Enhancive effects of Lewis y antigen on CD44-mediated adhesion and spreading of human ovarian cancer cell line RMG-I

Background

This study aimed to investigate the molecular structural relationship between cell adhesive molecule CD44 and Lewis y antigen, and determine the effects of Lewis y antigen on CD44-mediated adhesion and spreading of ovarian cancer cell line RMG-I and the Lewis y antigen-overexpressed cell line RMG-I-H.

Methods

The expression of CD44 in RMG-I and RMG-I-H cells before and after treatment of Lewis y monoclonal antibody was detected by immunocytochemistry; the expression of Lewis y antigen and CD44 was detected by Western Blot. The structural relationship between Lewis y antigen and CD44 was determined by immunoprecipitation and confocal laser scanning microscopy. The adhesion and spreading of RMG-I and RMG-I-H cells on hyaluronic acid (HA) were observed. The expression of CD44 mRNA in RMG-I and RMG-I-H cells was detected by real-time RT-PCR.

Results

Immunocytochemistry revealed that the expression of CD44 was significantly higher in RMG-I-H cells than in RMG-I cells (P < 0.01), and its expression in both cell lines was significantly decreased after treatment of Lewis y monoclonal antibody (both P < 0.01). Western Blot confirmed that the content of CD44 in RMG-I-H cells was 1.46 times of that in RMG-I cells. The co-location of Lewis y antigen and CD44 was confirmed by co-immunoprecipitation. The co-expression of CD44 and Lewis y antigen in RMG-I-H cells was 2.24 times of that in RMG-I cells. The adhesion and spreading of RMG-I-H cells on HA were significantly enhanced as compared to those of RMG-I cells (P < 0.01), and this enhancement was inhibited by Lewis y monoclonal antibody (P < 0.01). The mRNA level of CD44 in both cell lines was similar (P > 0.05).

Conclusion

Lewis y antigen strengthens CD44-mediated adhesion and spreading of ovarian cancer cells.     

Expression of Lewis y antigen and integrin αv, β3 in ovarian cancer and their relationship with chemotherapeutic drug resistance

Objective

This study investigates the expression of Lewis y antigen, integrin αv, β3 in epithelial ovarian cancer tissues. We further evaluate the relationship between their expression and chemotherapy resistance of ovarian cancer and its possible clinical significance.

Methods

Tissues of 92 patients with ovarian cancer meeting the inclusion criteria with complete follow-up data were enrolled and divided into chemotherapy resistant group and sensitive group. The expression and relationship of Lewis y antigen and integrin αv, β3 are assessed in paraffin sections using immunohistochemistry and double-labeling immunofluorescence method. Multivariate logistic regression analysis was used to investigate the relationship between age, clinical stage, differentiation, histologic subtype, Lewis y antigen and integrin αv, β3 expression in ovarian cancer patients.

Results

The expression rates of Lewis y antigen and integrin αv in the resistant group, significantly higher than the rates found in the sensitive group (p <0.05). Multivariate analysis showed that the expression of Lewis y antigen, integrin αv and ovarian cancer’s clinical stage were independent, drug resistance-related risk factors. The expression levels of Lewis y antigen and integrin αv, β3 were positively correlated with each other.

Conclusions

A close correlation between Lewis y antigen, integrin αv, β3 and ovarian cancer was observed. Lewis y antigen can influence the biological behavior of a tumor cell as an important composition of integrin αv, β3 by some signal pathway. And the expression of Lewis y antigen, integrin αv and ovarian cancer’s clinical stage are both independent, drug resistance-related risk factors.     

Membranous expressions of Lewis y and CAM-DR-related markers are independent factors of chemotherapy resistance and poor prognosis in epithelial ovarian cancer

