The expression and function of PD-L1 in CD133(+) human liver cancer stem-like cells / 中华肿瘤杂志

Objective: To investigate the expression of programmed death protein-ligand 1 (PD-L1) in liver cancer stem-like cells (LCSLC) and its effect on the characteristics of tumor stem cells and tumor biological function, to explore the upstream signaling pathway regulating PD-L1 expression in LCSLC and the downstream molecular mechanism of PD-L1 regulating stem cell characteristics, also tumor biological functions. Methods: HepG2 was cultured by sphere-formating method to obtain LCSLC. The expressions of CD133 and other stemness markers were detected by flow cytometry, western blot and real-time quantitative polymerase chain reaction (RT-qPCR) were used to detect the expressions of stemness markers and PD-L1. The biological functions of the LCSLC were tested by cell function assays, to confirm that the LCSLC has the characteristics of tumor stem cells. LCSLC was treated with cell signaling pathway inhibitors to identify relevant upstream signaling pathways mediating PD-L1 expression changes. The expression of PD-L1 in LCSLC was down regulated by small interfering RNA (siRNA), the expression of stem cell markers, tumor biological functions of LCSLC, and the changes of cell signaling pathways were detected. Results: Compared with HepG2 cells, the expression rate of CD133 in LCSLC was upregulated [(92.78±6.91)% and (1.40±1.77)%, P<0.001], the expressions of CD133, Nanog, Oct4A and Snail in LCSLC were also higher than those in HepG2 cells (P<0.05), the number of sphere-formating cells increased on day 7 [(395.30±54.05) and (124.70±19.30), P=0.001], cell migration rate increased [(35.41±6.78)% and (10.89±4.34)%, P=0.006], the number of transmembrane cells increased [(75.77±10.85) and (20.00±7.94), P=0.002], the number of cloned cells increased [(120.00±29.51) and (62.67±16.77), P=0.043]. Cell cycle experiments showed that LCSLC had significantly more cells in the G(0)/G(1) phase than those in HepG2 [(54.89±3.27) and (32.36±1.50), P<0.001]. The tumor formation experiment of mice showed that the weight of transplanted tumor in LCSLC group was (1.32±0.17)g, the volume is (1 779.0±200.2) mm(3), were higher than those of HepG2 cell [(0.31±0.06)g and (645.6±154.9)mm(3), P<0.001]. The expression level of PD-L1 protein in LCSLC was 1.88±0.52 and mRNA expression level was 2.53±0.62, both of which were higher than those of HepG2 cells (P<0.05). The expression levels of phosphorylation signal transduction and transcription activation factor 3 (p-STAT3) and p-Akt in LCSLC were higher than those in HepG2 cells (P<0.05). After the expression of p-STAT3 and p-Akt was down-regulated by inhibitor treatment, the expression of PD-L1 was also down-regulated (P<0.05). In contrast, the expression level of phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) in LCSLC was lower than that in HepG2 cells (P<0.01), there was no significant change in PD-L1 expression after down-regulated by inhibitor treatment (P>0.05). After the expression of PD-L1 was knockdown by siRNA, the expressions of CD133, Nanog, Oct4A and Snail in LCSLC were decreased compared with those of siRNA-negative control (NC) group (P<0.05). The number of sphere-formating cells decreased [(45.33±12.01) and (282.00±29.21), P<0.001], the cell migration rate was lower than that in siRNA-NC group [(20.86±2.74)% and (46.73±15.43)%, P=0.046], the number of transmembrane cells decreased [(39.67±1.53) and (102.70±11.59), P=0.001], the number of cloned cells decreased [(57.67±14.57) and (120.70±15.04), P=0.007], the number of cells in G(0)/G(1) phase decreased [(37.68±2.51) and (57.27±0.92), P<0.001], the number of cells in S phase was more than that in siRNA-NC group [(30.78±0.52) and (15.52±0.83), P<0.001]. Tumor formation in mice showed that the tumor weight of shRNA-PD-L1 group was (0.47±0.12)g, the volume is (761.3±221.4)mm(3), were lower than those of shRNA-NC group [(1.57±0.45)g and (1 829.0±218.3)mm(3), P<0.001]. Meanwhile, the expression levels of p-STAT3 and p-Akt in siRNA-PD-L1 group were decreased (P<0.05), while the expression levels of p-ERK1/2 and β-catenin did not change significantly (P>0.05). Conclusion: Elevated PD-L1 expression in CD133(+) LCSLC is crucial to maintain stemness and promotes the tumor biological function of LCSLC.

