NRP-1过表达对乳腺癌细胞MDA-MB-231、SK-BR-3生物学行为的影响

目的 通过上调乳腺癌细胞MDA-MB-231、SK-BR-3中NRP-1的表达,观察NRP-1对细胞增殖、凋亡、迁移及侵袭能力的影响。方法 构建pcDNA3.1-NRP-1表达载体,脂质体介导NRP-1 表达质粒转染MDA-MB-231、SK-BR-3细胞,用G418筛选出稳定转染的乳腺癌细胞株。利用RTqPCR、Western blot法分别检测NRP-1基因mRNA及其蛋白表达;CCK-8法、AnnexinⅤ-APC/7-AAD法、Transwell小室分别检测转染细胞增殖率、凋亡率及侵袭、迁移能力。结果 成功构建pcDNA3.1- NRP-1表达载体,转染MDA-MB-231、SK-BR-3细胞并筛选稳定表达系。与对照组相比,过表达组细胞的NRP-1 mRNA及蛋白表达水平明显升高（均P<0.05）;NRP-1过表达组的细胞较对照组增殖率增加、凋亡率降低、侵袭及迁移能力增强（均P<0.05)。结论 NRP-1在乳腺癌发展、浸润、转移中起着一定的作用,它可促进乳腺癌细胞增殖、迁移和侵袭,抑制其凋亡。     

CD44V6基因蛋白在乳腺癌中的表达及其意义

目的 探讨ＣＤ４４Ｖ６在乳腺癌中的表达及其意义。方法 应用免疫组织化学Ｓ－Ｐ法检测５４例乳腺癌组织中ＣＤ４４Ｖ６基因蛋白表达变化。结果 乳腺癌ＣＤ４４Ｖ６阳性率为５９．３％（３２／５４）,良性肿瘤的阳性率为２７．３％（３／１１）,Ｐ〈０．０５。有淋巴结转移者ＣＤ４４Ｖ６高表达者的阳性率为６９．２％（１８／２６）。在无淋巴结转移者中,浸润性癌高表达生率为５４．５％（１２／２２）,低表达阳性率为１３．     

Topical Application of <i>Houttuynia cordata Thunb</i> Ethanol Extracts Increases Tumor Infiltrating CD⁸⁺ /Treg Cells Ratio and Inhibits Cutaneous Squamous Cell Carcinoma <i>in vivo</i>

Houttuynia cordata Thunb (HCT) is a medicinal and edible herb that has beneficial effects on various diseases dueto its diuretic, anti-inflammatory, anti-oxidative, anti-microbial, anti-viral, anti-cancer and anti-diabetic properties. Most reports of its anti-cancer activity were conducted in vitro, and its effects on cutaneous squamous cellcarcinoma (SCC) have not been investigated yet. Using DMBA/TPA induced SCC mice model, we found thattopical treatment by HCT, as well as its bioactive ingredient monomer, efficiently inhibited tumor growth.Mechanistically, tumor infiltrating CD⁴⁺, Foxp³⁺ T regulatory cells (Tregs) were significantly reduced andCD⁸⁺/Treg cells ratio was largely increased in tumors after HCT treatment. In addition, several chemokineswhich function to recruit immune cells were largely reduced in SCC cancer cells treated by HCT in vitro. Ourresults demonstrate the therapeutic effects of HCT on cutaneous SCC and indicate it might inhibit cancer throughregulating tumor infiltrating lymphocytes and the tumor immune microenvironments.     