Background: Chemotherapy resistance is a common problem faced by patients diagnosed with epithelial ovarian cancer (EOC). Currently there are no specific or sensitive clinical biomarkers that maybe implemented to identify chemotherapy resistance and give insight to prognosis. The aim of this study is to investigate the roles of Lewis y antigen and the markers associated with cell-adhesion-mediated drug resistance (CAM-DR) in patients with EOC. Methods: 92 EOC patients who were treated with systemic chemotherapy after cytoreductive surgery were included in this analysis. Patients were divided into two groups, chemotherapy sensitive (n = 56) and resistant (n = 36). Immunohistochemical (IHC) staining for Lewis y and CAM-DR-related cell surface proteins including CD44, CD147, HE4 (Human epididymis protein 4), integrin α5, β1, αv and β3 were conducted on tissues collected during primary debulking surgery. Using multivariate logistic regressions, IHC results were compared to clinical variables and chemotherapy resistance to determine possible correlations. The relationships between IHC expression and progression-free survival (PFS) and overall survival (OS) were analyzed using Kaplan–Meier method and Cox regression analysis. Results: Membranous expression of Lewis y and all these CAM-DR-related markers were significantly higher in the resistant group than that of the sensitive group (all P < 0.01). Multivariate regression analysis revealed that high expression of Lewis y, CD44, HE4, integrin α5 and β1 as well as advanced FIGO stage were independent risk factors for chemotherapy resistance (all P < 0.05). Advanced FIGO stage, lymph node metastasis and high expression of Lewis y, CD44, CD147, HE4, integrin α5, β1 were associated with a shorter PFS and OS (all P < 0.05). Moreover, multivariate COX analysis demonstrated that the following variates were independent predictors of worse PFS and OS survival: late FIGO stage (P = 0.013, 0.049), high expressions of Lewis y (P = 0.010, 0.036), HE4 (P = 0.006, 0.013) and integrin β1 (PFS, P = 0.003), integrin α5 (OS, P = 0.019). Conclusion: Membranous expression of Lewis y and CAM-DR-related markers including CD44, CD147, HE4, integrin α5, β1, αv and β3 are associated with the development of chemotherapy resistance. High expression of Lewis y antigen and CAM-DR-related markers including CD44, CD147, HE4, integrin α5 and β1 are independent markers for PFS and OS, in which Lewis y and HE4 are the most significant.     

Expression and correlation of Lewis y antigen and TGF-β1 in ovarian epithelial carcinoma

Lewis y is a difucosylated oligosaccharide carried by glycoconjugates on the cell surface. Elevation of Lewis y is frequently observed in epithelial-derived cancers. This study aimed to detect the expression and clinical significance of the Lewis y antigen and TGF-β1 (transforming growth factor β1) in ovarian epithelial tumors, and to evaluate the correlation between them. Immunohistochemical staining was used to detect the expression of Lewis y antigen and TGF-β1 in 60 cases of ovarian epithelial malignant tumors, 20 cases of borderline ovary tumors, 20 cases of benign ovary tumors and 10 cases of normal ovarian tissues. An immunofluorescence double labeling method was also used to detect the correlation between Lewis y antigen and TGF-β1. The positive rates of Lewis y antigen in ovarian epithelial cancer tissues was 88.33%, significantly higher compared to those of borderline ovarian tumors (60.00%) (P<0.05), benign ovarian tumors (35.00%) (P<0.01) and normal ovarian tissues (0%) (P<0.01). Its expression was not associated with clinical parameters; the positive rates of TGF-β1 in ovarian epithelial cancers were 78.33%, significantly higher compared to those of benign ovarian tumors (65.00%) (P<0.05) and normal ovarian tissues (40.00%) (P<0.05); the positive rates of the TGF-β1 and Lewis y were not associated with metastasis of lymph nodes and histological types, differentiation degree and clinical stage (P>0.05). Expression of Lewis y antigen and TGF-β1 was significantly positively associated with epithelial carcinoma. Close correlation between Lewis y, TGF-β1 and ovarian cancer was observed. Altered expression of Lewis y antigen may cause changes in TGF-β1 expression. Lewis y can increase the growth of ovarian cancer cells and the invasion ability by promoting TGF-β1 abnormal expression and by promoting angiogenesis and a change in its signal transduction pathway. This study provides theoretical evidence for the development of ovarian cancer biological treatments.     

