Centrosome abnormalities in non-small cell lung cancer: correlations with DNA aneuploidy and expression of cell cycle regulatory proteins

The aims of this study were to investigate centrosome abnormalities in non-small cell lung cancer (NSCLC), and to assess their relationship with DNA aneuploidy, the expression of the cell cycle-associated proteins, and clinicopathological profiles. Tissue microarrays were constructed from 175 NSCLCs. We analyzed centrosome abnormalities and the expression of p16^INK4a, p53, and pRb using immunohistochemistry. Centrosome abnormalities were noted in 29% of the tumors and were even observed in the normal cells adjacent to the tumor. The frequency of DNA aneuploidy was significantly higher in the tumors containing centrosome abnormalities than in the tumors with a normal centrosome. p16^INK4a expression and loss of pRb expression, but not p53 expression, were significantly associated with centrosome abnormalities. Clinically, centrosome abnormalities were not found to have any prognostic value for NSCLCs. These results suggest that centrosome abnormalities may be associated with inactive pRb-pathway and contribute to pulmonary carcinogenesis by the level of increasing chromosome instability.     

The modulatory role of dendritic cell-T cell cross-talk in breast cancer: Challenges and prospects

Antigen recognition and presentation are highlighted as the first steps in developing specialized antigen responses. Dendritic cells (DCs) are outstanding professional antigen-presenting cells (APCs) responsible for priming cellular immunity in pathological states, including cancer. However, the diminished or repressed function of DCs is thought to be a substantial mechanism through which tumors escape from the immune system. In this regard, DCs obtained from breast cancer (BC) patients represent a notably weakened potency to encourage specific T-cell responses. Additionally, impaired DC-T-cell cross-talk in BC facilitates the immune evade of cancer cells and is connected with tumor advancement, immune tolerance, and adverse prognosis for patients. In this review we aim to highlight the available knowledge on DC-T-cell interactions in BC aggressiveness and show its therapeutic potential in BC treatment.     

Isolation and characterization of CD105+/CD90+ subpopulation in breast cancer MDA-MB-231 cell line

Background: The epithelial-mesenchymal transition (EMT) generates cells with properties of stem cells, if that happened, the stem cell should be with mesenchymal property. This study aimed to identify a group of cells with mesenchymal stem cell (MSC)-like characteristics in breast cancer bone metastatic cell line MDA-MB-231, moreover, the relevance between breast cancer stem cells and the EMT was observed. CD105 and CD90, identified as the standards of MSCs, were used for the identification. Methods: The CD105⁺/CD90⁺ and CD105^-/CD90^- subpopulation of MDA-MB-231 cells were detected and sorted by flow cytometry. MSC-like characteristics in cell proliferation, migration and cell cycle were investigated here by MTT asaay, transwell migration assay, and PI staining respectively. The expression profiles of some stem cell-associated genes were also observed by quantitative real time PCR. Results: Around 0.99% and 90.77% of parental cells were identified as CD105⁺/CD90⁺ and CD105^-/CD90^- cell subpopulations respectively. The CD105⁺/CD90⁺ cells exhibited stronger migratory capacity as compared to parental and CD105^-/CD90^- cells, while less CD105⁺/CD90⁺ cells were arrested in the S phase. Besides, pluripotent stem cell factors, like Oct-4, Nanog, Klf4 and Sox-2, were all upregulated in CD105⁺/CD90⁺ cells, with also proliferation increase, as compared with other two populations. Conclusion: The CD105⁺/CD90⁺ subpopulation from breast cancer MDA-MB-231 cells was proven to possess “mesenchymal stem cell-like” characteristics, and its high migratory ability might be associated with EMT. Moreover, using the surface markers of CD105 and CD90 for the identification of MSCs might provide new theoretical basis for the recurrence and metastasis of breast cancer.     

