重度子痫前期患者胎盘组织中CD44 mRNA和CD24 mRNA及蛋白表达水平的临床价值研究

目的探讨重度子痫前期(severe preeclampsia,SPE)患者胎盘中细胞表面跨膜糖蛋白分子(cell surface transmembrane glycoprotein molecules,CD)44 mRNA,细胞表面跨膜糖蛋白分子24(CD24)mRNA及蛋白表达水平表达的临床价值研究。方法选取2019年6月~2022年6月在聊城市第二人民医院接受剖宫产分娩的SPE患者,根据发病孕龄的不同,进一步将其分为早发型SPE组(孕龄≤34周,n=45)和晚发型SPE组(孕龄>34周,n=55)。选取同期产检正常者100例为对照组。采用荧光定量PCR和免疫组织化学检测SPE患者胎盘中CD44和CD24表达,Pearson法分析其表达水平差异以及与SPE疾病临床特征的相关性,多因素Logistic回归分析发生SPE的影响因素。结果相较于对照组,SPE胎盘组织中CD44 mRNA(0.55±0.12 vs 1.02±0.33),CD24 mRNA的表达水平(0.68±0.19vs 1.05±0.11)均降低,差异具有统计学意义(t=13.385,16.853,P<0.05)。免疫组织化学染色结果显示,CD44,CD24在SPE组胎盘组织中多呈阴性表达或弱阳性表达,而在对照组中多呈阳性表达,且SPE胎盘组织中CD44,CD24阳性率低于对照组,差异具有统计学意义(χ^(2)=9.696,14.346,P<0.05)。相较于早发型SPE组,晚发型SPE胎盘组织中CD44(0.65±0.17 vs 0.42±0.11),CD24(0.77±0.23 vs 0.58±0.13)mRNA的表达水平均较高,差异具有统计学意义(t=7.830,4.932,P<0.05)。相较于对照组,SPE组BMI,收缩压、舒张压、尿蛋白、Cr,LDH和BUN均显著升高,差异有统计学意义(t=5.360~30.241,均P<0.05);SPE组分娩孕周较早、MPV,ALB较低、新生儿出生身长较短和体质量较轻均低于对照组,差异具有统计学意义(t=3.232~11.109,均P<0.05)。且SPE胎盘组织中CD44与CD24表达呈正相关(r=0.698,P<0.05),SPE胎盘组织中CD44的表达分别与CD24,分娩孕周、MPV和新生儿出生身长呈正相关(r=0.611,0.639,0.612,0.465,均P<0.05);与收缩压、尿蛋白和LDH呈负相关(r=-0.604,-0.569,-0.593,均P<0.05)。CD24的表达分别与分娩孕周、MPV和新生儿出生身长呈正相关(r=0.605,0.584,0.640,均P<0.05);与收缩压、尿蛋白和LDH呈负相关(r=-0.637,-0.593,-0.561,均P<0.05)。经Logistic回归分析结果显示,MPV(95%CI:1.429~4.350),尿蛋白(95%CI:1.529~2.709),LDH(95%CI:1.425~3.932)均是发生SPE的独立危险因素(均P<0.05)。高水平CD44(95%CI:0.561~0.940),CD24(95%CI:0.495~0.814)是发生SPE的独立保护因素(均P<0.05)。结论SPE患者胎盘中CD44,CD24表达水平较低,高水平CD44,CD24均是SPE发生的独立保护因素,可为后续SPE的治疗提供方向。

Ardisia gigantifolia ethanolic extract inhibits cell proliferation and targets cancer stem cells in gastric cancer

Objective:To evaluate the effects of ethanol extract from Ardisia gigantifolia leaves on cell proliferation and cancer stem cell(CSC)number in gastric cancer.Methods:The inhibitory effect of Ardisia gigantifolia extract on the proliferation of MKN45 and MKN74 gastric cancer cells was assessed using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay.Non-adherent culture(3D)model was used to evaluate the effect of the extract on tumorsphere size and number.Moreover,the expression of CD44,ALDH,and p21 was determined by immunofluorescence analysis.Flow cytometric analysis was performed to evaluate cell cycle arrest and the expression of gastric CSC markers CD44 and ALDH.Real-time PCR analysis was also carried out to assess the effect of the extract on the expression of cell cycle-regulated genes.Results:Ardisia gigantifolia extract effectively inhibited cell proliferation with an IC_(50)of 55.7μg/m L in MKN45 cells and 123.6μg/m L in MKN74 cells.The extract also arrested cell cycle in the G_(0)/G_(1)phase as well as significantly reduced the size and number of tumorspheres.The markedly increased expression of p21 was observed at both m RNA and protein levels in the extract-treated adherent cells and tumorspheres.In addition,Ardisia gigantifolia extract significantly reduced the number of CD44-and/or ALDH-expressing gastric CSC.Conclusions:The development of gastric CSC can be inhibited by the ethanol extract of Ardisia gigantifolia.