Lewisy抗原及IGF-1R在卵巢黏液性肿瘤中的表达及意义

目的：探讨Lewis y抗原及胰岛素样生长因子受体-1（IGF-1R）在卵巢黏液性肿瘤中表达规律及其相关性。方法：用免疫组化法检测62例卵巢黏液性肿瘤和20例正常卵巢组织中Lewis y抗原与IGF-1R的表达,并分析其表达与卵巢癌病理分级、临床分期及淋巴结转移的关系。结果：Lewis y在恶性、交界性、良性和正常组的阳性表达率分别是90.0%、70.6%、53.3%和0%,呈下降趋势,其中恶性与交界性,良性和正常组比较差异有统计学意义（P〈0.05）;IGF-1R在恶性、交界性、良性和正常组的阳性表达率分别是93.3%、70.6%、46.7%和40.0%,呈下降趋势,其中恶性与交界性,良性和正常组比较差异有统计学意义（P〈0.05）;Lewis y抗原或IGF-1R的阳性表达率与卵巢黏液癌临床病理参数无关（P均〉0.05）;Lewis y抗原和IGF-1R的表达水平随临床分期的增加,但无统计学意义（P〉0.05）、随分化程度的降低而增加（P〈0.05）,与有无淋巴结转移无关（P〉0.05）;Lewis y抗原和IGF-1R在卵巢黏液性囊腺癌中的表达呈显著正相关（r=0.711,P〈0.005）。结论：随着卵巢黏液性肿瘤的恶性进展Lewis y抗原及IGF-1R的表达上调,且两者的表达呈显著性正相关。提示Lewis y抗原及IGF-1R对卵巢黏液性囊腺癌的转移、分化起促进作用。     

HE4与Lewis在卵巢上皮性肿瘤中的表达及相关性研究

目的:研究HE4与Lewis y抗原在卵巢上皮性肿瘤中的表达、相关性及临床意义。方法:免疫组化法检测卵巢上皮性恶性肿瘤、卵巢上皮性交界性肿瘤、卵巢上皮性良性肿瘤及正常卵巢组织中HE4与Lewis y抗原的表达。应用免疫荧光双标记方法检测卵巢上皮性肿瘤中HE4与Lewis y的结构关系。结果:HE4在卵巢恶性肿瘤组的阳性率最高,明显高于良性及正常卵巢组（P均<0.05）。Lewis y表达与HE4相似,在卵巢恶性上皮性肿瘤中,Lewis y抗原的阳性表达率明显高于交界性、良性及正常卵巢组。相关性分析显示:HE4与Lewis y呈直线相关(r=0.813,P<0.05)。在激光共聚焦显微镜下可见HE4与Lewis y抗原有空间位置上的重叠。结论:HE4与Lewis y抗原在卵巢恶性肿瘤中均呈现明显高表达,且两者在卵巢上皮性肿瘤中的表达呈正相关。     

Lewis y antigen promotes the proliferation of ovarian carcinoma-derived RMG-I cells through the PI3K/Akt signaling pathway

Background

Lewis y antigen is difucosylated oligosaccharide and is carried by glycoconjugates at cell surface. Elevated expression of Lewis y has been found in 75% of ovarian tumor, and the high expression level is correlated to the tumor''s pathological staging and prognosis. This study was to investigate the effect and the possible mechanism of Lewis y on the proliferation of human ovarian cancer cells.

Methods

We constructed a plasmid encoding α1,2-fucosyltransferase (α1,2-FT) gene and then transfected it into ovarian carcinoma-derived RMG-I cells with lowest Lewis y antigen expression level. Effect of Lewis y on cell proliferation was assessed after transfection. Changes in cell survival and signal transduction were evaluated after α-L-fucosidase, anti-Lewis y antibody and phosphatidylinositol 3-kinase (PI3K) inhibitor treatment.

Results

Our results showed that the levels of α1,2-FT gene and Lewis y increased significantly after transfection. The cell proliferation of ovarian carcinoma-derived RMG-I cells sped up as the Lewis y antigen was increased. Both of α-L-fucosidase and anti-Lewis y antibody inhibited the cell proliferation. The phosphorylation level of Akt was apparently elevated in Lewis y-overexpressing cells and the inhibitor of PI3K, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, dramatically inhibited the growth of Lewis y-overexpressing cells. In addition, the phosphorylation intensity and difference in phosphorylation intensity between cells with different expression of α1,2-FT were attenuated significantly by the monoantibody to Lewis y and by the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002.

Conclusions

Increased expression of Lewis y antigen plays an important role in promoting cell proliferation through activating PI3K/Akt signaling pathway in ovarian carcinoma-derived RMG-I cells. Inhibition of Lewis y expression may provide a new therapeutic approach for Lewis y positive ovarian cancer.     

Breast cancer humoral immune response: involvement of Lewis y through the detection of circulating immune complexes and association with Mucin 1 (MUC1)

Background

In cancer patients, MUC1 glycoprotein may carry Lewis y which could be involved in immune response. Purposes: 1- to evaluate the presence of Lewis y and MUC1 in circulating immune complexes (Lewis y/CIC and MUC1/CIC, respectively) and their correlation; 2- to analyze the possible presence of Lewis y in carbohydrate chains of tumoral MUC1 glycoprotein and 3- to correlate serum and tissue parameters considered.