CD44+/CD24-/low在乳腺癌中的表达及其与含蒽环类药物化疗方案敏感度的关系

目的研究乳腺癌中CD44+/CD24-/low的表达及其与含蒽环类药物化疗方案敏感度的关系。方法 91例乳腺癌接受含蒽环类药物的术前新辅助化疗,2～4个疗程后进行效果评价;采用双染免疫组织化学方法检测化疗前后CD44+/CD24-/low的表达,t检验分析化疗前后CD44+/CD24-/low细胞比例的变化,χ2检验分析乳腺癌CD44+/CD24-/low表型与乳腺癌临床病理参数及化疗疗效的关系。结果乳腺癌中CD44+/CD24-/low阳性表达率为39.6%(36/91),在接受含蒽环类药物的新辅助化疗后乳腺癌中CD44+/CD24-/low细胞比例较化疗前明显增加(P=0.028)。CD44+/CD24-/low阳性组ER阳性率明显低于CD44+/CD24-/low阴性组(25.0%vs.47.3%,P=0.033)。三阴性乳腺癌中CD44+/CD24-/low阳性率明显高于非三阴性乳腺癌(61.9%vs.32.9%,P=0.017)。CD44+/CD24-/low表型与年龄、肿瘤大小、临床分期、病理类型、组织学分级等乳腺癌临床病理参数无明显关系(P>0.05)。CD44+/CD24-/low阳性组的总有效率高于CD44+/CD24-/low阴性组,但两组之间差异无统计学意义(75%vs.69.1%,P=0.542);CD44+/CD24-/low阳性组的病理完全缓解率明显高于CD44+/CD24-/low阴性组(38.9%vs.18.2%,P=0.028)。结论 CD44+/CD24-/low细胞仅存在于部分乳腺癌,CD44+/CD24-/low表型与ER(﹣)、三阴性乳腺癌相关,CD44+/CD24-/low表型与乳腺癌对含蒽环类药物化疗方案的敏感度相关,CD44+/CD24-/low表型可能成为乳腺癌临床化疗疗效的预测指标之一。     

Double Heterozygosity in the <i>BRCA1/2</i> Genes in a Turkish Patient with Bilateral Breast Cancer: A Case Report

BRCA1 and BRCA2 tumor suppressor genes are responsible for a quarter of hereditary breast cancers. Double heterozygous (DH) pathogenic variant carrier status in these genes is an extremely rare condition, especially in non-Askenazi individuals. We report a woman patient with bilateral breast cancer that carries DH disease-causing variants in BRCA1/2 genes. The 45-year-old patient who was followed up with the diagnosis of metachronous bilateral breast cancer was diagnosed with cancer at the age of 39 and 43, respectively. BRCA1/2 genes of the patient were evaluated using Next-Generation Sequencing. In the patient, the c.2800C>T (p.Gln934Ter) pathogenic variant in BRCA1 and the c.9648+1G>C likely pathogenic variant in BRCA2 were detected as DH. Segregation analysis in family members revealed that her two healthy siblings available for testing were heterozygous for either BRCA1 or BRCA2 variants, but her mother, who had a past diagnosis of ovarian cancer, was heterozygous for both BRCA1 and BRCA2 variants. Germline double heterozygosity in inherited cancer is a rare condition, and as far as we know it is reported for the first time from patient population in Turkey. Large-scale patient series are needed to determine the impact of double heterozygosity on diseases course, such as prognosis and treatment responses.     

CD44v6、p53在乳腺癌中的表达与意义

目的　探讨CD44v6及p5 3在乳腺癌组织中的表达及临床意义 ;分析CD44v6与p5 3的表达有无相关关系。方法　应用免疫组织化学PictureTM通用型二步法检测 5 5例乳腺癌组织、15例癌旁正常乳腺组织中CD44v6和p5 3的表达情况。结果　CD44v6、p5 3的阳性表达率在乳腺癌组织中均明显高于正常乳腺组织 ,有淋巴结转移组明显高于无淋巴结转移组。结论　CD44v6和p5 3是两个独立因子 ,在乳腺癌的发生、发展、转移过程中发挥调控作用 ,可作为判断预后的重要生物学指标。     

CD44v6、E-cadhesion表达与乳腺浸润性癌预后的关系

目的检测CD44v6、E-cad在乳腺浸润性癌中的表达情况,探讨它们与乳腺浸润性癌浸润和转移的关系。方法采用免疫组化SP法检测103例乳腺浸润性癌中CD44v6、E-cad的表达,结合病理指标和临床指标分析其与预后的关系。结果CD44v6蛋白表达在细胞浆和(或)细胞膜上,阳性表达率为76.7%(79/103)。E-cad蛋白表达主要见于细胞膜,阳性表达率为35.0%(36/103)。CD44v6、E-cad蛋白表达与乳腺浸润性癌组织学类型无关(P>0.05),与组织学分级及组织浸润程度有关(P<0.05)。在56例有随访记录的患者中,CD44v6、E-cad蛋白表达与患者年龄无关(P>0.05),与腋窝淋巴结转移、局部复发、远处转移均有关(P<0.05)。两者在同一癌组织中的表达呈负相关(P<0.01)。结论CD44v6、E-cad蛋白表达与腋窝淋巴结转移、局部复发、远处转移有关(P<0.05),因此CD44v6、E-cad蛋白表达可作为判断乳腺浸润性癌的浸润程度、预测预后的参考指标之一。     