Endothelial adhesion of synchronized gastric tumor cells changes during cell cycle transit and correlates with the expression level of CD44 splice variants

AIM To study adhesion capacity and CD44 expression of human gastric adenocarcinoma MKN45 cells at different stages of a first cell cycle. METHODS MKN45 cells were synchronized by aphidicolin and assayed for adhesion to an endothelial cell （HUVEC） monolayer. Surface expression of CD44 and CD44 splice variants on MKN45 cells was evaluated by flow cytometry. Functional relevance of CD44 adhesion receptors was investigated by blocldng studies using anti CD44 monodonal antibodies or by hyaluronan digestion. RESULTS： Adhesion of MKN45 to HUVEC was increased during G2/M transit, after which adhesion returned to baseline levels with cell cycle completion. In parallel, CD44 splice vadants CD44v4, CD44v5, and CD44v7 were all upregulated on MKN45 during cell cycle progression with a maximum effect in G2/M. The function of CD44 surface receptors was assessed with specific receptor blocking monoclonal antibodies or removal of hyaluronan by digestion with hyaluronidase. Both strategies inhibited tumor cell adhesion to HUVEC by nearly 50%, which indicates that MKN45-HUVEC-interaction is CD44 dependent. CONCLUSION CD44 expression level is linked to the cell cycle in gastrointestinal tumor cells, which in turn leads to cell cycle dependent alterations of their adhesion behaviour to endothelium.

Effect of CD44 Suppression by Antisense Oligonucleotide on Attachment of Human Trabecular Meshwork Cells to HA

The effects of suppression of CD44 by CD44-specific antisense oligonucleotide on attachment of human trabecular meshwork cells to hyaluronic acid (HA) were observed and the possible relationship between CD44 and primary open-angle glaucoma (POAG) investigated. CD44-specific antisense oligonucleotide was delivered with cationic lipid to cultured human trabecular meshwork cells. The expression of CD44 suppressed by CD44-specific antisense oligonucleotide was detected by RT-PCR and Western blotting. The effect of CD44 suppression by specific antisense oligonucleotide on attachment of trabecular meshwork cells to HA was measured by MTT assay. Results showed that expression of CD44 was suppressed by CD44-specific antisense oligonucleotide. Antisense oligonucleotide also suppressed the adhesion of human trabecular meshwork cells to HA in a concentration dependent manner. It was concluded that attachment of human trabecular meshwork cells to HA was decreased when CD44 was suppressed by specific antisense oligonucleotide. CD44 might play a role in pathogenesis of POAG by affecting the adhesion of trabecular meshwork cells to HA.

PEI-PEG as a siRNA Genetic Vector Demonstrating Interference in the Expression of CD44v6 Protein in Gastric Cancer Cells

