Comparison of fluorescence <Emphasis Type="Italic">in situ</Emphasis> hybridization and immunohistochemistry for assessment of HER-2 status in breast cancer patients

The accurate assessment ofa proto-oncogene, human epidermal growth factor receptor-2 gene （HER-2）, is extremely important for the therapy and prognosis of breast cancer. Currently, immunohistochemistry （IHC） is the method widely used for the detection of HER-2 protein. Fluorescence in situ hybridization （FISH） has been suggested to be a golden standard assay for HER-2 amplification. This study examined the expression and amplification of HER-2 in paraffin-embedded sections of breast cancer tissues, and compared the two methods on the measurement of HER-2 status. HER-2 gene and protein were determined in breast cancer samples from 52 Chinese women by FISH and IHC respectively. The findings indicated that the HER-2 gene amplification was found in 18 cases （34.6%） by FISH and the HER-2 protein over-expression （score 3＋） in 15 cases （28.8%） by IHC. hnmunohistochemically, 28.6% of the cases scored as 2＋ and 93.3% of the cases scored as 3＋ were HER-2-positive by FISH. There was a significant correlation between the HER-2 gene amplification and HER-2 protein over-expression in breast cancer （P〈0.005）. No correlation was noted between the HER-2 gene amplification and any of the clinicopathological parameters examined, including age, menopausal status, menarche age, tumor size, histological tumor type, histological grade, lymph node status, and the expression of ER and PR. It was concluded that the detection of HER-2 gene amplification in breast cancer by FISH is valuable and can compare with HER-2 protein detection by IHC.     

ESTROGEN RECEPTOR-NEGATIVE BREAST CANCER CELLS TRANSFECTED WITH ESTROGEN RECEPTOR EXHIBIT DECREASED TUMOUR PROGRESSION ANDSENSITIVITY TO GROWTH INHIBITION BY ESTROGEN

Breast cancer containing estrogen receptors (ER) are responsive to antiestrogen treatment and have a better prognosis compared with ER negative tumors. The loss of estrogen receptors appears to be associated with a progression to less-differentiated cells. We transfected the human ER into the ER-negative breast cancer cell line MDA-MB-231 cells. We found that expression of adequate ER is strong associated with the ability of human breast cancer cell growth inhibition and progression. Compared with nontransfected or mock-transfected cells,ER-transfected cells exhibited growth slower, forming smaller colonies in soft agar and growth inhibited by estrogen and tamoxifen. Therefore reactivation or transfection of the estrogen receptor gene can be considered as therapeutic approaches to hormone-lndependent breast cancer.     

Inhibition of cell proliferation by siRNA targeting hPRLR in breast cancer MCF-7 cell line

Objective： To study the inhibition of proliferation of breast cancer by small interfering RNA（siRNA） targeting human prolactin （hPRLR） and the underlying mechanisms. Methods：The siRNA targeting hPRLR was chemically synthesized and transfected into MCF-7 cells, the expression of hPRLR was analyzed by real-time quantitive PCR, cell growth inhibition was measured with MTT assay, cell cycle of the transfected cells was examined by flow cytometry, meanwhile, expression of cyclin D1 was tested by semi-quantitative RT-PCR, Results：24 h after transfection with 100 nmol/L siRNA-PRLR, the expression of hPRLR mRNA was suppressed by 65%, cells in G1 phase increased, but cells in S phase decreased. Down regulated hPRLR expression exhibited significant inhibition in cell proliferation. And the expression of cyclin D 1 was down regulated. Conclusion：The results indicate that siRNA-hPRLR is a useful tool for silencing hPRLR expression and inhibiting cell proliferation in breast cancer MCF-7 cell line, and it may be a possible new approach for breast cancer gene therapy.     