Clinical Characteristics and Molecular Patterns of <i>RET</i>-Rearranged Lung Cancer in Chinese Patients

RET rearrangement has been proven as an oncogenic driver in patients with lung cancer. However, the prevalence, clinical characteristics, molecular features, and therapeutic options in RET-rearranged patients remain unclear, especially in Chinese lung cancer patients. We retrospectively collected 6,125 Chinese lung cancer patients who have been profiled using next-generation sequencing (NGS). The clinical demographics and molecular features of RET rearrangement-positive patients were analyzed. RET rearrangements were identified in 84 patients with a proportion of 1.4% in our cohort. The median age at diagnosis was 58 years, and it mainly occurred in females with adenocarcinoma histology. KIF5B-RET was the most frequent fusion type and accounted for 53.8% (57/106) of all RET fusions identified, with K15-R12 as the most frequent variant (71.9%). Among 47 RET+ patients profiled with larger panels, 72.3% (34/47) harbored concurrent alterations. TP53 ranked as the most common concurrent alteration, and concomitant EGFR oncogenic alterations were identified in seven patients. Moreover, an adenocarcinoma patient harboring concurrent RET fusion and EGFR L858R responded to combinatorial treatment of cabozantinib and osimertinib, with a progression-free survival of 5 months. Our study improved knowledge of clinical characteristics and molecular features of RET-rearranged lung cancers in China. It might be helpful to guide clinicians for more effective personalized diagnostic and therapeutic approaches.     

Prognostic Value of CD44 Variant exon 6 Expression in Non-Small Cell Lung Cancer: a Meta-analysis

Background: CD44v6 (CD44 variant exon 6) is the chief CD44 variant isoform regulating tumor invasion, progression, and metastasis. The prognostic value of CD44v6 expression in non small cell lung cancer (NSCLC) has been evaluated in many studies, but the results have remained controversial. Thus, we performed a metaanalysis of currently available studies to investigate the prognostic value of CD44v6 expression in NSCLC patients and the relationship between the expression of CD44v6 and clinicopathological features. Materials and Methods: Two independent reviewers searched the relevant literature in Pubmed, Medline and Embase from 1946 to January 2014. Overall survival (OS) and various clinicopathological features were collected from included studies. This meta-analysis was accomplished using STATA 12.0 and Revman 5.2 software. Pooledhazard ratios (HRs) with 95% confidence intervals (95%CIs) were calculated to estimate the effects. Results: A total of 921 NSCLC patients from ten studies met the inclusion criteria. The results showed that CD44v6 high expression was a prognostic factor for poor survival (HR=1.91, 95%CI=1.12-3.26, p<0.05). With respectto clinicopathological features, CD44v6 high expression was related to histopathologic type (squamous cell carcinoma versus adenocarcinoma: OR=2.72, 95%CI=1.38-5.38, p=0.004), and lymph node metastasis (OR=3.02,95%CI=1.93-4.72, p<0.00001). Conclusions: Our results suggested CD44v6 high expression as a poor prognostic factor for NSCLC, and CD44v6 expression is associated with lymph node metastasis and histopathologic type. Therefore, CD44v6 expression can be used as a novel prognostic marker in NSCLC cases.     