Methods

Pretreatment serum and tissue breast samples from 76 adenocarcinoma, 34 benign and 36 normal specimens were analyzed. Anti-MUC1 and anti-Lewis y MAbs were employed. To detect Lewis y/CIC and MUC1/CIC, ELISA tests were developed; serum samples containing MUC1 were previously selected by Cancer Associated Serum Antigen (CASA). Immunoprecipitation (IP) was performed in 9 malignant, benign and normal samples and analyzed by SDS-PAGE and Western blot. Lewis y and MUC1 expression was studied by immunohistochemistry (IHC). Statistical analysis was performed employing principal component analysis (PCA), ANOVA, Tukey HSD, Chi square test and classical correlation (p < 0.05).

Results

By ELISA, Lewis y/IgM/CIC levels showed statistically significant differences between breast cancer versus benign and normal samples; mean ± SD values expressed in OD units were: 0.525 ± 0.304; 0.968 ± 0.482 and 0.928 ± 0.447, for breast cancer, benign disease and normal samples, respectively, p < 0.05. Lewis y/IgG/CIC did not show any statistically significant difference. MUC1/IgM/CIC correlated with Lewis y/IgM/CIC. By CASA, 9 samples with MUC1 values above the cut off were selected and IP was performed, followed by SDS-PAGE and Western blot; bands at 200 kDa were obtained with each MAb in all the samples. By IHC, with C14 MAb, 47.5%, 31% and 35% of malignant, benign and normal samples, respectively, showed positive reaction while all the samples were positive with anti-MUC1 MAb; in both cases, with a different pattern of expression between malignant and non malignant samples.

Conclusion

Our findings support that in breast cancer there was a limited humoral immune response through Lewis y/IgM/CIC levels detection which correlated with MUC1/IgM/CIC. We also found that Lewis y might be part of circulating MUC1 glycoform structure and also that Lewis y/CIC did not correlate with Lewis y expression.     

High expression of S100P is associated with unfavorable prognosis and tumor progression in patients with epithelial ovarian cancer

Accumulating evidence has demonstrated that S100P is involved in the tumorigenesis and progression of multiple cancers. In the current study, we evaluated the expression of S100P in epithelial ovarian cancer and assessed its relevance to clinicopathological characteristics. Moreover, we investigated the biological effects of S100P using A2780 and SKOV3 cells. S100P expression was significantly increased in epithelial ovarian cancer specimens compared with fallopian tube tissues and normal ovary tissues. And high expression of S100P in epithelial ovarian cancer samples was significantly associated with tumor stage (P<0.001), serum CA125 level (P=0.026), residual tumor (P<0.001), ascites (P<0.001) and lymph nodes metastasis (P<0.001). Multivariate Cox analysis showed that S100P expression was an independent prognostic factor of overall survival (OS) and progression free survival (PFS) (P=0.017 and 0.031, respectively). Functional assays showed that overexpression of S100P promoted cell proliferation and cell cycle progression but did not affect cell migration and invasion in A2780 and SKOV3 cells. These data suggest that S100P may contribute to tumor development in epithelial ovarian cancer and could be a useful marker for the prognosis of epithelial ovarian cancer patients.     

Lewis y抗原及整合素α_5、β_1在卵巢浆液性肿瘤中的表达及意义

目的：研究Lewis y抗原及整合素α5、β1在卵巢浆液性肿瘤中的表达及其意义。方法：应用免疫组织化学方法检测30例卵巢浆液性囊腺癌、11例交界性卵巢浆液性囊腺瘤、13例卵巢浆液性囊腺瘤、11例正常卵巢组织中Lewis y抗原及整合素α5、β1的表达情况。结果：Lewis y抗原在卵巢浆液性囊腺癌中的阳性表达率为90.0%,明显高于交界性（63.6%）、良性卵巢浆液性囊腺瘤（38.5%）及正常卵巢组织（0）中的表达（P均〈0.05）。整合素α5、β1在卵巢浆液性囊腺癌中的阳性表达率分别为86.7%和83.3%,高于两者在交界性（72.7%,72.7%）、良性卵巢浆液性囊腺瘤（69.2%,53.8%）及正常卵巢组织（36.4%,36.4%）中的表达（P均〈0.05）。Lewis y抗原及整合素α5、β1在卵巢浆液性囊腺癌中的表达呈显著正相关（P均〈0.01）。结论：Lewis y抗原及整合素α5、β1在卵巢浆液性囊腺癌中的阳性表达率普遍增高,且两者的表达呈显著性正相关。提示Lewis y抗原及整合素α5、β1在卵巢浆液性囊腺癌的发生、发展中起着重要作用。     