Targeted killing effect of bispecific CAR-T cells on EGFRvⅢ+/CD133+ glioma stem cells / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：制备双特异性CAR-T（bsCAR-T）细胞，观察其对表达表皮生长因子Ⅲ型突变阳性（EGFRvⅢ+，简称vⅢ+）和CD133+胶质瘤干细胞的靶向杀伤作用。方法：基于前期研制的vⅢ/CD133双特异性微抗体和二代CAR构建的双特异性CAR（bsCAR），制备慢病毒载体转染人外周血T细胞，FCM和WB法检测bsCAR转染效率和表达水平。bsCAR-T细胞和vⅢ+/CD133+ U87胶质瘤干细胞共培养，乳酸脱氢酶（LDH）释放实验、IFN-γ分泌实验检测其特异性杀伤作用和对IFN-γ分泌的促进作用。制备裸鼠vⅢ+/CD133+ U87干细胞移植瘤模型检测bsCAR-T细胞对移植瘤生长的抑制作用。结果：vⅢscFv和CD133scFv通过重叠PCR无缝连接入二代CAR表达框（S-vⅢscFv/CD133scFv-Hinge-TM-CD137-CD3z）中，然后克隆入pCDH-MSCV-MCS-EF1-copGFP载体的EcoRⅠ和BamHⅠ位点（pbsCAR）。3种质粒（pVSV-G、pCMV-dR8.9和pbsCAR）共转染HEK293T细胞制备慢病毒载体，转染外周血T细胞，FCM检测bsCAR表达率为71.1%，WB法结果显示bsCAR表达正确。bsCAR-T细胞和vⅢ+/CD133+ U87干细胞共培养检测结果显示，bsCAR-T细胞对胶质瘤干细胞具有特异性杀伤作用，与效靶比呈正比；IFN-γ分泌量为（2 350.6±92） pg·mL-1，明显高于对照组（P<0.01）。裸鼠移植瘤动物模型显示，bsCAR-T细胞在体内具有明显的移植瘤抑制作用（P<0.01）。结论： bsCAR-T细胞能够特异性靶向杀伤vⅢ+/CD133+胶质瘤干细胞，实验结果为促进实体瘤的细胞免疫治疗提供了实验依据。

Correlation between CD10,CA9,CD133 and prognosis of mccRCC treated with sorafenib or sunitinib as first-line therapy / 现代泌尿外科杂志

【Objective】 To analyze the correlation between the expressions of CD10,CA9 and CD133 and the prognosis of patients with metastatic renal clear cell carcinoma (mccRCC) treated with sorafenib or sunitinib. 【Methods】 A total of 80 mccRCC patients who received sorafenib or sunitinib as first-line therapy were retrospectively enrolled. Immunohistochemical staining (IHC) was performed for CD10,CA9 and CD133 in tumor tissue samples to analyze the correlation between the expression of each marker and clinicopathologic variables. Univariate and multivariate Cox proportional risk models were used to analyze prognostic factors of progression free survival (PFS) and overall survival (OS),and Kaplan-Meier survival analysis was performed for CA9 expression and PFS,OS in the treatment subgroups. 【Results】 Altogether 37 patients (46.25%) had PFS,and the median PFS (mPFS) was 24.9 months (95%CI:16.5-33.2 months),while 55 patients (68.75%) died and the median OS (mOS) was 44.2 months (95%CI:14.6-73.7). Low expression of CD10 was correlated with high Fuhrman grade (χ2=6.241,P=0.012),lymph node metastasis (χ2=5.952,P=0.015),and the number of metastatic organs ≥2 (χ2=8.205,P=0.004). Univariate analysis showed that Fuhrman grade,number of metastatic organs and lymph node metastasis were the prognostic factors of PFS (P<0.05),while the number of metastatic organs,lymph node metastasis and CA9 expression were the prognostic factors of OS (P<0.05). Multivariate analysis showed that Fuhrman grade was an independent factor of PFS (HR=2.457,95%CI:1.126-5.365,P=0.024),and the number of metastatic organs was an independent prognostic factor of OS (HR=1.857,95%CI:1.048-3.290,P=0.034). Survival analysis in subgroups showed that high CA9 expression in the sorafenib group was associated with longer OS (HR=0.401,95%CI:0.204-0.787,P=0.008). 【Conclusion】 Low expression of CA9 is an non-independent risk factor for OS,while CD10 and CD133 cannot be used as prognostic factors for mccRCC patients. Since mccRCC patients with low CA9 expression have less survival benefit from sorafenib and sunitinib,they can choose target therapy combined with immunotherapy or dual immunotherapy according to the guidelines to improve prognosis.

RGD peptide inhibits tumor growth by affecting angiogenesis related signaling pathway of laryngeal cancer stem cells / El péptido RGD inhibe el crecimiento tumoral al afectar la vía de señalización relacionada con la angiogénesis de las células madre del cáncer de laringe

SUMMARY: This study is to investigate the role and mechanism of RGD peptide in laryngeal cancer stem cells (CSCs). Laryngeal cancer CD133+Hep-2 CSCs were sorted by flow cytometry. RGD peptide was co-cultured with sorted laryngeal CSCs. Cell proliferation was detected with CCK-8 assay. The mRNA levels of VEGF/VEGFR2/STAT 3/HIF-1α were detected with RT-PCR. The proteins of VEGF/ VEGFR2/STAT 3/HIF-1α were detected with Western blot. The sorted CSCs were inoculated into nude mice. Tumor volume was measured. Integrin αvβ3 expression in tumor tissues was analyzed with immunohistochemistry. The results showed that the ratio of CD133+ CSCs to the total number of cells was 1.34±0.87 %, while CD133-non-tumor stem cells accounted for 95.0±5.76 %. The sorted cancer stem cells grew well. The RGD peptide significantly inhibited the proliferation of CD133+Hep-2 laryngeal CSCs in a dose-dependent manner. The RGD peptide significantly inhibited the mRNA of VEGFR2, STAT3 and HIF-1α in laryngeal CSCs in a concentration-dependent manner. Consistently, the RGD peptide significantly inhibited the protein expression of VEGFR2, STAT3 and HIF-1α in laryngeal CSCs in a dose-dependent manner. At the same time, in vivo tumor experiments showed that the RGD peptide significantly inhibited tumor volume but not the body weight. Furthermore, RGD peptide significantly inhibited the expression of tumor angiogenesis-related protein integrin αvβ3. Our findings demonstrate that RGD peptide inhibits tumor cell proliferation and tumor growth. The underlying mechanism may that RGD inhibits tumor angiogenesis-related signaling pathways, thus affecting the tumor angiogenesis, and decreasing the progression of human laryngeal CSCs.