miR-21 targets Fas ligand-mediated apoptosis in breast cancer cell line MCF-7

Over-expression of Fas ligand （FasL） on tumor cell surface can induce the apoptosis of spe- cific activated tumor infiltrating lymphocytes （TILs） via the Fas/FasL pathway, leading to the formation of a site of immune privilege surrounding the tumor mass for escaping immune surveillance and pro- moting tumor proliferation, invasion and metastasis. The blocking effect of miR-21 on FasL-mediated apoptosis in breast cancers was investigated in this study. The expression levels of miR-21 and FasL in human breast carcinoma cell lines were detected by using RT-PCR and Western blotting. FasL as a tar- get gene of miR-21 was identified by Luciferase assay. The apoptosis of Jurkat T lymphocytes induced by MCF-7 cells was determined by flow cytometry. It was found that in four human breast cancer cell lines, FasL expression level in MCF-7 cells was the highest, while miR-21 was down-regulated the most notably. After miR-21 expression in MCF-7 cells was up-regulated, FasL was identified as a target gene of miR-21. When the effector/target （E/T） ratio of MCF-7 cells and Jurkat cells was 10：1, 5：1 and 1：1, the inhibitory rate of apoptosis of Jurkat T lymphocytes induced by MCF-7 cells was 95.81%, 93.16% and 91.94%, respectively. It is suggested that in breast cancers miR-21 expression is negatively associ- ated with FasL expression, and FasL is a target gene of miR-21, miR-21 targeting and regulating FasL-mediated apoptosis will bring us the possibility of a new tumor immunotherapy via breaking tu- mor immune privilege.     

Inhibition of Notch1 increases paclitaxel sensitivity to human breast cancer

Background Paclitaxel （PAC） is the first-line chemotherapy drug for most breast cancer patients,but clinical studies showed that some breast cancer patients were insensitive to PAC,which led to chemotherapy failure.It was reported that Notch1 signaling participated in drug resistance of breast cancer.Here,we show whether Notch1 expression is related to PAC sensitivity of breast cancer.Methods We employed Notch1 siRNA and Notch1 inhibitor,N-[N-（3,5-difluorophenacetyl）-1-alanyl]-S-phenylglycine t-butylester （DAPT）,to down regulate Notch1 expression in human breast cancer cells MDA-MB-231,and detected the inhibition effect by Western blotting and reverse trans cription-polymerase chain reaction,respectively.After 24 hours exposure to different concentration of PAC （0,1,5,10,15,20,and 25 μg/ml）,the viability of the control group and experimental group cells was tested by MTT.We also examined the expression of Notch1 in PAC sensitive and nonsensitive breast cancer patients,respectively by immunohistochemistry （IHC）.The PAC sensitivity of breast cancer patients were identified by collagen gel droplet embedded culture-drug sensitivity test （CD-DST）.Results Down regulation of Notch1 expression by Notch1siRNA interference or Notch1 inhibitor increased the PAC sensitivity in MDA-MB-231 cells （P ＜0.05）.Also,the expression of Notch1 in PAC sensitive patients was much lower than that of PAC non-sensitive patients （P ＜0.01）.Conclusion Notch1 expression has an effect on PAC sensitivity in breast cancer patients,and the inhibition of Notch1 increases paclitaxel sensitivity to human breast cancer.     

Effects of notch-1 down-regulation on malignant behaviors of breast cancer stem cells

This study examined the effect of Notch-1 signaling on malignant behaviors of breast cancer cells by regulating breast cancer stem cells （BCSCs）. BCSCs were enriched by using serum-free me- dium and knocked out of Notch-1 by using a lentiviral vector. Real-time polymerase chain reaction （RT-PCR） and Western blotting were used to detect the Notch-1 expression levels in breast cancer cell lines and BCSCs, and fl0w cytometry to detect the proportion of BCSCs in BCSC spheres. The BCSC self-renewal, migration, invasion, and tumorigenicity were examined by the tumor microsphere-forming assay and transwell assay and after xenotransplantation. The results showed that the Notch-1 silencing reduced the number of BCSC spheres, the proportion of BCSCs, and the number of cells penetrating through the transwell membrane. It also decreased the size of tumors that were implanted in the nude mice. These results suggest that Notch-1 signaling is intimately linked to the behaviors of BCSCs. Blocking Notch-1 signaling can inhibit the malignant behaviors of BCSCs, which may provide a prom- ising therapeutical approach for breast cancer.     