OBJECTIVE To investigate the effect of polyethylene imine glycol （PEI-PEG）/siRNA nanocomposites in the in vitro transfection of human gastric cancer SGC7901 cell lines and the down-regulation of gene expression of the adherence factor CD44v6. METHODS PEI-PEG/siRNA nanoparticles, in different N/P ratios, were synthesized and transfected into gastric cancer cells. Lipo2000/siRNA was used in the control group. The transfection efficiencies were observed under fluorescence microscope. The cytotoxicity of the nanoparticles was measured using the MTT assay （mononuclear cell direct cytotoxicity assay）, and the down-regulation effect of siRNA on CD44v6 gene was evaluated by Western blot. Based on the different N/P ratios, PEI-PEG/siRNA composites were synthesized and transfected into gastric cancer cells. Lipo2000/siRNA was used in the controls. The transfection efficiency was observed under fluorescence microscope. The cytotoxicity of the nanoparticles was measured using the MTT assay and the down-regulation effect of siRNA on CD44v6 gene was evaluated by Western blot. RESULTS After transfection, the transfection efficiency of the PEI-PEG/siRNA nanocomposites increased incrementally in N/P ratio value. The transfection efficiency improved with an increase in N/P ratio. When the N/P value was 15, fluorescence became more intense in the PEI-PEG/siRNA group than in the Lipo2000/siRNA group. At the same time, cell viability was （80.4 ± 5.6）% in the MTT reduction assay, which was similar to that in the Lipo2000/siRNA group. The results of Western blot analysis showed that the expression level of CD44v6 protein decreased to （59.7 ± 3.0）% after siRNA-CD44v6 was inhibited. CONCLUSION PEI-PEG could effectively form the nanocomposite in combination with siRNA, be transfected into the SGC7901 gastric cancer cell lines and inhibit CD44v6 protein expression. Moreover, as a genetic carrier, PEI-PEG copolymer has greater advantages, including high transfection e. ciency, less cytotoxicity and an easily alterable vector structure.

CD44蛋白在胃癌中的表达情况及临床意义分析

目的:分析胃癌中白细胞分化抗原44(CD44)蛋白的表达情况与临床意义。方法:回顾性分析2019年1月—2022年1月于苏州市立医院接受手术治疗的胃癌患者139例的临床资料,并采用免疫组织化学法检测患者胃癌组织病理标本中CD44蛋白表达情况。分析CD44蛋白表达情况与临床病理特征及预后的关系。结果:胃癌组织中CD44蛋白表达率高于正常胃黏膜组织,差异有统计学意义(P<0.001)。阳性病例中,低分化胃癌患者CD44蛋白免疫组化评分高于高/中分化胃癌患者,肿瘤最大径≥4 cm胃癌患者CD44蛋白免疫组化评分高于肿瘤最大径<4 cm胃癌患者,差异有统计学意义(P<0.001);CD44蛋白阳性与CD44蛋白阴性胃癌患者性别、年龄、Lauren分型比较,差异无统计学意义(P>0.05);CD44蛋白阳性胃癌患者肿块最大径≥4 cm、低分化、血管侵犯阳性、淋巴结转移阳性、TNM分期为Ⅲ或Ⅳ期占比高于CD44蛋白阴性者,差异有统计学意义(P<0.05);CD44阳性表达者生存期短于阴性表达者,差异有统计学意义(P=0.003)。结论:不同分化程度、肿瘤最大径的胃癌患者CD44蛋白表达存在明显差异,CD44蛋白阳性与CD44蛋白阴性胃癌患者肿块最大径、分化程度、血管侵犯情况、淋巴结转移情况、TNM分期存在明显差异,可将CD44蛋白作为胃癌浸润、转移及判读预后的生物学标记物。

The expression and clinical significance of β-catenin and colorectal cancer stem cells marker EpCAM^(high)/CD44^+ in colorectal cancer

Objective： The aim of the study was to explore the role of Wnt βcatenin signalling pathway in the maintenance, invasion and metastasis of colorectal cancer stem cells. Methods： Double immunohistochemical staining was used to detect the expression of EpCAMhigh/CD44~ which is regarded as the marker of colorectal cancer stem cells in 80 cases of colorectal cancer and their corresponding liver metastases. The SP method of immunohistochemistry was used to detect the expression of the key protein βcatenin in the Wnt pathway in these tissue. The expression and correlation of ^-catenin and EpCAMh^gh/ CD44＋ in colorectal cancer were analyzed and their role on the biological behavior of colorectal cancer was explored. Results： The abnormal expression of βcatenin was significantly higher in colorectal cancer than in the paraneoplastic normal intes- tinal mucosa [55% （44/80） vs 10% （2/20）, P 〈 0.05]. The positive expression of EpCAMhigh/CD44＋ was significantly higher in colorectal cancer than in the paraneoplastic normal intestinal mucosa [66.25% （53/80） vs 0% （0/20）, P 〈 0.05]. In the 80 cases of colorectal cancer, the abnormal expression of ^-catenin has no correlation with gender （P = 0.079）, age （P = 0.416） and the magnitude （P = 0.816） of the tumor （P 〉 0.05）, but it was significantly correlated with degree of differentiation （P = 0.001）, depth of invasion （P = 0.001）, clinical stage （P = 0.000） and metastasis （P = 0.000）. In the colorectal cancer, the expression of EpCAMhi^h/CD44~ cells has no correlation with gender （P = 0.934） and the magnitude （P = 0.160） of the tumor （P 〉 0.05）, but was significantly correlated with age （P = 0.021）, degree of differentiation （P = 0.013）, depth of invasion （P = 0.000）, clinical stage （P = 0.000） and metastasis （P = 0.000）. In the corresponding liver metastases, we could also detecte EpCAMhih/CD44＋ cells. In cases with abnormal expression of βcatenin, the positive expression rate of EpCAMhigh/CD44＋ was significantly higher than those with normal expression of β-catenin （84.1% vs 44.4%）, and the difference was statistically significant （P 〈 0.05）. Conclusion： The abnormal activation of Wnt β-catenin signalling pathway may prompt the abnormal proliferation of the colorectal cancer stem cells, which leads to the recurrence and metastasis of the cancer.