Este estudio se realizó para investigar el papel y el mecanismo del péptido RGD en las células madre del cáncer de laringe (CSC). Las CSC CD133+Hep-2 de cáncer de laringe se clasificaron mediante citometría de flujo. El péptido RGD se cocultivó con CSC laríngeas clasificadas. La proliferación celular se detectó con el ensayo CCK-8. Los niveles de ARNm de VEGF/VEGFR2/ STAT 3/HIF-1α se detectaron con RT-PCR. Las proteínas de VEGF/ VEGFR2/STAT 3/HIF-1α se detectaron con Western blot. Las CSC clasificadas se inocularon en ratones nudos. Se midió el volumen del tumor. La expresión de integrina αvβ3 en tejidos tumorales se analizó con inmunohistoquímica. Los resultados mostraron que la proporción de CSC CD133+ con respecto al número total de células fue de 1,34 ± 0,87 %, mientras que las células madre no tumorales CD133 representaron el 95,0 ± 5,76 %. Las células madre cancerosas clasificadas crecieron bien. El péptido RGD inhibió significativamente la proliferación de CSC laríngeas CD133+Hep-2 de una manera dependiente de la dosis. El péptido RGD inhibió significativamente el ARNm de VEGFR2, STAT3 y HIF-1α en CSC laríngeas de manera dependiente de la concentración. De manera consistente, el péptido RGD inhibió significativamente la expresión proteica de VEGFR2, STAT3 y HIF-1α en CSC laríngeas, de manera dependiente de la dosis. Al mismo tiempo, los experimentos con tumores in vivo mostraron que el péptido RGD inhibía significativamente el volumen del tumor pero no el peso corporal. Además, el péptido RGD inhibió significativamente la expresión de la proteína integrina αvβ3 relacionada con la angiogénesis tumoral. Nuestros hallazgos demuestran que el péptido RGD inhibe la proliferación de células tumorales y el crecimiento tumoral. El mecanismo subyacente puede ser que RGD inhiba las vías de señalización relacionadas con la angiogénesis tumoral, afectando así la angiogénesis tumoral y disminuyendo la progresión de las CSC laríngeas humanas.

Effects of Monoammonium Glycyrrhizinate on Stem Cell-like Characteristics, Oxidative Stress and Mitochondrial Function of Acute Promyelocytic Leukemia NB4 Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of monoammonium glycyrrhizinate on the stem cell-like characteristics, oxidative stress and mitochondrial function of acute promyelocytic leukemia cells NB4.@*METHODS@#CCK-8 method was used to detect the viability of acute promyelocytic leukemia cells NB4, and the appropriate dose was screened; Cloning method was used to detect the proliferation rate of NB4 cell; Western blot was used to detect the expression of cell cycle-related protein; flow cytometry was used to detect cell apoptosis and sort NB4 stem cells positive (CD133+); Stem cell markers (Oct4, ABCG2, Dclk1) were detected by RT-PCR; ROS was detected by fluorescence; The kit was used to detect the level of oxidative stress markers (MDA); The flow cytometry was used to detect the change of mitochondrial membrane potential; Western blot was used to detect the expression of mitochondrial damage index-related proteins (Bax/BCL-2).@*RESULTS@#Compared with the control group, if the concentration of MAG was less than 5 μmol/L, the cell NB4 viability showed no significant difference; if the concentration was higher than 5 μmol/L, the inhibitory effect on the growth of cell NB4 increased and showed significant difference (P<0.05), according to the results of CCK-8 experiment, four groups were set based on the concentration of MAG 0 μmol/L, MAG 5 μmol/L, MAG 10 μmol/L, and MAG 20 μmol/L; compared with the control group (MAG 0 μmol/L), the cells in MAG 5 μmol/L group showed no significant difference, while the proliferation rate, cyclin expression, mitochondrial membrane potential, stem cell CD133+ ratio, and marker mRNA level ( Oct4, ABCG2, Dclk1) of NB4 cell were significantly reduced (P<0.05); the apoptosis rate, reactive oxygen species, MDA content and Bax/BCL-2 expression of NB4 cell significantly increased (P<0.05).@*CONCLUSION@#Monoammonium glycyrrhizinate has a significant inhibitory effect on acute promyelocytic leukemia cells NB4, which may be related to the regulation of stem cell-like characteristics, oxidative stress and mitochondrial function.