Annexin A2 promotes choroidal neovascularization by increasing vascular endothelial growth factor expression in a rat model of argon laser coagulation-induced choroidal neovascularization

Background Choroidal neovascularization （CNV） is a common cause of visual loss in the elderly patients with age-related macular degeneration and represents the growth of subretinal new vessels in the macular region. This study aimed to investigate the relationship between annexin A2 （ANXA2） and vascular endothelial growth factor （VEGF） in CNV.Methods In a rat model of argon laser coagulation-induced CNV, the mRNA expressions of the annexins and VEGF protein expression in the retina were detected using fluorescent real-time polymerase chain reaction （PCR） and immunohistochemistry, respectively. The interactions between ANXA2 and VEGF in both a retinal pigment epithelial cell line RPE-J and the rat model of CNV were examined by means of RNA interference, real-time PCR, Western blotting, enzyme-linked immunosorbent assay （ELISA） and histopathological examinations. Results Fundus fluorescein angiography （FFA） showed that argon laser coagulation of the retina induced stable CNV models in the rats. Two to three weeks after the coagulation, ANXA2. and VEGF expressions in the coagulated area in the retina and choroid increased to the peak level, while the other annexin members （ANXA4, ANXA5, ANXA7 and ANXA11） showed no obvious changes. In RPE-J cells and the CNV model, RNA interference of ANXA2 gene significantly lowered the VEGF protein and mRNA expressions, and application of an adenoviral vector containing ANXA2 gene markedly increased VEGF expressions in the rat model of CNV, but produced no significant effects on the expressions of the kinase insert domain-containing receptor （KDR） or the fms-like tyrosine kinase （Fit-l）. The expression of KDR inhibited the increment in ANXA2 expression, but VEGF and Rt-1 did not directly affect ANXA2 expression. Conclusion Besides the role as a plasminogen and the receptor of tissue plasminogen activator, ANXA2, which is under regulation of KDR via a negative feedback mechanism, also participates in neovascularization by regulating VEGF expression through a positive feedback mechanism.     

Clinicopathological classification and individualized treatment of breast cancer

Background The clinicopathological classification was proposed in the St. Gallen Consensus Report 2011. We conducted a retrospective analysis of breast cancer subtypes, tumor-nodal-metastatic （TNM） staging, and histopathological grade to investigate the value of these parameters in the treatment strategies of invasive breast cancer.     

Effect of neoadjuvant chemotherapy on expressions of estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2, and Ki-67 in breast cancer

Background This study was designed in an attempt to determine the influence of neoadjuvant chemotherapy on estrogen receptor （ER）, progesterone receptor （PR）, human epidermal growth factor receptor 2 （Her-2）, and Ki-67 expressions in patients with breast cancer. Methods Pre- and post-neoadjuvant chemotherapy, paired-tumor specimens from 103 patients with breast cancer administrated with anthracycline or anthracycline combined taxane regimen were collected. Immunohistochemical staining for ER, PR, Her-2, and Ki-67 was performed by the DAKO EnVision method. Results Among the 103 cases, five patients （4.9%） had a complete response （CR）, 82 （79.6%） partial response （PR）, 15 （14.6%） stable disease （SD）, and one （0.9%） progressive disease （PD）, yielding an overall response rate （CR ＋ PR） of 84.5%. Nine patients achieved pathological CR. There was a significant decrease in the average index of Ki-67 post- neoadjuvant chemotherapy, compared with that before chemotherapy （24.1% vs. 39.7%, P 〈0.001）. After neoadjuvant chemotherapy, the changes of Ki-67 in different subtypes of breast cancer were different （P 〈0.001）, and these changes correlated with response to neoadjuvant chemotherapy （P 〈0.001）. No significant changes in immunohistochemical expression were observed for ER, PR and Her-2. Conclusions Neoadjuvant chemotherapy apparently reduced Ki-67 index in primary breast carcinomas, but profiles for ER, PR and Her-2 were not significantly different before and after neoadjuvant chemotherapy. The change of Ki- 67 correlated with molecular subtypes and response to neoadjuvant chemotherapy, suggesting that Ki-67 index was a surrogate marker to predict the treatment response of neoadjuvant chemotherapy.     