RhoC is a major target of microRNA-93-5P in epithelial ovarian carcinoma tumorigenesis and progression

Background

An increasing amount of evidence has revealed that microRNAs regulate various biological processes, including cell differentiation, cell proliferation, apoptosis, drug resistance, and fat metabolism. Studies have shown that miR-93’s targetome in cancer has not been fully defined. Moreover, the role of miR-93 in epithelial ovarian carcinoma (EOC) remains largely unknown.

Methods

MIR-93 mRNA expression in normal ovarian tissue, benign tumors, borderline tumors, primary ovarian carcinomas, and metastatic omentum was quantified. The ovarian carcinoma cell lines OVCAR3, SKOV3/DDP, and HO8910-PM were transfected with miR-93-5P, after which cell phenotype and expression of relevant molecules were assayed. Dual-luciferase reporter assay and a xenograft mouse model were used to examine miR-93 and its target gene RHOC (Ras homolog gene family member C).

Results

MIR-93 mRNA expression was significantly lower in ovarian carcinomas and borderline tumors than in normal ovarian tissues (p < 0.05), and was lower in metastatic omentum than in relative primary ovarian carcinomas (p < 0.05). MIR-93 mRNA expression was also negatively associated with differentiation (well vs. poor and moderate) and International Federation of Gynecology and Obstetrics staging (FIGO stage I/II vs. stage III/IV) in ovarian carcinoma (p < 0.05), besides, miR-93 was higher expressed in mucinous adenocarcinoma than the other types (p < 0.05). MiR-93-5P overexpression reduced proliferation (p < 0.05); promoted G₁ or S arrest and apoptosis (p < 0.05); suppressed migration and invasion (p < 0.05); and reduced RhoC, P70S6 kinase, Bcl-xL, matrix metalloproteinase 9 (MMP9) mRNA or protein expression; conversely, it induced P53 and cleaved PARP expression (p < 0.05). Dual-luciferase reporter assay indicated that miR-93 directly targeted RhoC by binding its 3′ untranslated region. MiR-93-5P transfection also suppressed tumor development and RhoC expression (determined by immunohistochemistry) in vivo in the xenograft mouse model (p < 0.05).

Conclusions

This is the first demonstration that miR-93-5P may inhibit EOC tumorigenesis and progression by targeting RhoC. These findings indicate that miR-93-5P is a potential suppressor of ovarian cellular proliferation. The involvement of miR-93-5P–mediated RhoC downregulation in inhibiting EOC aggressiveness may provide extended insight into the molecular mechanisms underlying cancer aggressiveness.     

Clinicopathological significance of Fas and Fas ligand expressions in esophageal cancer

Esophageal carcinomas have recently been shown to express Fas ligand (FasL) and down-regulate Fas to escape from host immune surveillance. However, the prognostic importance of Fas/FasL and their correlation with clinicopathological characteristics are yet to be delineated in this highly malignant carcinoma. Specimens from 106 esophageal squamous cell carcinoma patients were used for immuno-histochemical evaluation of Fas, FasL, and CD8 expressions. Fifty-two (49%) and 34 (32%) patients were positive for FasL and Fas, respectively. There were no associations between FasL expression and clinicopathological characteristics except lymph vessel invasion. Strong FasL expression correlated with significant (P < 0.001) decrease in tumor nest CD81 cells. However, neither FasL nor CD81 had any impact on patient survival. Strong Fas expression was correlated with depth of invasion (40.3% in pT1, T2 versus 20.5% in pT3, T4; P5 0.0308), histological differentiation (45.7% in well versus 25.4% in nonwell; P < 0.05), and lymph node metastasis (22.6% in positive versus 45.5% in negative; P < 0.01). Fas expression was one of the independent favorable prognosticators for patients’ survival (risk ratio, 3.26; P < 0.01) in esophageal SCC. Fas expression was an independent prognosticator for recurrencefree survival, whereas FasL expression did not influence the survival in esophageal squamous cell carcinoma. Down-regulation of tumor Fas may be the hallmark of immune privilege for the tumor, thus causing the patients’ poorer outcome. Tumor FasL may counterattack the host immune cells to such an extent that the prognosis is not affected.     