Targeted killing of CD133+ lung cancer stem cells using paclitaxel-loaded PLGA-PEG nanoparticles with CD133 aptamers / 南方医科大学学报

OBJECTIVE@#To construct a polylactic acid-glycolic acid-polyethylene glycol (PLGA-PEG) nanocarrier (N-Pac-CD133) coupled with a CD133 nucleic acid aptamer carrying paclitaxel for eliminating lung cancer stem cells (CSCs).@*METHODS@#Paclitaxel-loaded N-Pac-CD133 was prepared using the emulsion/solvent evaporation method and characterized. CD133+ lung CSCs were separated by magnetic bead separation and identified for their biological behaviors and gene expression profile. The efficiency of paclitaxel-loaded N-Pac-CD133 for targeted killing of lung cancer cells was assessed in vitro. SCID mice were inoculated with A549 cells and received injections of normal saline, empty nanocarrier linked with CD133 aptamer (N-CD133), paclitaxel, paclitaxel-loaded nanocarrier (N-Pac) or paclitaxel-loaded N-Pac-CD133 (n=8, 5 mg/kg paclitaxel) on days 10, 15 and 20, and the tumor weight and body weight of the mice were measured on day 40.@*RESULTS@#Paclitaxel-loaded N-Pac-CD133 showed a particle size of about 100 nm with a high encapsulation efficiency (>80%) and drug loading rate (>8%), and was capable of sustained drug release within 48 h. The CD133+ cell population in lung cancer cells showed the characteristic features of lung CSCs, including faster growth rate (30 days, P=0.001) and high expressions of tumor stem cell markers OV6(P < 0.001), CD133 (P=0.001), OCT3/4 (P=0.002), EpCAM (P=0.04), NANOG (P=0.005) and CD44 (P=0.02). Compared with N-Pac and free paclitaxel, paclitaxel-loaded N-Pac-CD133 showed significantly enhanced targeting ability and cytotoxicity against lung CSCs in vitro (P < 0.001) and significantly reduced the formation of tumor spheres (P < 0.001). In the tumor-bearing mice, paclitaxel-loaded N-Pac-CD133 showed the strongest effects in reducing the tumor mass among all the treatments (P < 0.001).@*CONCLUSION@#CD133 aptamer can promote targeted delivery of paclitaxel to allow targeted killing of CD133+ lung CSCs. N-Pac-CD133 loaded with paclitaxel may provide an effective treatment for lung cancer by targeting the lung cancer stem cells.

The expression differences and prognostic value of four cancer stem cell markers in hepatocellular carcinoma tissues and circulating tumor cells in peripheral blood / 中华检验医学杂志

Objective:To investigate the expression of four cancer stem cell (CSC) markers (EpCAM, CD133, CD90 and CD24) in hepatocellular carcinoma tissues and peripheral blood circulating tumor cells (CTC),their value in the prognosis of patients with hepatocellular carcinoma.Methods:A total of 50 hepatocellular carcinoma tissues and 29 peripheral blood sample from 50 patients with hepatocellular cancer treated in Zhongshan Hospital Fudan University from October 2013 to September 2014 were collected and analyzed by flow cytometry or qRT-PCR to examine the expression of EpCAM, CD133, CD90 and CD24. The clinical data of patients were collected, including tumor size, tumor number, satellite lesions, vascular invasion, Edmondson stage, BCLC stage and liver cirrhosis, etc. The correlation between the expression of four markers in hepatocellular carcinoma tissues and CTC with the clinical data and survival time of patients were compared.Results:The positive expression rates of EpCAM, CD133, CD90 and CD24 in hepatocellular carcinoma tissues were 66% (33/50), 18% (9/50), 60% (30/50) and 56% (28/50); the positive expression rates in CTC were 55% (16/29), 38% (11/29), 31% (9/29) and 59% (17/29). CD90 expression in hepatocellular carcinoma tissue was positively correlated with the occurrence HCC liver cirrhosis ( P<0.05), while CD133 expression was negatively correlated with the 5-year survival rate of patients ( P<0.05). The expression of EpCAM and CD24 in peripheral blood CTC were closely related to the patient′s Edmondson stage ( P<0.05). The survival time of patients with CD133 positive expression in hepatocellular carcinoma tissue was lower than those without CD133 expression ( P<0.05); the survival rate of patients with EpCAM expressed in either tissue or peripheral blood CTC was lower than that of patients with EpCAM double negative expression ( P<0.05). The survival rate of patients with CD90 negative in HCC tissue and positive in peripheral blood was lower than that in patients with double negative/double positive in tissue and peripheral blood or patients positive in hepatocellular carcinoma tissue and negative in peripheral blood ( P<0.01). Conclusion:Different expression characteristics of four markers in cancer tissues and peripheral blood CTC might provide useful information about predicting prognosis of hepatocellular carcinoma. The expression of CD133 in tissues can be used as an important survival predictor of hepatocellular carcinoma patients. The differential expression of cancer markers in tissue samples and blood samples can provide more clinical prognostic information.

Expression of CD133 in triple negative breast cancer and its clinical significance / 中华内分泌外科杂志