Inhibition of all-trans retinoic acid on MDM2 gene expression in astrocytoma cell line SHG-44

Objective To investigate the impact of all-trans retinoic acid （ATRA） on MDM2 gene expression in astrocytoma cell line SHG-44, and to provide basic data for further research on the progression mechanism and gene therapy of human astrocytoma. Methods The differential expressions of MDM2 gene and protein in SHG-44 cells were detected by cDNA microarray and Western blot, respectively, before and after treatment of ATRA. The expressions of MDM2 protein in WHO grade Ⅱ and grade Ⅳ astrocytomas were determined by immunohistochemical streptavidin-peroxidase method. Some differentially expressed genes were selected randomly for Northern blot analysis. Results The intensity ratio of ATRA-treated to untreated SHG-44 cell was 0.37 in the cDNA microarray, suggesting that the expression of MDM2 gene was down-regulated in SHG-44 cells after treatment with ATRA. Some genes differentially expressed in the microarray were confirmed by Northern blot. Western blot demonstrated that the optical density ratios of MDM2 to β-actin in ATRA-treated and untreated SHG-44 were 14.02±0.35 and 21.40±0.58 （t = 24.728, P = 0.000）, respectively, suggesting that the expression of MDM2 protein was inhibited in ATRA-treated SHG-44 cells. Moreover, the percentages of MDM2-positive protein were 24.00% （6/25） and 56.52% （13/23） （x^2 = 5.298, P = 0.021） in WHO grade Ⅱ and grade Ⅳ astrocytomas, respectively, suggesting that the expression of MDM2 protein may increase along with the elevation of astrocytoma malignancy. Conclusion ATRA can inhibit MDM2 gene expression in SHG-44 cells, and MDM2 is related to astrocytoma progression.

Expression of cell adhesion molecule CD44 in gastric adenocarcinoma and its prognostic importance

AIM: To evaluate the relation of cluster of differentiation 44 (CD44) expression with clinicopathological features of gastric adenocarcinoma, and also its effect on prognosis with an emphasis on the differences between intestinal and diffuse types. METHODS: From 2000 to 2006, 100 patients with gastric adenocarcinoma, who had undergone total or subtotal gastrectomy without any prior treatment, were studied. Haematoxylin & eosin (HE) staining was used for histological evaluation, including the type (Lauren's classifi cation) and grading of the tumor. The expression of CD44 in the gastric adenocarcinoma mucosa and the adjacent mucosa were determined by immunohistochemistry. The survival analysis was obtained using the Kaplan-Meier test. RESULTS: Of 100 patients, 74 (74%) patients were male. The tumors were categorized as intestinal type (78%) or diffuse type (22%). Sixty-five percent of patients were CD44-positive. CD44 expression was not detected in normal gastric mucosa. Rather, CD44 was more commonly expressed in the intestinal subtype (P = 0.002). A signifi cant relation was seen between the grade of tumor and the expression of CD44 (P = 0.014). The survival analysis showed a poor prognosis of patients with CD44-positive tumors (P = 0.008); and this was more prominent in the intestinal (P = 0.001) rather than diffuse type. CONCLUSION: Cell adhesion molecule CD44 is highly expressed in gastric adenocarcinoma. CD44 expression is correlated with a poor prognosis in patients with the intestinal type of gastric adenocarcinoma. CD44 can, therefore, be utilized as a prognostic marker for this group of patients.