乳腺癌中Wnt/β-catenin信号通路相关蛋白的表达及意义

目的:本研究旨在探讨乳腺癌组织和细胞中Wnt/β-catenin信号通路相关蛋白Wnt-1和β-catenin的表达及意义。方法:选取我院于2015年1月~2017年1月间收治的150例乳腺癌患者作为研究对象。150例乳腺癌的肿瘤组织样本(实验组)来源于手术切除的、临床病理资料保存完整的乳腺癌石蜡切块;相应的临近非肿瘤组织样本(对照组)取自距离肿瘤组织0.5~1 cm的上述乳腺癌患者相应癌旁组织。采用real-tme PCR和Western blot法检测Wnt-1和β-catenin的表达情况。将乳腺癌细胞MCF-7分为3组:阴性对照组(MCF-7细胞)、激动剂组[MCF-7细胞+Wnt3a(1 mg/L)]和拮抗剂组[MCF-7细胞+DKK1(16μmol/L)]。采用real-time和Western blot检测Wnt-1和β-catenin表达情况。结果:与对照组相比,实验组患者Wnt-1和β-catenin的mRNA和蛋白表达量均升高(P0.05)。实验组患者肿瘤组织的Wnt-1表达阳性率和β-catenin表达阳性率均高于对照组(P0.05)。Wnt-1表达与乳腺癌患者肿瘤转移(x~2=5.352,P=0.021)、肿瘤分期(x~2=9.412,P=0.002)及肿瘤直径(x~2=9.412,P=0.002)密切相关(P0.05)。β-catenin表达与乳腺癌患者肿瘤转移(x~2=9.851,P=0.002)、肿瘤分期(x~2=5.661,P=0.017)密切相关(P0.05)。与对照组相比,激动剂组Wnt-1和β-catenin mRNA和蛋白表达量升高(P0.05);与对照组相比,拮抗剂组Wnt-1和β-catenin mRNA和蛋白表达量降低(P0.05)。结论:乳腺癌中Wnt/β-catenin信号通路相关蛋白Wnt-1和β-catenin表达上调,且其表达与肿瘤恶性状态密切相关。     

TIP30/CC3 expression in breast carcinoma: relation to metastasis, clinicopathologic parameters, and P53 expression

Metastasis is the most frequent cause of death in patients with breast cancer. Tip30/CC3 gene is a putative metastasis suppressor gene, which was first identified by a differential display analysis of messenger RNA from the highly metastatic human variant small cell lung carcinoma (SCLC) versus less metastatic classic SCLC cell lines. The aim of this study was to analyze the relationship between expression of TIP30/CC3 and clinical prognosis in 87 patients with surgically removed breast carcinoma. Tumor tissues were stained immunohistochemically with anti-TIP30/CC3 antibody. We demonstrated that the expression of TIP30/CC3 was inversely associated with axillary lymph node metastasis (P = .0008) and vascular invasion (P = .0016). Expression of TIP30/CC3 was not correlated with tumor grade, estrogen, progesterone, and P53 expression. Inhibition of TIP30/CC3 expression by RNA-mediated interference greatly enhanced breast cancer cell invasion through the extracellular matrix, whereas overexpression of TIP30/CC3 by adenovirus vector suppressed invasion through the extracellular matrix. These data supported the theory that the expression of TIP30/CC3 had a suppressive function on tumor metastasis. In summary, the decrease in expression of TIP30/CC3 is related to metastasis and may represent a new prognosticator in breast carcinoma.     

RP215 single chain fragment variable and single domain recombinant antibodies induce cell cycle arrest at G0/G1 phase in breast cancer

Background

Targeted therapy is an attractive approach to avoid the side effects of cancer treatment. Based on antibody-targeted superantigens, single chain variable fragment (scFv) and single domain (sdAb) antibodies, characterized by a low molecular weight, low immunogenicity and a high tumor penetration compared to monoclonal antibodies (mAb), have been increasingly used in gene-targeted therapy for cancer. In the present study, we aimed to develop the novel recombinant scFv-RP215 and sdAb-RP215 antibodies based on the variable regions of the RP215 monoclonal antibody (RP215-mAb) against CA215, a pan cancer marker expressed in various human tumor tissues, and to examine their biological activity in breast cancer cell lines.

Methods

The VH and VL genes were amplified from hybridoma cells secreting RP215-mAb by RT-PCR and joined with a linker using splicing by overlap extension PCR (SOE-PCR) to obtain the RP215-scFv gene, whereas the VH gene was used to generate the RP215-sdAb. Gene fragments of antibodies were subcloned into the pET32a(+) vector and expressed in Escherichia coli BL21. Western blot, indirect immunofluorescence (IF), ELISA and competitive ELISA were used to detect the immunoreactivity of scFv-RP215, sdAb-RP215, and RP215-mAb. The CCK-8 assay and cell cycle analysis were used to assess antibodies function.

Results

The novel recombinant scFv-RP215 and sdAb-RP215 antibodies were successfully developed based on the variable regions of the monoclonal antibody RP215 (RP215-mAb) against CA215. We verified that scFv-RP215 and sdAb-RP215 recognize CA215 on the surface of breast cancer cells (MB231, MCF7, MB468, SK-BR-3 and BT549) and characterized their activity and specificity. Our findings also indicate that scFv-RP215 and sdAb-RP215 induce cell cycle arrest at the G0/G1 phase in breast cancer cells.