BCL6与Lewisy在卵巢浆液性肿瘤中的表达及其临床意义

目的：检测 B 细胞淋巴瘤－6（B cell lymphoma 6,BCL6）与 Lewis y 抗原在卵巢浆液性肿瘤组织中的表达,探讨其表达与卵巢浆液性囊腺癌的临床病理参数、预后的关系及其临床意义。方法：采用免疫组织化
　　学 SP 法检测卵巢浆液性囊腺癌、卵巢交界性肿瘤、卵巢良性肿瘤以及正常卵巢组织中 BCL6与 Lewis y 的表达,分析其与卵巢浆液性囊腺癌的临床病理参数和预后的关系以及二者相关性。结果：BCL6蛋白在卵巢浆液性囊腺癌、卵巢交界性肿瘤、卵巢良性瘤以及正常卵巢组织中的高表达率之间均有明显的差异（P 均＜0．05）。Lewis y 抗原在卵巢浆液性癌中的表达明显高于卵巢良性瘤及正常卵巢组织（P 均＜0．05）。随着卵巢肿瘤恶性程度的逐步增高,BCL6和 Lewis y 的表达明显增高,且二者存在正相关性（r ＝0．359,P ＝0．023）。BCL6的表达随着 FIGO 分期增加而逐渐增加（P ＜0．05）。BCL6为影响卵巢癌预后的危险因素（P ＜0．05）。结论：随着卵巢肿瘤恶性程度的逐步增高,BCL6蛋白和 Lewis y 的表达明显增高,且二者存在正相关性。BCL6的表达与临床期别、淋巴结转移、分化等相关,并可以用于患者的预后监测。     

Clinical relevance of stem cell surface markers CD133, CD24, and CD44 in colorectal cancer

Colon cancer stem cells (CSC) identified by cell surface markers CD133, CD24, and CD44, have been shown to be involved with tumor formation, chemotherapy resistance, and the progression of metastatic disease. Using an in silico translational approach, we hypothesize that a combination of these CSC markers has prognostic value in a large cohort of patients with colorectal cancer. Clinicopathologic and RNA expression data from a total of 594 colorectal cancer (CRC) patients from TCGA were analyzed. The expression of CD133, CD24, and CD44 was individually defined as “high” or “low” based on the median expression. Disease specific survival (DSS) and overall survival (OS) were not associated with tumors that are CD133-high or CD44-high alone. Patients with CD24-high tumors have significantly better DSS (P<0.001) and OS (P = 0.043). CD24-high, CD44-high and CD133-high tumors were associated with significantly greater EGFR, KRAS and Ki67 expression (all P<0.001). CD133, CD24 and CD44-high tumors were independently enriched for conventional stemness-related signaling pathways such as Wnt/β-catenin and Hedgehog signaling pathways. There was no survival difference linked to CD133-high/CD44-low patients, but CD44-high/CD24-low patients have worse DSS (P = 0.005) compared with CD44-low/CD24-high patients. CD133-high/CD24-low tumors show significant negative enrichment of MYC targets, E2F targets, G2M checkpoint and mitotic spindle gene sets, suggesting less cell proliferation in these tumors. Patients with CD133-high/CD24-low tumors have worse DSS (P = 0.004) and OS (P = 0.044), and are more likely to have early and late recurrences. In conclusion, we demonstrated that CD133-high/CD24-low tumors may predict colorectal cancer prognosis.     

TGFBI promoter hypermethylation correlating with paclitaxel chemoresistance in ovarian cancer

The purpose of this study is to determine the methylation status of Transforming growth factor-beta-inducible gene-h3 (TGFBI) and its correlation with paclitaxel chemoresistance in ovarian cancer. The methylation status of TGFBI was examined in ovarian cancer and control groups by methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP). The TGFBI expression and cell viability were compared by Quantitative Real-Time PCR, Western Blotting and MTT assay before and after demethylating agent 5-aza-2''-deoxycytidine (5-aza-dc) treatment in 6 cell lines (SKOV3, SKOV3/TR, SKOV3/DDP, A2780, 2780/TR, OVCAR8). In our results, TGFBI methylation was detected in 29/40 (72.5%) of ovarian cancer and 1/10 (10%) of benign ovarian tumors. No methylation was detected in normal ovarian tissues (P < 0.001). No statistical correlation between RUNX3 methylation and clinicopathological characteristics was observed. A significant correlation between TGFBI methylation and loss of TGFBI mRNA expression was found (P < 0.001). The methylation level of TGFBI was significantly higher in paclitaxel resistant cell lines (SKOV3/TR and 2780/TR) than that in the sensitive pairs (P < 0.001). After 5-aza-dc treatment, the relative expression of TGFBI mRNA and protein increased significantly in SKOV3/TR and A2780/TR cells. However, no statistical differences of relative TGFBI mRNA expression and protein were found in other cells (all P > 0.05), which showed that re-expression of TGFBI could reverse paclitaxel chemoresistance. Our results show that TGFBI is frequently methylated and associated with paclitaxel-resistance in ovarian cancer. TGFBI might be a potential therapeutic target for the enhancement of responses to chemotherapy in ovarian cancer patients.     