Establishment of novel rat models for premalignant breast disease

Background Breast cancer has become one of the most common malignant tumors among females over the past several years.Breast carcinogenesis is a continuous process,which is featured by the normal epithelium progressing to premalignant lesions and then to invasive breast cancer （IBC）.Targeting premalignant lesions is an effective strategy to prevent breast cancer.The establishment of animal models is critical to study the mechanisms of breast carcinogenesis,which will facilitate research on breast cancer prevention and drug behaviors.In this study,we established a feasible chemically-induced rat model of premalignant breast cancer.Methods Following the administration of the drugs （carcinogen,estrogen,and progestogen） to Sprague-Dawley （SD） rats,tumors or suspicious tumors were identified by palpation or ultrasound imaging,and were surgically excised for pathological evaluation.A series of four consecutive steps were carried out in order to determine the carcinogen：7,12-dimethylbenzaanthracene （DMBA） or 1-methyl-1-nitrosourea,the route of carcinogen administration,the administration period of estrogen and progestogen,and the DMBA dosage.Results Stable premalignant lesions can be induced in SD rats on administration of DMBA （15 mg/kg,administered three times） followed by administration of female hormones 5-day cycle.Results were confirmed by ultrasound and palpation.Conclusion Under the premise of drug dose and cycle,DMBA combined with estrogen and progestogen can be used as a SD rat model for breast premalignant lesions.     

Idiopathic pulmonary fibrosis will increase the risk of lung cancer

Objective To review the studies investigating the increased risk of lung cancer in patients with idiopathic pulmonary fibrosis （IPF）.Data sources Data cited in this review were obtained mainly from PubMed and Medline from 1999 to 2013 and highly regarded older publications were also included.Study selection We identified,retrieved and reviewed the information on the frequency,risk factors,anatomical features,histological types,clinical manifestations,computed tomography findings and underlying mechanisms of lung cancer in IPF patients.Results The prevalence rates of lung cancer in patients with IPF （4.8％ to 48％） are much higher than patients without IPF （2.0％ to 6.4％）.The risk factors for lung cancer in IPF include smoking,male gender,and age.Lung cancers often occur in the peripheral lung zones where fibrotic changes are predominant.Adenocarcinoma and squamous cell carcinoma are the most common types of lung cancer in patients with IPF.Radiologic features of these patients include peripherally located,ill-defined mass mimicking air-space disease.The underlying mechanisms of the development of lung cancer in patients with IPF have not been fully understood,but may include the inflammatory response,epithelial injury and/or abnormalities,aberrant fibroblast proliferation,epigenetic and genetic changes,reduced cell-to-cell communication,and activation of specific signaling pathways.Conclusions These findings suggest that IPF is associated with increased lung cancer risk.It is necessary to raise the awareness of lung cancer risk in IPF patients among physicians and patients.     

Tumorigenesis and spontaneous metastasis by luciferase-labeled human xenograft osteosarcoma cells in nude mice

Background  Osteosarcoma (OS) is the most common primary malignant tumor of bone. Mouse models of human OS can invariably provide greater insight into the complex mechanisms that underlie the development and pathogenesis of this aggressive tumor. Bioluminescence technology favored tracing cancer cells in vivo. In this study, an OS model was described and evaluated using human OS cell line, Saos2, labeled with luciferase (Saos2-luc).

Methods  Saos2 cells were infected by lentivirus loading a firefly luciferase gene. Luciferase expression of Saos2-luc cells was characterized both in vitro and in vivo. Specific biologic and oncologic features of Saos2-luc cells were analyzed. The OS was established as orthotopic xenografts in nude mice. Both orthotopic tumors and spontaneous lung metastasis were analyzed.

Results  Tumorigenesis and spontaneous lung metastasis in nude mice could be monitored in vivo through in vivo imaging system. The enhancement in proliferation, migration and invasion abilities and the attenuation in adhesion ability were observed in Saos2-luc cells compared with Saos2 cells. Furthermore, there were the up-regulation of Osteocalcin, CCR10, CXCR1 and ID1 and the down-regulation of ALP, collagen I, CCR1, CCR3, CXCR3, NID and N-cadherin in Saos2-luc cells compare to Saos2 cells. The rate of spontaneous lung metastasis in Saos2-luc cells was higher than that in Saos2 cells, although without significant difference.

Conclusions  Lentivirus transfection may cause alteration of gene expression profiles and further biological functions. This model can be used in the elucidation of molecular mechanisms of tumorigenesis and the screening of new therapeutic agents.