Expression of human chorionic gonadotropin, CD44v6 and CD44v4/5 in esophageal squamous cell carcinoma

AIM： To study the relationship between the expression of human chorionic gonadotropin （HCG）, CD44v6, CD44v4/5 and the infiltration, metastasis of esophageal squamous cell carcinoma. METHODS： By labeled streptavidin-biotin technique, the expressions of HCG, CD44v6, and CD44v4/5 in 42 patients with esophageal squamous cell carcinoma were examined. RESULTS： The positive rate of HCG expression in patients with lymph node metastasis was 85.71% （18/21）, higher than that （57.14%, 12/21） in those without lymph node metastasis （P〈0.05）. The positive rate of CD44v6 expression was 71.43% （15/21） in lymph node metastasis group, and 38.09% （8/21） in nonmetastasis group; there was a significant difference between the two groups （P〈0.05）. The positive rate of CD44v4/5 expression was 76.19% （16/21） in lymph node metastasis group, and 42.86% （9/21） in non-metastasis group; there was also a significant difference between them （P〈0.05）. From grade Ⅰ to grade Ⅲ in differentiation, the positive rate of HCG expression was 84.62% （11/13）, 70.59% （12/17） and 58.33% （7/12）, respectively, there was no significant difference among them （P〉0.05）. The positive rate of CD44v6 expression in grades Ⅰ-Ⅲ of cancer tissues was 76.92% （10/13）, 52.94% （9/17）, and 33.33% （4/12） respectively; there was no significant difference among them. The positive rate of CD44v4/5 expression in grades Ⅰ-Ⅲ of cancer tissues was 69.23% （9/13）, 64.71% （11/17）, and 41.67% （5/12） respectively; there was no significant difference among the three groups. There was no correlation between the positive rates of HCG and CD44v6, CD44v4/5 expression. Cancer cells in carcinomatous emboli and those infiltrating into vascular wall strongly expressed HCG, CD44v6, and CD44v4/5. CONCLUSION： Expression of HCG, CD44v6, and CD44v4/5 in esophageal squamous cell carcinoma is related to its infiltration and metastasis. HCG, CD44v6, and CD44v4/5 have different effects on the infiltration and metastasis of esophageal squamous cell carcinoma.

Sox2 enhances the tumorigenicity and chemoresistance of cancer stem-like cells derived from gastric cancer

Gastric cancer stem-like cells（GCSCs） have been identified to possess the ability of self-renewal and tumor initi-ation.However,the mechanisms involved remain largely unknown.Here,we isolated and characterized the GCSCs by side population（SP） sorting procedure and cultured sphere cells（SC） from human gastric cancer cell lines SGC-7901,BGC-823,MGC-803,HGC-27 and MKN-28.The sorting and culture assay revealed that SP cells proliferated in an asymmetric division manner.In addition,SP cells exhibited a higher potential of spheroid colony formation and greater drug resistance than non-SP cells（NSP）.Moreover,the SC were found with enhanced capabilities of drug resistance in vitro and tumorigenicity in vivo.Sox2 mRNA and protein was highly and significantly overex-pressed in the SP cells and SC.Importantly,downregulation of Sox2 with siRNA obviously reduced spheroid colony formation and doxorubicin efflux,as well as increased apoptosis rate in sphere cells in vitro and suppressed tumori-genicity in vivo.These results suggest that both SP cells and cultured SC enrich with GCSCs and that Sox2 plays a pivotal role in sustaining stem cell properties and might be a potential target for gastric cancer therapy.

Effects of salvianolic acid B on in vitro growth inhibition and apoptosis induction of retinoblastoma cells