Correlation Between Expression of Cell Adhesion Molecules CD44 v6 and E-cadherin and Lymphatic Metastasis in Nonsmall Cell Lung Cancer

Objective: To explore the relationship between expressions of cell adhesion molecules CD44 v6 and E-cadherin(E-cad) and lymphatic metastasis in non-small cell lung cancer (NSCLC). Materials and Methods: Eightyseventissue samples obtained from patients with primary NSCLC were collected in our hospital from Dec.,2007 to Dec., 2012, and the expressions of CD44 v6 and E-cad gene proteins in these samples were detected byimmunohistochemical method. Results: In the tissue without lymphatic metastasis, the positive expression rateof CD44 v6 was significantly lower, whereas the normal expression rate of E-cad was notably higher than thatwith lymphatic metastasis (55.6% vs. 78.4%, 47.2% vs. 21.6%), and both differences had statistical significance(P<0.05). Besides, CD44 v6 and E-cad expressions had a significant correlation in the NSCLC tissue with lymphaticmetastasis (P<0.05). Conclusions: The positive expression of CD44 v6 and abnormal expression of E-cad mayplay a very important role in promoting lymphatic metastasis of NSCLC, with synergistic effect. Hence, detectionof CD44 v6 and E-cad expressions is conductive to judging the lymphatic metastasis in NSCLC.     

RUNX3基因在乳腺癌组织和乳腺癌细胞系中的表达及意义

[目的]探讨乳腺癌组织和细胞系中RUNX3基因mRNA与蛋白表达及在乳腺癌发生中的作用。[方法]运用RT-PCR、WesternBlot方法研究5种乳腺癌细胞系、1种正常乳腺细胞系和30对新鲜乳腺癌及癌旁对照组织中RUNX3mRNA和蛋白表达。[结果]5种乳腺癌细胞系中有3种(T47D、MCF7和SKBR3)RUNX3mRNA和蛋白表达阴性。30例新鲜的乳腺癌组织中RUNX3mRNA和蛋白表达明显低于癌旁正常对照组织,差异具有统计学意义。RUNX3蛋白表达与乳腺癌临床分期、淋巴结转移相关,而与患者的年龄、病理分级无关。[结论]RUNX3基因可能作为一个抑癌基因参与乳腺癌的发生。     

The TP53 intron 6 G13964C Polymorphism and Risk of Thyroid and Breast Cancer Development in the Iranian Azeri Population

Background: TP53 mutations are the most common genetic alterations in human cancers. There are alsoseveral polymorphisms in both exons and introns of TP53 that may influence its anti-tumor functions and increasethe risk of cancer development. Associations of the TP53 intron 6 G13964C polymorphism with increased risk ofdevelopment of several cancers have been investigated in numerous studies, but the results were controversialand conflicting. In this study, we aimed to investigate the probable association of this polymorphism with riskof both thyroid and breast cancers among the Iranian-Azeri population. Materials and Methods: We performedtwo separate case control studies on associations of the intron 6 polymorphism with two different kinds ofcancer. In one case-control study, a total of 75 patients with thyroid carcinoma and 180 controls were analyzedand the other study included 170 patients with breast cancer and 135 healthy women. The intron 6 genotypewas determined by RFLP-PCR and the SPSS 16 program was applied for data analysis. Results: For thyroidcancer, the frequencies of GG genotype were 96.0% in patients and 93.3% in controls. The GC genotype had afrequency of 4.0 % in patients and 6.7% in controls. In the study on breast cancer, the frequency of GG and GCgenotypes in patients were 95.3% and 4.7%, respectively. In breast related control group, the frequency of GGgenotype was 93.3 % and the frequency of GC genotype was 6.7%. None of the cases and controls had the CCgenotype. Conclusions: There was no significant association between the TP53 intron 6 G13964C polymorphismand risk of development of both thyroid and breast cancer in Iranian-Azeri patients.     

