PGRMC1参与调控乳腺癌细胞增殖及化疗敏感度的实验

目的探讨孕激素膜受体1(progesterone receptor membrane component-1,PGRMC1)是否参与乳腺癌细胞增殖及化疗药物敏感度的调控,为乳腺癌的化疗提供参考依据。方法设计合成以PGRMC1为靶标的siRNA1、2,用lipofectin2000转染人乳腺癌高转移细胞株MDA-MB-231(ER-,PR-)和低转移细胞株MCF-7(ER+,PR+),QRT-PCR检测转染siRNAs后PGRMC1基因mRNA水平变化,Western blot法检测蛋白表达变化,以siRNA-GFP为阳性对照,未处理组为空白对照。CCK8法检测转染前后DTX敏感度。应用1/2的IC50DTX作用转染组与对照组,流式细胞仪检测各组细胞周期变化,Annexin V/PI阳性细胞百分比,对比各组细胞凋亡率、死亡率,双氢二氯荧光黄染色阳性率判定各组细胞内ROS水平。JC-1染色后,免疫荧光显微镜下观察各组细胞线粒体膜电位变化。结果以PGRMC1为靶标设计的siRNA1、2对该基因mRNA及蛋白表达显著抑制在70％以上,细胞增殖受抑,对化疗药物敏感度增高,与非转染组相比G2期细胞比例明显增加,凋亡细胞比率明显增加,细胞内ROS水平也随之升高,线粒体膜电位下调。 结论PGRMC1参与了乳腺癌细胞增殖及化疗敏感度的调控。     

PGRMC1在宫颈癌组织中的表达及对细胞增殖的影响

目的:检测孕激素受体膜成分1（PGRMC1）在宫颈鳞状细胞癌、腺癌、高级别上皮内病变（high-grade squamous intra-epithelial lesions,HSIL）以及对应正常宫颈组织中的表达情况,并分析其对宫颈癌细胞增殖的影响。方法:利用免疫组化法检测PGRMC1在151例宫颈鳞状细胞癌、18例宫颈腺癌、26例HSIL以及对应正常宫颈组织中的表达情况;利用CCK-8法分析PGRMC1对宫颈癌SiHa细胞增殖的影响。结果:PGRMC1在宫颈鳞状细胞癌中的表达高于癌旁正常鳞状上皮中的表达（P＜0.000 1）,在宫颈腺癌中的表达高于癌旁宫颈腺上皮中的表达（P=0.015 7）,在HSIL中的表达也高于瘤旁宫颈鳞状上皮中的表达（P=0.003 2）。PGRMC1的表达与宫颈鳞状细胞癌患者的年龄、脉管侵犯、肿瘤分化、肿瘤浸润深度、肿瘤直径、淋巴结转移以及FIGO分期均无关（P均>0.05）。CCK-8分析显示干扰PGRMC1可抑制宫颈癌SiHa细胞的增殖。结论:PGRMC1在宫颈鳞状细胞癌、宫颈腺癌以及HSIL中的表达均高于正常宫颈组织中的表达。干扰PGRMC1可抑制宫颈癌SiHa细胞的增殖。     

过度活化Notch1信号促进肾癌细胞增殖的机制研究

目的 研究Notch1信号过度活化对肾癌细胞增殖的影响及其可能的分子机制。方法 首先应用免疫组织化学法检测120例肾透明细胞癌患者的肿瘤组织中Notch1的表达水平,并结合患者的临床病理特征进行比较。然后在肾癌细胞系ACHN中开展体外的机制和功能研究。用Western blot检测Notch1过度活化对PI3K/Akt信号通路的调控。并进一步分析Notch1和PI3K/Akt信号通路对细胞增殖和细胞周期的影响。结果 120例肾透明细胞癌组织标本中,有64例高表达Notch1,占53.3%。Notch1在直径≥7cm的肿瘤中的表达强度要高于直径＜7cm的肿瘤(P = 0.005)。与局限期(TNM Ⅰ～Ⅱ)肾癌相比,进展期(TNM Ⅲ～Ⅳ期)肾癌有高表达Notch1的趋势(P = 0.062)。结论 肾癌细胞中过度活化的Notch1信号,通过上调PI3K/Akt信号通路的活性,加快了肿瘤细胞的增殖,进而促进肾癌的发生、发展。该发现提供了一种可能的肾透明细胞癌发生的新机制和潜在分子药靶。     