Conclusion

Our results showed that scFv-RP215 and sdAb-RP215 have excellent immunoreactivity and localize accurately to breast cancer cells in membrane-bound form, suggesting their potential as tumor targeting antibodies for breast cancer therapy.     

Expression of proliferating cell nuclear antigen,Ki-67 antigen,estrogen receptor protein,and tumor suppressor p53 gene in cytologic samples of breast cancer: An immunochemical study with clinical,pathobiological, and histologic correlations

Sixty-six unselected breast cancers were analyzed in cytologic smears and histologic sections for the expression of Ki-67, proliferating cell nuclear antigen (PCNA). estrogen receptor protein (ERP), and p53 protein using a standard immunochemical method. The results, expressed as both positive cases and labelling index (LI), were compared with clinical and pathobiological variables. Ki-67 and PCNA immunostaining was seen in all cases, whereas ERP was detectable in 46/63 cases and p53 protein in 20/66 cases. The expression of these markers was generally lower in cytology than in histology, though the differences were not statistically significant. PCNA-LI and Ki-67-LI were closely correlated (P < 0.001), the mean PCNA:Ki-67 ratio being 0.92 ± 0.57. Occasional discrepancies, however, were found. PCNA and Ki-67 expression was associated with an increase in histologic grade and a decrease in ERP content of tumors, whereas p53 was statistically associated with no clinical or pathobiological variables. The data suggest that proliferative activity and oncogene overexpression may be reliably evaluated in breast cancer by FNA cytology, though PCNA is not a suitable indicator for cell proliferation. The results do not resolve the issue as to whether immunostaining for p53 protein constitutes a dedifferentiation product of the tumor, or is a fundamental aspect of the malignant progression. Survival studies in a larger series of tumors are thus needed to elucidate this point. Diagn Cytopathol 1994; 11:131–140. © 1994 Wiley-Liss, Inc.     

Comprehensive immunohistochemical analysis of Her-2/neu oncoprotein overexpression in breast cancer: HercepTest™ (Dako) for manual testing and Her-2/neuTest 4B5 (Ventana) for Ventana BenchMark automatic staining system with correlation to results of fluorescence in situ hybridization (FISH)

Overexpression of Her-2/neu-oncoprotein is used as marker for Herceptin® therapy. To investigate the sensitivity and specificity of automatic immunohistochemistry (Benchmark, Ventana), we compared the results to the manual testing (Dako) in 130 breast carcinomas and validated the results by fluorescence in situ hybridization (FISH). Manual and automatic immunohistochemistry of Her-2/neu-oncoprotein using two different antibodies (HercepTest?, Her-2/neuTest 4B5) was analyzed. FISH was performed in all cases with uncertain or strong overexpression in either immunohistochemical stainings or with different immunohistochemical results. Same immunohistochemical results were seen in 73.8%. Two cases with overexpression, detected with Her-2/neuTest 4B5 and confirmed by FISH, showed no overexpression using HercepTest?. From 21 cases with 2+ by Her-2/neuTest 4B5, 15 cases had no gene amplification (two of them with 3+ HercepTest?); three cases showed a gene amplification (one of them with failing overexpression by HercepTest?); two other cases were polysomic; one could not be analyzed. Ventana immunohistochemistry seems to be of same reliability like Dako with a little better concordance to FISH in our study.     

Interleukin 1-induced cancer cell/endothelial cell adhesion in vitro and its relationship to metastasis in vivo: role of vessel wall 13-HODE synthesis and integrin expression

Previously, we have demonstrated that stimulation of endothelial cells (ECs) with interleukin-la (IL-l) enhances the synthesis and expression of the vitronectin receptor (VnR), promotes VnR-dependent adhesion of human A549 adenocarcinoma cells to ECs, and is associated with decreased EC 13-hydroxyoctadecadienoic acid (13-HODE) synthesis in vitro. To determine whether these observations are relevant in vivo, we examined the acute retention and subsequent metastasis of intravenously-injected B16F10 melanoma cells in murine lungs, in relation to vessel wall 13-HODE. In C57BL/6 mice pretreated with IL-l, vessel wall 13-HODE was decreased and B16F10 lung entrapment and metastasis were increased. The latter two events were blocked by pretreating the animals with the GRGDS peptide. These data suggest a relationship between vessel wall 13-HODE synthesis, adhesion molecule expression, and adhesion of B16F10 cells to the endothelium.