Objective:To investigate the correlation between CD133 expression and clinicopathological features in triple negative breast cancer (TNBC) patients, and the impact of CD133 on prognosis in these patients.Methods:Data of 70 patients who received surgical treatment in our center from Jan. 2008 to Dec. 2012 were collected. Immunohistochemistry was used to examine the expression of CD133. Patients were divided into two groups according to CD133 expression. Univariate analysis, Cox and Logistic regression multivariate analysis were used in order to investigate the correlation between CD133 expression and clinicopathological features. Kaplan-Meier curve and Log-rank analysis were used to evaluate DFS (disease-free survival) and OS (overall survival) .Results:CD133 was expressed in cytomembrane and cytoplasm with expression rate of 95.71% (67/70) . Of which, 64.29% (45/70) of patients were low CD133 expression and 35.71% (25/70) were high expression. High CD133 expression was significantly correlated with younger age (≤50) ( P=0.007) and larger tumor size (>2 cm) ( P=0.020) . Tumor size ( P=0.035) , axillary status ( P=0.001) , Ki67 ( P=0.005) and CD133 expression ( P=0.014) were independent predictors of recurrence and metastasis in TNBC patients. Axillary status was independent predictor of death event ( P=0.008) . Increased CD133 was associated with poor prognosis. Compared with high expression, patients with low CD133 expression had better DFS ( P=0.002) and OS ( P=0.088) , while OS did not reach significant difference. Conclusion:CD133 expression was correlated with age and tumor size in TNBC patients. High expression was associated with recurrence, metastasis and poor prognosis. Thus, CD133 may be a potential biomarker in predicting prognosis in TNBC.

MMR-proficient and MMR-deficient colorectal cancer cells: 5-Fluorouracil treatment response and correlation to CD133 and MGMT expression

Cancer stem cells (CSCs) from colorectal cancer (CRC), characterized by CD133 expression, have beenassociated with 5-fluorouracile (5-FU) chemoresistance. DNA repair mechanisms, such as O6-alkylguanineDNA alkyltransferase (MGMT) and mismatch repair (MMR) systems, have also been correlated to 5-FUresistance in CRC. The aim of this study was to evaluate the modulation of CD133 and MGMT in MMRproficient and MMR-deficient CRC cells under 5-FU treatment and the effect of this drug in CSCs. CD133 andMGMT methylation status were determined in MMR-proficient (SW480 and HT29) and MMR-deficient (RKOand HCT116) cell lines by methylation-specific PCRs. SW480 and RKO were selected to determine modulation of CD133, MGMT and MMR expression after 5-FU treatment by qPCR. In addition, CD133, MGMTand MMR were analyze in SW480 and RKO CSCs. No association between promoter methylation and MGMTand CD133 expression was found. 5-FU treatment increased CD133 expression independently to MMR statusin SW480 and RKO and was able to increase hMLH1 expression in RKO, a MMR-deficient cell line. RKO/CSCs overexpressed CD133 and MMR (hMSH2 and hMSH6) while SW480/CSCs showed a significantincrease in CD133, MMR (hMLH1, hMSH2 and hMSH6) and MGMT, moreover 5-FU resistance thanparental cell lines. Thus, although CSCs 5-FU chemoresistance appears to be independently to MMR status,hMLH1 might play a key role in CSC response to 5-FU. New drugs exploding these differences could benefitthe prognostic of patients with CRC.

Detection of cancer stem cell-related markers in different stages of colorectal carcinoma patients of Indian origin by immunohistochemistry

Context: Although the incidence rate of colorectal cancer (CRC) in all Indian cancer registries is very close to the lowest rate in the world, westernization has shown an increasing trend in the recent years. Recurrence is reported in CRC because the slowly proliferating stem cells escape the chemotherapeutic regimen. Aim: To detect the presence of CD133 and CD44 in human CRC specimens and to correlate the level of marker expression with tumor staging. Materials and Methods: We included 26 colorectal carcinoma patients between 20 and 70 years of age. Histological and immunohistochemical analysis of CD133 and CD44 was done in sections of 5 μm prepared from paraffin-embedded blocks with most representative areas. Statistical Analysis: All analyses were performed using Microsoft Excel 2010 and SPSS version 22. Results: CD133 expression was seen exclusively on the cell membrane at the glandular luminal surface with dot-like cytoplasmic staining. In the normal mucosa, CD44 expression was seen in the superficial region of the cell, whereas in most of the carcinomas, the staining was localized in the basolateral region of the cell. Both CD133 and CD44 showed significant correlation with tumor stage. Conclusions: In the present study, CD133 and CD44 show significant correlation with tumor staging. Cancer stem cell markers have shown similar pattern of expression in the patients of Indian origin. Using combination of markers for staging is preferred as it increases the sensitivity and specificity

Treatment of triple-negative breast cancer with anti CD133 antibody-modified shikonin-loaded microemulsion / 中草药