乳腺癌内分泌治疗中激素受体问题

雌激素受体(ｅｓｔｒｏｇｅｎｒｅｃｅｐｔｏｒ,ＥＲ)已被作为乳腺癌内分泌治疗和预后评估的一个重要指征。近年来的研究已取得显著进展,特别是有关实际应用中存在的一些问题,许多已有较明确的解释。现就一些有关新认识、新进展做一概要综述。一、雌激素、激素受体与乳腺癌1896年Ｂｅｎｔｓｏｎ发现乳腺细胞的增生及癌变与激素密切相关,并观察到切除卵巢可使进展期乳腺癌消退。1967年Ｊｅｎｓｅｎ发现人类乳腺癌中含有ＥＲ。这一发现把乳腺癌内分泌治疗推向了崭新的阶段,使内分泌治疗变得有的放矢,疗效明显提高。研究证明,有的肿瘤细胞恶变…     

Sharp1在乳腺癌中作用机制的研究进展

乳腺癌是女性最常见的恶性肿瘤之一,其发病率在全球范围内逐年上升,且死亡率位于女性恶性肿瘤的前列,因此乳腺癌的治疗备受关注［1］。分化型胚胎软骨发育基因（ Sharp1,the en-hancer of split and hairy related protein-1）,又名DEC2（differentia-ted embryo-chondrocyte expressed gene）,在肿瘤的发生和进展中均发挥重要作用。新近研究显示Sharp1在不同类型乳腺癌的组织和细胞中表达不同,提示其可能在不同类型的乳腺癌中发挥的作用及机制也各不相同。目前全球范围内关于Sharp1与乳腺癌的相关实验研究较少,且多集中在体外细胞和动物实验水平,有报道提示Sharp1的表达情况可能与乳腺癌的发生发展具有一定的相关性,但具体通过何种方式和分子机制尚待深入研究。因此,检测Sharp1在不同分子类型乳腺癌组织中的表达情况,阐明其分子作用机制,对今后乳腺癌的临床诊治和预后评估都具有重要意义,作为新靶点Sharp1也有望成为乳腺癌临床治疗的新的希望。     

SCD1抑制剂对乳腺癌细胞增殖凋亡的影响及其机制

目的 检测硬脂酰辅酶A去饱和酶1（stearoyl-CoA desaturase 1, SCD1）在乳腺癌组织中的表达,分析抑制SCD1的活性对乳腺癌MCF-7细胞增殖凋亡的影响及其机制。方法 采用免疫组织化学法检测乳腺癌及癌旁正常组织中SCD1蛋白的表达。应用MTS法测定SCD1抑制剂MF-438对MCF-7细胞增殖的抑制率。应用Hoechst33342染色荧光显微镜观察细胞形态并计算凋亡指数,PI染色流式细胞术检测细胞凋亡率。蛋白质印迹法检测凋亡相关蛋白Bcl-2和Bax的表达。结果 乳腺癌组织中SCD1阳性表达率显著高于正常乳腺组织（P<0.05）,并且三阴性乳腺癌中的SCD1表达水平低于其他亚型（P<0.05）。MF-438在0.1~100 μmol/L浓度范围内,对MCF-7细胞显示出显著的剂量依赖性的增殖抑制作用,并在低血清条件下更为敏感。MCF-7细胞在5 μmol/L MF-438作用48 h后,表现出典型的细胞凋亡特征性变化,细胞凋亡指数及细胞凋亡率显著高于对照组（P<0.05）;同时,MF-438下调MCF-7细胞抑凋亡蛋白Bcl-2的表达,并上调促凋亡蛋白Bax的表达。结论 抑制SCD1能抑制乳腺癌细胞增殖并诱导凋亡,对SCD1的深入研究将有望为乳腺癌的分子靶向治疗提供一个新的靶标。     