Dendritic cells recognize tumor-specific glycosylation of carcinoembryonic antigen on colorectal cancer cells through dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin

Dendritic cells play a pivotal role in the induction of antitumor immune responses. Immature dendritic cells are located intratumorally within colorectal cancer and intimately interact with tumor cells, whereas mature dendritic cells are present peripheral to the tumor. The majority of colorectal cancers overexpress carcinoembryonic antigen (CEA), and malignant transformation changes the glycosylation of CEA on colon epithelial cells, resulting in higher levels of Lewis(x) and de novo expression of Lewis(y) on tumor-associated CEA. Dendritic cells express the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) that has high affinity for nonsialylated Lewis antigens, so we hypothesized that DC-SIGN is involved in recognition of colorectal cancer cells by dendritic cells. We show that immature dendritic cells within colorectal cancer express DC-SIGN and that immature dendritic cells but not mature dendritic cells interact with tumor cells. DC-SIGN mediates these interactions through binding of Lewis(x) and Lewis(y) carbohydrates on CEA of colorectal cancer cells. In contrast, DC-SIGN does not bind CEA expressed on normal colon epithelium that contains low levels of Lewis antigens. This indicates that dendritic cells may recognize colorectal cancer cells through binding of DC-SIGN to tumor-specific glycosylation on CEA. Similar to pathogens that target DC-SIGN to escape immunosurveillance, tumor cells may interact with DC-SIGN to suppress dendritic cell functions.     

抗CD47单克隆抗体对卵巢癌细胞靶向治疗的体外研究

  目的   探讨抗CD47单克隆抗体在体外对巨噬细胞吞噬卵巢癌细胞作用的影响。  方法   收集天津医科大学附属肿瘤医院2011年6月至2011年12月手术切除的卵巢组织样本45例(包含卵巢癌组织样本39例, 正常卵巢组织样本6例), 获得的组织细胞通过流式细胞术分析CD47的表达水平, 通过细胞功能学实验观察抗CD47单克隆抗体在体外对小鼠巨噬细胞吞噬卵巢癌细胞的靶向治疗作用。  结果   流式细胞术分析显示, 卵巢癌细胞系SKOV-3细胞和卵巢癌组织细胞中CD47表达水平均明显高于正常人卵巢上皮细胞, 差异具有统计学意义(P < 0.01), 且与疾病的分化程度相关(P < 0.05)。细胞功能学实验结果显示, 抗CD47单克隆抗体在体外可促进巨噬细胞对卵巢癌细胞的吞噬作用, 各实验组(1μg/mL、5μg/mL、10μg/mL、20μg/mL)分别较空白对照组吞噬指数显著增加, 并且随抗体浓度的增加吞噬指数呈浓度依赖性, 差异均具有统计学意义(P < 0.01)。统计学分析显示, 10μg/mL浓度组吞噬指数达到峰值, 同20μg/mL浓度组吞噬指数无显著性差异(P > 0.05)。  结论   CD47高表达于卵巢癌组织细胞, 且与组织分化程度相关。抗CD47单克隆抗体在体外可促进巨噬细胞对卵巢癌肿瘤细胞的吞噬作用, 为卵巢癌的靶向治疗提供了实验依据。      

Increased microvessel density in mucinous compared with malignant serous and benign tumours of the ovary.