AIM: To observe the effects of salvianolic add B (SalB) on in vitro growth inhibition and apoptosis induction of retinoblastoma HXO-RB44 cells. METHODS: The effects of SalB on the HXO-RB44 cells proliferation in vitro were observed by MTT colorimetric method. The morphological changes of apoptosis before and after the treatment of SalB were observed by Hoechst 33258 fluorescent staining method. Apoptosis rate and cell cycle changes of HXO-RB44 cells were detected by flow cytometer at 48 hours after treated by SalB. The expression changes of Caspase-3 protein in HXO-RB44 cells were detected by Western Blot. RESULTS: SalB significantly inhibited the growth of HXO-RB44 cells, while the inhibition was in a concentration-and time-dependent manner. The results of fluorescent staining method indicated that HXO-RB44 cells showed significant phenomenon of apoptosis including karyorrhexis, fragmentation and the formation of apoptotic bodies, etc. after 24, 48 and 72 hours co-culturing of SalB and HXO-RB44 cells. The results of flow cytometer showed that the apoptosis rate and the proportion of cells in S phase were gradually increased at 48 hours and 72 hours after treated by different concentrations of SalB. Western Blot strip showed that the expression of Caspase-3 protein in HXO-RB44 cells was gradually increased with the increase of the concentration of SalB. CONCLUSION: SalB can significantly affect on HXO-RB44 cells growth inhibition and apoptosis induction which may be achieved through the up-regulation of Caspase-3 expression and the induction of cell cycle arrest.

DAPT suppresses the proliferation of human glioma cell line SHG-44

Objective:To explore the suppressing effect ofγ-secretase inhibitor DAPT on proliferation of human glioma cell line SHG-44 in vitro and its mechanism.Methods:The SHG-44 cell was treated by DAPT with different concentration.The proliferation of cells was detected by MTT assay;cell cycle and TSC of CD133^+were determined by flow cytometry analysis technique;the key factor in Notch signaling pathway(Notch-1,Delta-1,Hes-1)was measured by reverse transcrip tase-polymerase chain reaction and western blotting.Results:DAPT inhibited the growth and proliferation of SHG-44 cells significantly(P<0.05).And the inhibiting effect on SHG-44 cells produced by DAPT showed a dose-dependent manner.DAPT increased the rate of cells in G_0/G_1 phase of SHG-44 cells,while it decreased the rate of cells in S phase.TSC of CD133^+was significantly reduced after DAPT treated SHC-44 cells.The expression of protein and mRNA of Notch-1,Delta-1 and Hes-1 were gradually downregulated with the increase of DAPT doses.Conclusions:DAPT can downregulate these key factor in Notch signaling pathway,reduce the TSC of CD133+and inhibit the proliferation of SHC-44 cells.

HIF-2α regulates CD44 to promote cancer stem cell activation in triple-negative breast cancer via PI3K/AKT/mTOR signaling

BACKGROUND Breast cancer is a common malignant tumor that seriously threatens women’s health.Breast cancer stem cell(CSC)-like cell population may be the main factor for breast cancer metastasis.Therefore,targeted therapy for CSCs has great potential significance.Hypoxia-inducible factor is a transcription factor widely expressed in tumors.Studies have shown that down-regulation of the hypoxia signaling pathway inhibits tumor stem cell self-renewal and increases the sensitivity of stem cells to radiotherapy and chemotherapy mediated by hypoxiainducible factor-2α(HIF-2α).However,the specific mechanism remains unclear and further research is necessary.AIM To investigate the effect of HIF-2αdown-regulation on stem cell markers,microsphere formation,and apoptosis in breast cancer cell line MDA-MB-231 under hypoxia and its possible mechanism.METHODS Immunohistochemistry was used to detect the expression of HIF-2αand CD44 in triple-negative breast cancer(TNBC)and non-TNBC tissues.Double-labeling immunofluorescence was applied to detect the co-expression of HIF-2αand CD44 in MDA-MB-231 cells and MCF-7 cells.HIF-2αwas silenced by RNA interference,and the expression of CD44 and transfection efficiency were detected by real-time fluorescent quantitative PCR.Further,flow cytometry,TdT-mediated X-dUTP nick end labeling,and mammosphere formation assays were used to evaluate the effect of HIF-2αon CSCs and apoptosis.The possible mechanisms were analyzed by Western blot.RESULTS The results of immunohistochemistry showed that HIF-2αwas highly expressed in both TNBC and non-TNBC,while the expression of CD44 in different molecular types of breast cancer cells was different.In in vitro experiments,it was found that HIF-2αand CD44 were expressed almost in the same cell.Compared with hypoxia+negative-sequence control,HIF-2αsmall interfering ribonucleic acid transfection can lower the expression of HIF-2αand CD44 mRNA(P<0.05),increase the percentage of apoptotic cells(P<0.05),and resulted in a reduction of CD44+/CD24−population(P<0.05)and mammosphere formation(P<0.05)in hypoxic MDA-MB-231 cells.Western blot analysis revealed that phosphorylated protein-serine-threonine kinase(p-AKT)and phosphorylated mammalian target of rapamycin(p-mTOR)levels in MDA-MB-231 decreased significantly after HIF-2αsilencing(P<0.05).CONCLUSION Down-regulation of HIF-2αexpression can inhibit the stemness of human breast cancer MDA-MB-231 cells and promote apoptosis,and its mechanism may be related to the CD44/phosphoinosmde-3-kinase/AKT/mTOR signaling pathway.