Objective To evaluate the feasibility and advantages of therapy of triple-negative breast cancer with Anti CD133 antibody-modified shikonin-loaded microemulsion (Anti CD133Ab-SKN-MEs). Methods Anti CD133Ab-SKN-MEs were prepared by a classic EDC/NHS conjugation technique. The drug loading efficiency and density of modified antibody were optimized using average particle size, Zeta potential and entrapment efficiency as indicators. The cell proliferation of MDA-MB-231 cells was investigated by MTT method. The cellular uptake of various formulations was qualitatively and quantitatively investigated using FITC as a probe. MDA-MB-231 cellular apoptosis induced by various treatments was evaluated by the Annexin V-PE/7-amino actinomycin D (Annexin V-PE/7-AAD) assay kit. MDA-MB-231 breast cancer stem cells (MDA-MB-231 CSC) was enriched by a suspension culture technique, and the cell morphology and proportion of CD133-positive cells were studied after treatment with various SKN formulations. The model of MDA-MB-231 tumor-bearing nude mice was established, and then injected five times every other day with saline, shikonin (SKN), SKN-MEs, and Anti CD133Ab-SKN-MEs at a dose of 4 mg/kg, to observe the tumor volume, survival time, tumor inhibition and CD133+ cells ratio during/after the treatment. Results The optimal mass ratio of SKN to total carrier was 1.0% in the preparation of Anti CD133Ab-SKN-MEs, and the optimal density of modified antibody was 0.025%.The particle was spherical with a particle size of (31.4 ± 2.1) nm, a potential of (-18.7 ± 2.5) mV and an encapsulation efficiency of (93.6 ± 2.8) %. The IC50 of Anti CD133Ab-SKN-MEs against MDA-MB-231 cells was (1.53 ± 0.43) μg/mL, the cell uptake of Anti CD133Ab-SKN-MEs was significantly higher than that of SKN-MEs and SKN, and 8 h incubation induced (67.9 ± 4.2)% cell apoptosis. Anti CD133Ab-SKN-MEs can significantly inhibit the globularity of MDA-MB-231 CSC, with a decrease in the number of CD133-positive cells. The in vivo tumor inhibition rate of Anti CD133Ab-SKN-MEs-treated mice was 78.5%, and 12.5% of tumor-bearing nude mice still survived at day 69. Moreover, the ratio of CD133-positive tumor cells within the tumor tissues was significantly reduced. Conclusion Anti CD133Ab-SKN-MEs has obvious advantages in treatment of triple-negative breast cancer, which might be related to the inhibition of tumor cells differentiation.

Research advances in glioma stem cell surface marker CD133 and related targeted therapy / 医学研究生学报

Glioma is the most common intracranial malignant tumor of the nervous system. It is highly invasive, resistant to conventional treatment, and easy to relapse. The main treatment strategy is surgery plus radiotherapy, but the prognosis is still poor. Glioma stem cells (GSCs) are a group of cells with neural stem cell-like properties in glioma. As the starting cells of glioma, they are considered to be the key factors for tumorigenesis and recurrence. CD133 is considered to be a biomarker for glioblastoma and is used as a marker for GSCs. Although its biological significance is currently controversial, more and more studies have shown that CD133 is involved in GSCs-mediated tumor formation and recurrence. This article mainly reviews the GSCs surface marker CD133 and its related targeted therapies.

Proliferation,Self-renewal and Metastasis Ability of CD133+Cells in Cutaneous Squamous Cell Carcinoma / 中山大学学报(医学科学版)

[Objective]To observe the biological characteristics of CD133 positive cells derived from cutaneous squa-mous cell carcinoma(cSCC),and to investigate the mechanism of self-renewal of cancer stem cells(CSC)and the malig-nant behavior in cSCC.[Methods]The flow cytometry was applied to purify CD133+cells and CD133-from cutaneous squa-mous cell carcinoma cell line Colo-16.The effects of CD133 on the cell proliferative,self renew and migratory activity of two group cells were detected by MTT assay,Sphere formation assay and Transwell assay.Western blot and quantitative flu-orescent PCR were performed to investigate the expression of cancer stem cell associated gene CD44,OCT4 and SOX2 in two groups of cells.Examined the expression level of CD133 in actinic keratosis,squamous cell carcinoma in situ and cSCC by immunohistochemistry.[Result]The CD133+Colo-16 cells exhibited significantly stronger proliferation,self-renewal and metastasis ability(P<0.05)and present higher level protein and mRNA expression on CD44,SOX2 and OCT4(P<0.05). In addition,the expression level of CD133 was dynamic rise during the continuous evolution of actinic keratosis,squamous cell carcinoma in situ and cSCC.[Conclusions]The expression level of CD133 may be related to the tumorigenesis of cSCC and the malignant behavior including proliferative,self renew and migratory activity,and CD133+cSCC cells show the properties of cancer stem cells.The occurrence and development of cSCC may be related to the CSC,which express CD133.

The expression and significance of CD276 and CD133 in colorectal cancer and precancerous lesions / 中华内科杂志

In order to study the significance of CD276 and CD133 in the development and progression of colorectal cancer (CRC),the expression of CD276 and CD133 was detected by immunohistochemistry in CRC and precancerous lesions.The results showed that the intensity of CD276 and CD133 in CRC samples was higher than that in adenoma group and non-adenoma group.CD276 and CD133 single and double positive expression were significantly correlated with CRC lymph node metastasis,distant metastasis and survival.CD276 and CD133 are significantly correlated to the development and progression of CRC and associated with poor prognosis.

Expression of CD133-2 during the Courses of Acute Leukemia and Its Clinical Significance / 现代检验医学杂志

Objective To explore the expression of CD133-2 during the treatment course of acute leukemia(AL) and its clinical significance.Methods Used flow cytometry with direct immunofluorescence staining to analyze CD133-2 of 67 acute leukemia patients with different treatment courese.Results The CD133-2 positive rate (52.4％)and expression rate (23.9％±21.5％) in AL were significantly higher than those in control (0,2.2％ ±3.9％).The CD133-2 positive rates of cases for primary treatment group,CR group and recurrence group were 52.4 ％,0 and 40.0 ％ respcctively,and expression rates were 23.9％±21.5％,5.0％±6.0％ and 28.4％±25.6％ respectively.There were significant difference in the positive rate and expression rate of CD133-2 among the three group (x2 =12.777,F=5.906,P＜0.05).The CD133-2 positive rates and expression rates in primary treatment group and recurrence group were significantly higher than those in complete remission cases.CD133-2 positive rate of CD34 + group was obviously higher than that of CD34-group (40.5％ vs 7.1％,x2=8.636,P＜0.05),and the CR rate of CD133-2-/CD34-group was significantly higher than that of CD133-2+/CD34 +group (83.3％ vs 33.3％,x2=6.078,P＜0.05).Conclusion The expression of CD133-2 was correlated with CD34,and CD133/CD34 co-overexpression might be a bad prognostic factor of AL.CD133-2 can be used as one of the indicator of predicting recurrence and monitoring MRD.