Microvessel density of benign, borderline and malignant ovarian tumours was studied immunohistochemically using antibodies to the endothelial cell markers CD31, CD34 and factor VIII-related antigen. Microvessel density was compared in tumours of different histological subtype, stage and patient outcome. CD31-immunostained sections were examined and regions of high and average microvessel density were selected. Identical regions were located on CD34- and factor VIII-related antigen-immunostained serial sections and microvessel counts obtained and converted to vessels mm(-2). CD31 and CD34 immunostaining revealed increased microvessel density in both the high and average vessel density regions of mucinous (222.4 +/- 24.8; 79.9 +/- 8.5) compared with serous (105.4 +/- 20.7; 33.3 +/- 6.8) and benign (84.4 +/- 19.4; 20.4 +/- 4.4) tumours (P < 0.001). CD31 and CD34 immunostaining also revealed increased microvessel density in early-stage mucinous tumours (234.6 +/- 28.2; 87.8 +/- 9.2) compared with that observed in both early- (72.8 +/- 15; 12.9 +/- 2.4) and late- (115.6 +/- 26.5; 29.8 +/- 8.5) stage serous tumours (P < 0.001). No differences in microvessel density in samples from patients with differing outcomes were observed (P > 0.05). Reduced factor VIII-related antigen compared with CD31 and CD34 immunostaining was observed in both borderline and malignant mucinous and serous tumours (P < 0.02) but not in benign tumours (P > 0.05). Our results contradict the putative association between increased microvessel density and poor prognosis and suggest that the level and control of angiogenesis may differ between ovarian tumour types.     

Drug resistance gene expression and chemotherapy sensitivity detection in Chinese women with different molecular subtypes of breast cancer

Objective:The aim of the study was to identify specific chemosensitivity drugs for various molecular subtypes of breast tumors in Chinese women, by detecting the expression of drug resistance genes and by using the drug sensitivity test on different molecular subtypes of breast cancers.Methods:The expression of drug resistance genes including Topo II, GST-π, P-gp, LRP, and CD133 were detected with immunohistochemistry in a tissue microarray. Drug sensitivity tests included those for paclitaxel, epirubicin, carboplatin, vinorelbine, and fluorouracil and were conducted on primary cancer tissue cells and cell lines, including the T47D, BT-474, and MDA-MB-231 cells and human breast cancer xenografts in nude mice.Results:The different drug resistant genes Topo II, GST-π, P-gp, and LRP were differentially expressed among different molecular subtypes of breast cancers (P < 0.05). Positive expression of CD133 was highest in basal-like breast cancer (P < 0.05). Kaplan-Meier survival analysis showed that positive expressions of Topo II and CD133 both correlated with shorter disease-free survival (DFS) (P < 0.05) and overall survival (P < 0.05), and positive expression of LRP correlated only with shorter DFS (P < 0.05). BT-474 showed chemosensitivity to paclitaxel and epirubicin, while MDA-MB-231 showed chemosensitivities to paclitaxel, epirubicin, carboplatin, and fluorouracil (T/C ≤ 50%). The basal-like and HER2+ breast cancer primary cells showed chemosensitivities to paclitaxel and epirubicin with significant differences compared with luminal breast cancer primary cells (P < 0.05).Conclusions:The differential expression of drug resistance genes and the differential chemosensitivities of drugs in different molecular subtype of breast cancers suggested that individual treatment should be given for each type of breast cancer.     

Co-expression of MET and CD47 is a novel prognosticator for survival of luminal-type breast cancer patients

Although luminal-type primary breast cancer can be efficiently treated, development of metastatic disease remains a significant clinical problem. We have previously shown that luminal-type circulating tumor cells (CTCs) co-expressing the tyrosine-kinase MET and CD47, a ligand involved in cancer cell evasion from macrophage scavenging, are able to initiate metastasis in xenografts. Here, we investigated the clinical relevance of MET-CD47 co-expression in 255 hormone receptor positive breast tumors by immunohistochemistry and found a 10.3-year mean overall-survival difference between MET-CD47 double-positive and double-negative patients (p<0.001). MET-CD47 co-expression defined a novel independent prognosticator for overall-survival by multivariate analysis (Cox proportional hazards model: HR: 4.1, p<0.002) and CD47 expression alone or in combination with MET was strongly associated with lymph node metastasis. Furthermore, flow cytometric analysis of metastatic patient blood revealed consistent presence of MET⁺CD47⁺ CTCs (range 0.8 – 33.3% of CTCs) and their frequency was associated with increased metastatic spread. Finally, primary uncultured CTCs with high MET⁺CD47⁺ content showed an enhanced capacity to initiate metastasis in mice. Detection and targeting of MET and CD47 may thus provide a rational basis for risk stratification and treatment of patients with luminal-type breast cancer.