Construction of cell lines with CD44 cDNA and its application in hepatocellular carcinoma

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Yi Qi Jie Du Formula and Salinomycin Combination Treatment Mediates Nasopharyngeal Carcinoma Stem Cell Proliferation,Migration and Apoptosis via CD44/Ras Signaling Pathway

Objective To assess the effects of Yi Qi Jie Du Formula(YQJDF)combined with salinomycin(SAL)on nasopharyngeal carcinoma stem cells(NPC-SCs)and investigate the underlying molecular mechanisms.Methods Cell counting methods,the CCK-8 assay,transwell migration assay and JC-1 staining,were used to observe the effects of the combination on the proliferation,migration and apoptosis of NPC stem cells,respectively.Western blot was used to detect the levels of protein in NPC-SCs.Results YQJDF combined with SAL had a synergistic effect on the inhibition of proliferation and migration and induction of NPCSC apoptosis.Mechanistically,YQJDF combined with SAL synergistically upregulated the levels of apoptotic proteins,including cleaved Caspase-3,cleaved Caspase-7 and cleaved Caspase-9.Moreover,YQJDF combined with SAL synergistically decreased the levels of CD44,p-c-Src,Ras,p-PKCδ,p-MEK,p-c-Raf,p-ERK1/2 and p-AKT proteins.Conclusions The combination of YQJDF and SAL has a synergistic effect on the inhibition of NPC-SC proliferation and migration and induction of apoptosis,which may be closely related to the downregulation of the CD44/Ras signaling pathway.

CD34^(+)CD38^(-)subpopulation without CD123 and CD44 is responsible for LSC and correlated with imbalance of immune cell subsets in AML

Acute myeloid leukemia(AML)is regarded as a stem cell disease.However,no one unique marker is expressed on leukemia stem cells(LSC)but not on leukemic blasts nor normal hematopoietic stem cells(HSC).CD34^(+)CD38^(-)with or without CD123 or CD44 subpopulations are immunophenotypically defined as putative LSC fractions in AML.Nevertheless,markers that can be effectively and simply held responsible for the intrinsical heterogeneity of LSC is still unclear.In the present study,we examined the frequency of three different LSC subtypes(CD34^(+)CD38^(-),CD34^(+)CD38^(-)CD123^(+),CD34^(+)CD38^(-)CD44^(+))in AML at diagnosis.We then validated their prognostic significance on the relevance of spectral features for diagnostic stratification,immune status,induction therapy response,treatment effect maintenance,and long^(-)term survival.In our findings,high proportions of the above three different LSC subtypes were all significantly characterized with low complete remission(CR)rate,high relapse/refractory rate,poor overall survival(OS),frequent FLT3^(-)ITD mutation,the high level of regulatory T cells(Treg)and monocytic myeloid^(-)derived suppressor cells(M^(-)MDSC).However,there was no significant statistical difference in all kinds of other clinical performance among the three different LSC groups.It was demonstrated that CD34^(+)CD38^(-)subpopulation without CD123 and CD44 might be held responsible for LSC and correlated with an imbalance of immune cell subsets in AML.

PRIMARY PLASMA CELL LEUKEMIA(A COMPREHENSIVE ANALYSIS OF 44 CASES)