Effects of hypoxia on the expression of autophagy-related molecules in human colon cancer cells / 基础医学与临床

Objective To explore the mechanism of hypoxia on SW480 cell autophagy.Methods We screened the CD133+cell with the characteristics of stem cell by magnetic cell separation. Then SW480 cell and CD133+cell were incubated in hypoxic conditions. Subsequently,we detected HIF-1α expression by immunofluorescence(IF), LC3 expression by Western blot and HIF-2α and Beclin 1 expression in 94 colon cancer tissue samples by immuno-histochemistry. Results Hypoxia induced the activation of HIF-1α in SW480 cell. After hypoxia 24 h, LC3 ex-pression was increased in both SW480 cell and CD133+cell with incubation time. The conversion from LC3Ⅰto LC3Ⅱwas increased in CD133+cell,but decreased in SW480 cell. In olon cancer tissue samples,HIF-2α and Be-clin 1 expression in poorly differentiated group were higher than that of well-differentiated group.HIF-2α expression in non-lymph node metastasis group was higher than high lymphatic metastasis group. However the Beclin expression was in the opposite regulation. Conclusions Hypoxia can induce the SW480 apoptosis and a few of CD133+cell autophagy that can protect the cell from apoptosis by inducing the HIF expression and increasing LC3.

Effects of bone marrow-derived mesenchymal stem cells on invasion and metastasis of gastric cancer cells and its mechanism / 国际外科学杂志

Objective To investigate the role of bone marrow mesenchymal stem cells (BM-MSCs) in the invasion and metastasis of gastric cancer cells and to explore its mechanism.Methods SGC7901 and KATO-Ⅲ gastric cancer cells were co-cultured with BM-MSCs respectively,and the invasion ability of SGC7901 and KATO-Ⅲ gastric cancer cells were detected by Transwell assay.Secondly,CD133 + and CD133-cells were sorted from KATO-Ⅲ gastric cancers and co-cultured with BM-MSCs respectively to compare their changes in invasiveness.Meanwhile,the expressions of p-AKT and epithelial-mesenchymal transition (EMT) relative factors in gastric cancer cells were detected by Western-blot.The role of CD133 in BM-MSCs affecting the ability of invasion of gastric cancer cells was further vertified by the overexpression of CD133 in SGC7901 cells.SPSS17.0 software was used for statistical processing,and the stand deviation of the measurement data were expressed as the standard deviation,independent sample t test was conducted.Results The invasiveness of co-cultured SGC7901 and KATO-Ⅲ cells was significantly enhanced.The invasive ability of KATO-Ⅲ CD133+ cells co-cultured with BM-MSCs tended to increase more significantly than that of co-cultured CD133 cells[(259.0 ± 24.0)vs (58.0 ±5.6),P ＜ 0.001].The expressions of p-AKT,Snail and N-cadherin were significantly increased in co-cultured CD133+ cells (P =0.003,P =0.003,P =0.002),while the expression of E-cadherin was reduced (P =0.021).After co-cultured with BM-MSCs,the expression of E-cadherin was also reduced in CD133-cells (P =0.005),but the expressions of p-AKT,Snail and N-cadherin were no significantly changes (P =0.744,P =0.277,P =0.295).SGC7901 co-cultured with BM-MSC after overexpression of CD133 showed higher i nvasiveness than blank control group[(239.3 ± 24.0) vs (103.3 ± 15.5),P ＜ 0.001].The expressions of p-AKT,Snail and N-cadherin were significantly increased when co-cultured with BM-MSCs in the group of CD133 overexpression (P =0.001,P =0.001,P =0.001),while the expression of E-cadherin was significantly decreased(P =0.003).The expressions of Snail and N-cadherin were also significantly increased after co-cultured with BM-MSCs in the blank control group (P =0.001,P =0.004),and the expression of E-cadherin was significantly decreased (P =0.018),while the expression of p-AKT was not significantly changed (P =0.193).Conclusions BM-MSCs can enhance the invasion and metastasis of gastric cancer cells by promoting the EMT of gastric cancer cells.CD133 may be involved in the regulation of EMT in gastric cancer cells through the PI3K/AKT signaling pathway.