3-甲基腺苷对芹菜素诱导乳腺癌T47D细胞系自噬和凋亡的影响

研究自噬抑制剂3-甲基腺苷（3-methyladenine,3-MA）对芹菜素诱导乳腺癌T47D细胞系自噬和凋亡的影响。方法：常规培养人乳腺癌T47D细胞,分为对照组、3-MA组、芹菜素组、3-MA+芹菜素组。MTT法检测各组的细胞增殖抑制率;GFP-LC3质粒转染观察各组细胞自噬情况;Hochest/Mito-Red/YO-PRO-1染色法观察各组细胞凋亡形态;AnnexinV/PI双染法流式细胞仪检测各组细胞凋亡率;Western blot法检测LC3及PARP的变化。结果：MTT示3-MA+芹菜素组的增殖抑制率明显高于其他各组（P<0.05）;GFP-LC3质粒转染结果显示：对照组,3-MA组及3-MA+芹菜素组自噬不明显,而芹菜素组与其他各组相比自噬明显增多（P<0.05）;3-MA+芹菜素组的凋亡细胞增多,各组凋亡率分别为（12.73±0.05）%,（18.46±0.03）%,（23.27±0.07）%,（34.14±0.05）%,与对照组相比有统计学意义（P<0.05）;Western blot结果显示,芹菜素组LC3-Ⅱ增高,而3-MA组及3-MA+芹菜素组的LC3-Ⅱ显著减少,3-MA+芹菜素组PARP的剪切带相比其他各组明显增加。结论：自噬抑制剂3-MA抑制细胞自噬后,能够明显增强芹菜素对乳腺癌T47D细胞系的凋亡诱导效应。     

Computational high-throughput screening and <i>in vitro</i> approaches identify CB-006-3; A novel PI3K-BRAF^V600E dual targeted inhibitor against melanoma

Malignant melanoma is characterized by both genetic and molecular alterations that activate phosphoinositide 3-kinase (PI3K), and RAS/BRAF pathways. In this work, through diversity-based high-throughput virtual screening we identified a lead molecule that selectively targets PI3K and BRAF^V600E kinases. Computational screening, Molecular dynamics simulation and MMPBSA calculations were performed. PI3K and BRAF^V600E kinase inhibition was done. A375 and G-361 cells were used for in vitro cellular analysis to determine antiproliferative effects, annexin V binding, nuclear fragmentation and cell cycle analysis. Computational screening of small molecules indicates compound CB-006-3 selectively targets PI3KCG (gamma subunit), PI3KCD (delta subunit) and BRAF^V600E. Molecular dynamics simulation and MMPBSA bases binding free energy calculations predict a stable binding of CB-006-3 to the active sites of PI3K and BRAF^V600E. The compound effectively inhibited PI3KCG, PI3KCD and BRAF^V600E kinases with respective IC₅₀ values of 75.80, 160.10 and 70.84 nM. CB-006-3 controlled the proliferation of A375 and G-361 cells with GI₅₀ values of 223.3 and 143.6 nM, respectively. A dose dependent increase in apoptotic cell population and sub G₀/G₁ phase of cell cycle were also observed with the compound treatment in addition to observed nuclear fragmentation in these cells. Furthermore, CB-006-3 inhibited BRAF^V600E, PI3KCD and PI3KCG in both melanoma cells. Collectively, based on the computational modeling and in vitro validations, we propose CB-006-3 as a lead candidate for selectively targeting PI3K and mutant BRAF^V600E to inhibit melanoma cell proliferation. Further experimental validations, including pharmacokinetic evaluations in mouse models will identify the druggability of the proposed lead candidate for further development as a therapeutic agent for treating melanoma.     

Prognostic Significance of Circulating Tumor Cells and SerumCA15-3 Levels in Metastatic Breast Cancer,Single CenterExperience,Preliminary Results

Background: Breast cancer is the second leading cancer causing death in women. Circulating tumor cellsare among the prognostic factors while tumor markers are of diagnostic value and can be used for follow-up.The aim of this study was to investigate the correlation between the prognostic significance of the serum CA15-3levels, number of circulating tumor cells and histopathological tumor factors. Materials and Methods: Thirtypatients recently diagnosed with breast cancer were included in the study. Number of circulating tumor cells andserum CA15-3 level were assessed when metastasis was detected and diagnostic value was assessed. Presence ofassociations with estrogen and progesterone receptors, c-erbB2, Ki-67 proliferation index and histological gradewere also evaluated. Results: Median overall survival of the patients with serum CA15-3 levels of >108 ng/dlwas 19 months whereas for those with a low serum level it was 62 months. Median overall survival for CTC≥5vs CTCConclusions: Prognostic significance of the CTC count and CA15-3 levels in metastaticbreast cancer patients was demonstrated.     

