imuno-histoquímica. O fenótipo CD44+/CD24- foi identificado e correlacionado com os subtipos moleculares e os fatores prognósticos clássicos, como idade, grau histológico, tamanho do tumor e status do linfonodo. RESULTADOS: Dezoito (14%) casos foram positivos para o fenótipo CD44+/CD24- (CD44+/CD24- ou CD44v6+/CD24-), sendo 11, 1%, 27, 8%, 38, 9% e 22, 2% dos subtipos luminal, luminal B-human epidermal growth factor receptor 2 (HER2), HER2 e triplo negativo, respectivamente. O fenótipo CD44+/CD24- foi mais comum no subgrupo HER2 (p = 0, 0197). CONCLUSÃO: O fenótipo CD44+/CD24- foi correlacionado com os subtipos moleculares do câncer de mama. A maior expressão do fenótipo CD44+/CD24- foi encontrada em pacientes com doença HER2+, subtipo molecular associado a um comportamento mais agressivo e a um pior prognóstico.

Research Progress in the Therapeutic Strategy Based on Targeting at Lung Cancer Stem Cell / 中国肺癌杂志

With high morbidity and mortality, lung cancer is a major threat to human health and one of the focuses of tumor researches. Lung cancer stem cells (LCSCs) are regarded as a subpopulation of cells within lung cancer tissues with the capacity of self-renewal and differentiation, and might be related to tumorigenesis and heterogeneity of lung cancer. Tumor recurrence, metastasis and drug resistance of lung cancers could be clarified by LCSC hypothesis. Thus it's therapeutically prospective to target at these cells. This review summarizes the biomarkers of LCSCs and their aberrant signal pathways, as well as the therapeutic strategies targeting at LCSCs.

Tumorsphere formation and cancer stem cell characterization of REM134 canine mammary carcinoma cells / 대한수의학회지

Canine mammary tumors are among the most frequently observed cutaneous tumors in female dogs. Cancer stem cells (CSCs), referred to as tumor-initiating cells, are thought to have properties similar to normal stem cells such as the ability to self-renewal and to differentiate into various cell types. Biological understanding of CSCs and the critical pathways involved in their maintenance are important in research and therapy for mammary tumors. We conducted the present study on sphere formation from REM134 cells by using methylcellulose to produce tumorspheres on a large scale and compared the specific markers of the spheres-formed and plating-cultured REM134 cells. The results revealed that the tumorspheres cultured in methylcellulose had higher seeding density and improved morphology compared to those produced in normal sphere formation medium. Expression levels of stemness markers and CSC-related markers were higher in tumorsphere-forming cells than in plating-cultured cells. Subsequently, we transplanted the tumorsphere-forming and plating-cultured cells into female nude mice to examine their tumorigenic potential. Tumor volume increased rapidly in mice transplanted with tumorsphere-derived cells compared to plating-cultured cells. We observed a novel sphere-forming condition for REM134 cells and showed that REM134 cell tumorspheres can exhibit improved CSC properties.

Expression of CD44 according to Clinicopathologic Characteristics of Gastric Cancer / 이화의대지

OBJECTIVES: Cancer stem cells are defined as focal cluster of cells within a tumor that possess the capacity for self-renewal and differentiation into phenotypically heterogeneous cells. Cluster of differentiation 44 (CD44) is considered one of the gastric cancer stem cell markers. We aimed to investigate how the expression of CD44 varies according to the clinicopathologic characteristics in gastric cancer. METHODS: For this study, 157 patients who received an operation due to gastric cancer between May 1998 and December 2009 were selected. CD44 immunohistochemistry was reviewed using the semi-quantitative scoring of intensity and proportion. The sum of the intensity and proportion scores was calculated, and a score of 2 or less was deemed ‘CD44-negative’ and 3 or more as ‘CD44-positive.’ RESULTS: Among the final 143 subjects, 69 (48.3%) were CD44 positive. Older age, intestinal type gastric cancer, lymphatic invasion, and lymph node metastasis were significantly correlated with expression of CD44. In the multivariate analysis, older age was the only independent factor associated with CD44 expression (P=0.028). CD44 expression was correlated with overall survival, 5-year survival, and disease-free survival. In the multivariate analysis, older age, male gender, and lymphatic invasion were independent predictors of poor overall survival. Also, older age and lymphatic invasion were significant factors in 5-year survival, and lymphatic invasion was an independent factor of poor disease-free survival. CONCLUSION: Older age (≥60 years) was independently associated with CD44 expression in gastric cancer patients. Also, CD44 expression was correlated with poor prognosis in gastric cancer patients.