     

MicroRNAs are implicated in the initiation and progression of gastric cancer

Objective Gastric cancer is a genetically heterogeneous disease that progresses via different oncogenes.MicroRNA （miRNA） can regulate oncogene expression at the post-translational level.In this review,we summarize the most commonly altered miRNAs and their possible roles in cancer initiation and progression in gastric cancer.Data sources Most articles were identified by searching PubMed online resources using the key terms of microRNA and gastric cancer.Study selection Mainly original milestone articles and critical reviews written by major pioneer investigators in the field were selected,and the 69 most important articles were cited finally.Results A set of miRNAs are consistently deregulated in gastric cancer,although there is no clear miRNA expression profiles,such as miR-21 and miR-17 （～92 clusters）.These deregulated miRNAs play important roles in promoting cell proliferation,tumor metastasis,and chemotherapeutic resistance in gastric cancer by targeting different oncogenes.Clinical relevance of these deregulated miRNAs is proved to be associated with TNM stages,metastasis,and prognosis of gastric cancer patients.In addition,circulating miRNAs are promising noninvasive biomarkers for gastric cancer.Conclusions miRNAs have produced a novel paradigm in research in gastric cancer.These small molecules play macroroles in gastric cancer initiation and progression.These results will help us improve management of gastric cancer in future.     

Antitumor effects of mutant endostatin are enhanced by Bcl-2 antisense oligonucleotides in UM-UC-3 bladder cancer cell line

Background Endostatin is a potent inhibitor of tumor angiogenesis. In the preliminary studies, we developed a mutant endostatin containing Arg-Gly-Asp-Arg-Gly-Asp (RGDRGD) sequences. In this study, we compared the antitumor effects of mutant endostatin and Bcl-2 antisense oligonucleotides both in combination and individually.

Methods The artificially synthesized Bcl-2 ASODN (antisense oligonucleotides) included a translation-initiation site and was transfected into the bladder cancer cells by Lipofectamine. Cell growth was investigated by the tumor cell growth chart, MTT assay, caspase-3 activity detection assay, AO/EB fluorescein stain, and the annexin V-FITC apoptosis detection assay. In the in vivo study, UM-UC-3 bladder cancer cells were subcutaneously implanted into nude mice and the growth of tumor was examined. The ultrastructure of the tumor tissues in the treated and control groups were observed.

Results The cell growth chart showed that the cell population of the treated combination group decreased by 52.04% compared to the control group. The inhibition rate of the treated combination group was (79.66±6.79)%, whereas those of the individual ASODN and ES groups were (53.39±3.22)% and (50.22±5.46)% respectively. In the caspase-3 activity detection using AO/EB fluorescein stain and annexin V-FITC apoptosis detection assay, the co-inhibitory effect was higher than the individual inhibitory effects (P<0.05). There were significant differences in the inhibition of the solid tumor growth in the in vivo study.

Conclusions Our findings indicated that Bcl-2 antisense oligonucleotides enhance the antitumor effects of mutant endostatin both in vitro and in vivo. We noted the synergistic effects of Bcl-2 antisense oligonucleotides combined with mutant endostatin.

     

果蝇EZH2与乳腺癌分子分型及临床病理特征的关系

摘要：目的  探讨果蝇Zeste基因增强子人类同源物2（EZH2）与乳腺癌分子分型及临床病理参数的关系。方法  根据新型乳腺癌分子分型标准,将乳腺癌进行分子分型;免疫组织化学方法检测乳腺癌组织石蜡切片中EZH2的表达;采用χ2检验分析EZH2表达与乳腺癌新型分子分型,以及临床病理参数之间的关系;采用Kaplan- Meier法分析EZH2表达与乳腺癌患者无病生存率（DFS）的关系。结果  13例管腔上皮A型乳腺癌患者中,3例为EZH2高表达（23.08%）;10例管腔上皮B型乳腺癌患者中,5例为EZH2高表达（50.00%）;7例人表皮生长因子受体2过表达型乳腺癌患者中,3例为EZH2高表达（42.86%）;12例三阴性型乳腺癌患者中,10例为EZH2高表达（83.33%）。乳腺癌各分子亚型间EZH2表达的比较,差异有统计学意义（P <0.05）。EZH2高表达与EZH2低表达患者DFS比较,差异有统计学意义（P <0.05）。结论  EZH2可能是预测乳腺癌预后的指标之一。