Expression and significance of Notch3, DLL1 and CD133 in colorectal adenocarcinoma / 重庆医学

Objective To investigate the expression of Notch3,DLL1 and CD133 in human colorectal adenocarcinoma and its clinical pathology meaning.Methods Immunohistochemical staining was used to detect the expression of Notch3,DLL1 and CD133 in 12 cases of normal colorectal mucosa tissue,30 cases of colorectal adenoma tissue and 50 cases of colorectal adenocarcinoma tissue,and the relationship between them and clinicopathological data wereanalyzed.Results The positive rates of Notch3,DLL1 and CD133 were 64.0 ％ (32/50),68.0 ％ (34/50) and 54.0 ％ (27/50) in colorectal carcinoma tissues,respectively,which were remarkably higher than those in colorectal adenoma tissue(26.7 ％,33.3％％,36.7 ％)and those in normal colorectal mucosa tissue (8.3％,16.7％,8.3％) (P＜0.05).There was no significant difference between the adenoma group and the normal group(P＞0.05).The expression levels of Notch3,DLL1 and CD133 were not correlated to age,gender,tumor location,degree of differentiation and the tumor size,except lymph node metastasis,in addition,the expression of Notch3 and DLL1 were also associated with Dukes staging,and The expression of DLL1 were also associated with tumor infiltration depth (P＜0.05).The expression of Notch3 protein was positively related to that of DLL1 protein(r=0.478,P=0.000).Conclusion The positive expression rates of Notch3,DLL1 and CD133 in colorectal adenocarcinoma tissue are remarkably higher than those in the normal colorectal mucosa and colorectal adenoma tissue,which prompts that the high expression of Notch3,DLL1 and CD133 may all participate in the process and metastasis of colorectal adenocarcinoma.Moreover Notch3 signalling pathway may act through the cancer stem cells and eventually regulate the initiation and development of colorectal adenocarcinoma.

Resistance of CD133+ cells in HepG2 cell line to doxorubicin and its mechanism / 中国肿瘤生物治疗杂志

@# Objective: To explore the resistance of CD133+ cells in HepG2 cell line to doxorubicin (DOX) and its mechanism. Methods: CD133+ cells were sorted by magnetic beads and CD133+ positive rate was detected by flow cytometry. MTT assay was used to detect the resistance to DOX-induced apoptosis of CD133+ cells. The expression of BCRP transporter mRNA was detected by RT-PCR. The expression of apoptosis-related proteins was detected by Western blotting. Immunofluorescence assay was used to detect the activation and transportation of P65 after DOX treatment. Results: Magnetic beads sorting could efficiently sort the CD133+ cells from HepG2 cells. MTT proliferation assay showed that CD133+ cells had stronger resistance to DOX than CD133- cells (P<0.05). Immunofluorescence showed that the activation rate and content of P65 in CD133+ cells were significantly higher than those in CD133- cells and HepG2 cells (P<0.05). The results of RT-PCR showed that the mRNA content of BCRP in CD133+ cells was significantly increased compared with CD133- cells and HepG2 cells (all P<0.05). Compared with HepG2 and CD133- groups, the expression of Bax and p53 in CD133+ cells was significantly decreased (P<0.05), while the expression of Bcl-2 and Survivin protein in CD133+ cells was significantly increased (P<0.05 or P<0.01). Conclusion: The molecular mechanism of high DOX-resistance of the CD133+ cell subsets in HepG2 cells is the high expression of the survival-related proteins NF-κB, Bcl-2, Survivin and the drug-resistance transporter BCRP, and low expression of apoptosis-promoting proteins p53 and Bax.

5-LOX inhibitor MK886 suppresses the stemness of human colon cancer HT-29 cells / 医学研究生学报

Objective It remains a controversy whether 5-lipoxygenase (5-LOX) is associated with colon cancer stem cells.This study was to investigate the effect of the 5-LOX inhibitor MK886 in maintaining the stemness of the human colon cancer cell line HT-29.Methods Using CCK-8 assay, we examined the inhibitory effects of different concentrations of MK886 (12.5, 25, 50, 75, 100, and 200 μmol/L) on the colon cancer HT-29 cells cultured in vitro and calculated its half-inhibitory concentration (IC50).Then, we detected the effects of MK886 IC50 on the clone-and sphere-forming abilities of the cells, determined the mRNA expressions of the stemness markers CD133, Lgr5, Oct4 and Ascl2 by real-time PCR after 24 and 48 hours of MK886 IC50 intervention, and measured their protein expressions by Western blotting after 24, 48 and 72 hours of MK886 IC50 intervention.Results The inhibition rates of MK886 on the HT-29 cells at 24 and 48 hours were significantly increased in a time-and dose-dependent manner ([14.99±3.06] and [19.98±0.57]％ at 12.5 μmol/L, [20.46±1.14] and [34.97±6.02]％ at 25 μmol/L, [50.76±5.94] and [66.90±5.74]％ at 50 μmol/L, [66.84±1.77] and [73.11±2.48]％ at 75 μmol/L, [72.67±2.36] and [77.78±3.30]％ at 100 μmol/L, [83.67±0.24] and [84.69±2.24] ％ at 200 μmol/L) as compared with the blank control (0％ and 0％) (P<0.05).The clone-forming rate and number of spheres formed were remarkably lower in the MK886 intervention than in the control group ([10.60±1.71] vs [44.67±3.21]％, P<0.05;6.00±1.60 vs 19.07±2.89, P<0.05).After 24 and 48 hours of MK886 intervention, the mRNA expression of CD133 in the HT-29 cells was markedly up-regulated in comparison with that at 0 hour (0.72±0.10 and 0.39±0.07 vs 1.66±0.33, P<0.05), and so were those of Lgr5, Oct4 and Ascl2 (P<0.05).Conclusion The 5-LOX inhibitor MK886 can inhibit the proliferation and clone-and sphere-forming abilities of human colon cancer HT-29 cells by down-regulating the expressions of the stemness markers and thus suppressing the stemness of the colon cancer stem cells.