Four cases of primary plasma cell leukemia (PPCL) admitted to Zhongshan Hospital from 1959 to 1987 are reported with a review on additional 40 cases reported in China. Comparing with the 57 cases of multiple myeloma (MM) treated in our hospital, the following features were observed in PPCL: (1) The age was younger, with a mean of 45.2 years, 34.1% of the patients were under 40 years. (2) Onset was abrupt. Duration from onset to diagnosis was 2 months or less in 77% patients but never beyond 6 months. (3) 81.8% patients had liver enlargement, 59.1% splenomegaly and 61.4% sternum tenderness. (4) All patients showed marked anemia with an average hemoglobin of 65 g/L. BPC count was less than 100 × 109/L in 76% patients and WBC was more than 10×109/L in 77%. (5) Plasma cell number in the marrow was markedly increased with an average of 69%, of which the blast cells and immature forms were predominant. (6) No destruction of bones was shown on X-ray film in 68.3% patients. (7) The response to chemotherapy was poor with a total response rate of 18% and a mean survival of 2 months. All the above-mentioned clinical features were significantly different from those of MM. In addition, these two diseases were also different in cytology, cytogenetics and ultrastructure. Therefore, PPCL should be considered as a special type of acute leukemia distinct from MM. High dose of alkylating agents in combination with autologous bone marrow transplantation might improve the prognoses.

Effects and Mechanism of Oxymatrine on Proliferation,Invasion and Migration of Hepatocellular Carcinoma Cells

[Objectives] To observe the effects of oxymatrine on the proliferation,invasion and migration of hepatocellular carcinoma cells,and to explore its possible molecular mechanism.[Methods]The hepatocellular carcinoma cell line Hep G2 was randomly divided into the negative control group and the low,medium and high concentration groups of oxymatrine.The negative control group was added to the cell culture medium,and the low,medium and high concentration groups of oxymatrine were added to the oxymatrine and cell culture medium with the final concentration of 1.0,2.0 and 4.0 mg/m L.The proliferation of each group was measured by MTT assay,and the inhibition rate of cell proliferation was calculated.The invasion and migration ability of each group was determined using Transwell chamber assay.The expression of E-cadherin and CD44 mRNA in each group was detected using Real-time PCR,while the expression of E-cadherin and CD44 protein was detected using Western blotting method.[Results] The inhibition rate of cell proliferation in the high concentration group of oxymatrine was higher than that in the medium and low concentration groups,and the inhibition rate of cell proliferation of medium concentration group was higher than that in the low concentration group.In the cell invasion and cell migration experiments,the number of transmembrane cells of low,medium and high concentration groups of oxymatrine was less than that in the negative control group(P < 0.05),the number of transmembrane cells of high concentration groups of oxymatrine was less than that in medium and low concentration groups,and the number of transmembrane cells of medium concentration group was less than in that of the low concentration group(P < 0.05).Compared with the negative control group,the expression of E-cadherin mRNA and protein increased in the low,medium and high concentration groups of oxymatrine,while the expression of CD44 mRNA and protein decreased(P < 0.05).[Conclusions] Oxymatrine has the effect of inhibiting the proliferation,invasion and migration of hepatocellular carcinoma cell line Hep G2 in vitro.The higher the concentration,the more significant the effect will be.The mechanism is possibly connected with the up-regulation of E-cadherin expression and down-regulation of CD44 expression.

Study of the Peripheral Blood CD44 Expression in the Patients with Non-Small Cell Lung Cancer

Previous study has demonstrated that the peripheral blood CD44 expression level is related with the clinical stage and lymph node metastasis of lung cancer. The present comment was to investigate the relationship between the peripheral blood CD44 expression level and clinic pathological change in 50 patients with non-small cell lung cancer (NSCLC) by flow cytometry method. The results showed that 1) the peripheral blood CD44 expression level in the NSCLC group was higher than that in the benign group (467 ± 15) or the normal group (448 ± 15);2) operation decreased the peripheral blood CD44 expression level in the NSCLC group (533 ± 10 vs. 324 ± 11);3) it also showed same results in NSCLC patients with and without lymph node metastasis (559 ± 12 vs. 477 ± 15) or before and after chemotherapy (550 ± 13 vs. 372 ± 10);4) there were significant differences in the peripheral blood CD44 expression level in non-small cell lung cancer patients of the clinical stage I, II, III and IV (474 ± 14, 526 ± 12, 528 ± 16 and 599 ± 20);And the peripheral blood CD44 expression level was not associated with the clinical pathology parameter including the patient age, gender and tumor size. The data suggested that the peripheral blood CD44 expression level was related with the NSCLC progress, lymphatic metastasis and clinical treatment, and the peripheral blood CD44 expression level as the clinical regular examination should evaluate the progress, lymphatic metastasis and clinical treatment for the patients with NSCLC.