Analysis of Cytosol CA15-3, Carcinoembryonic Antigen, Estrogen and Progesterone Receptors in Breast Cancer Tissues

Cytosol levels of carcinoembryonic antigen (CEA), CA15-3, estrogen receptor (ER) and progesterone receptor (PgR) of 194- primary breast cancer tissues were measured. ER and PgR determined by enzyme immunoassay methods correlated inversely with Page's grades of histological atypia and mitotic rate. Cytosol CA15-3 correlated inversely with the grades of tubular and nuclear atypia and positively with the mitotic rate. CA15-3 correlated positively with ER and PgR. Cytosol CEA showed no correlation with the pathological grade or hormone receptor status. These results indicate that hormone receptor-positive breast cancers tend to have more differentiated morphology and slower growth rate than those without those receptors and that the cytosol CA15-3 level may reflect some of the intrinsic malignant potency, particularly the growth rate, of breast cancer. Cytosol CA15-3 as well as the hormone receptor status may have prognostic value for patients with breast cancer.     

奥沙利铂对二甲肼诱导的结肠癌大鼠CD44V6、VEGF、survivin、Caspase-3和Caspase-7的影响

目的 研究奥沙利铂对结肠癌大鼠治疗的作用机制。方法 将42只SD大鼠均颈背部皮下注射二甲肼,每周注射 1次,连续注射5周。随机抽取2只大鼠切取直肠进行HE染色,病理学观察,确定结肠癌大鼠模型建立。将40只结肠癌大鼠随机分为模型组、奥沙利铂高、中、低剂量组,每组10只。另设正常组SD大鼠10只。奥沙利铂高剂量组（27.2 mg/kg）、中剂量组（13.6 mg/kg）、低剂量组（6.8 mg/kg）,给予1 ml 5%的葡萄糖注射液溶解,尾静脉注射给药,每3周给药一次,连续给药12周。正常组与模型组给予1 ml 5%的葡萄糖注射液代替。末次给药48 h后,通过颈脱位法处死大鼠。显微镜观察结肠组织中异变腺窝病灶(ACF)的个数。通过酶联免疫吸附（ELISA）法检测结肠癌大鼠血清中CD44V6和VEGF的含量变化。免疫蛋白印记（Western blot）法检测结肠癌组织中survivin、Caspase-3和Caspase-7的蛋白水平。结果 与模型组比较,奥沙利铂给药组结肠组织中ACF的数量明显减少。与正常对照组比较,模型组血清中CD44V6和VEGF的含量显著增加（P＜0.05）。与模型组相比,奥沙利铂高剂量组、中剂量组血清中CD44V6和VEGF的含量下降。其中奥沙利铂高剂量的效果最好,差异有统计学意义（P＜0.05）。与正常组相比,模型组、奥沙利铂给药组结肠癌组织中的survivin、Caspase-3和Caspase-7的蛋白水平均上升。与模型组相比,奥沙利铂给药组结肠癌组织中的survivin的蛋白水平降低;Caspase-3和Caspase-7的蛋白水平升高,差异有统计学意义（P＜0.05）。结论 奥沙利铂通过调控CD44V6、VEGF、survivin、Caspase-3和Caspase-7的水平,减少肿瘤血管的生成与转移、调节结肠癌组织中的细胞凋亡,从而达到治疗的目的。     

血清TPS和CA15-3水平变化与乳腺癌的关系及其临床意义

目的：探讨血清组织多肽特异性抗原（TPS）和癌抗原15－3（CA15－3）水平检测对乳腺癌的临床应用价值。方法：采用酶联免疫吸附法，分别在治疗前后测定173例乳腺癌和69例乳腺良性疾病患者血清TPS和CA15－3水平。结果：乳腺癌组患者TSP和CA15－3水平及检测阳性率均明显高于乳腺良性疾病和正常对照组（P＜0．01），CA15－3水平随临床分期的进展明显升高，而TPS即使在临床I期也有高水平的表达，乳腺癌患者经有效治疗后其血清TPS和CA15－3水平均明显降低，尤其是单纯根治术或加用放疗的患者降低幅度更为明显，乳腺癌复发患者血清TPS和CA15－3均明显升高，尤其是TPS升高幅度较为显著，结论：TPS和CA15－3联合检测可明显提高乳腺癌的阳性检出率，对乳腺癌早期诊断，病情判断，疗效评估和复发监测等均有重要价值，而动态监测其水平变化的临床价值更为可靠。