白藜芦醇抑制MCF-7乳腺癌细胞增殖的机制研究

研究白藜芦醇对MCF-7乳腺癌细胞抑制效应及其作用机制。方法：以人MCF-7乳腺癌细胞株为研究对象,利用MTT方法研究白藜芦醇抑制MCF-7乳腺癌细胞的生物学效应;观察在ERK1/2抑制剂PD98059预处理情况下,白藜芦醇抑制MCF-7乳腺癌细胞增殖效应的改变;利用免疫印迹方法观察白藜芦醇对MCF-7乳腺癌细胞中ERK1/2与AKT信号分子的蛋白表达。结果：白藜芦醇能够明显降低MCF-7乳腺癌细胞增殖能力,该作用呈一定的浓度依赖性关系。在ERK1/2抑制剂PD98059预处理情况下,白藜芦醇对MCF-7乳腺癌细胞增殖抑制效应能明显抑制,PD98059可明显减轻该效应。同时,白藜芦醇明显增加p-ERK1/2蛋白表达,降低p-AKT表达水平,但对ERK1/2与AKT蛋白表达无改变。结论：白藜芦醇能够有效抑制MCF-7乳腺癌细胞增殖,该效应与白藜芦醇对ERK1/2及AKT信号途径的调节有关。     

水飞蓟宾抑制乳腺癌细胞增殖和促进其凋亡的作用及机制

目的:探讨水飞蓟宾抑制乳腺癌细胞增殖和促进其凋亡的作用及机制。方法:MCF7细胞分为对照组和SIL组(处理浓度分别为10、20、40mg/L),各组细胞分别处理24、48、72h。MTT法检测不同浓度SIL对乳腺癌MCF7存活率的影响,TUNEL法（DNA断裂的原位末端标记法）检测各组细胞凋亡率,caspase-Glo⑧3/7定量试剂盒测定细胞caspase3/7的表达情况,Western blot法检测PUMA蛋白表达水平。结果:MTT结果显示SIL对MCF7细胞增殖有抑制作用（P＜0.01）,其中用40mg/L SIL处理MCF7细胞72h时抑制效果最明显,结果呈药物浓度时间依赖效应。TUNEL法显示（48h）SIL可以显著增加MCF7细胞的凋亡率（P＜0.01）。caspase3/7结果示SIL可以使caspase3/7活性显著升高（P＜0.01）。Western blot示SIL可以诱导PUMA蛋白的表达升高。结论:水飞蓟宾能抑制乳腺癌细胞增殖和促进其凋亡,其作用机制之一可能是诱导PUMA蛋白表达。     

Immunohistochemical evaluation of hormone receptor status for predicting response to endocrine therapy in metastatic breast cancer

BACKGROUND: The importance of establishing hormone receptor status of tumors for the treatment of women with hormone receptor-positive breast cancer has been emphasized, however, there is no general agreement as to how immunohistochemical assays should be evaluated. It is critical to evaluate hormone receptor status when considering response to endocrine therapy. METHODS: Estrogen receptor (ER) and progesterone receptor (PgR) expression was examined by immunohistochemistry using Allred's score for primary breast tumors from 75 metastatic breast cancer patients who received first-line treatment with endocrine therapy (56 patients received tamoxifen, 11 patients received aromatase inhibitors, and 8 patients received LH-RH agonist or other endocrine reagents) on relapse. Correlation between hormone receptor status and response to endocrine therapy as well as post-relapse survival was analyzed. RESULTS: The most significant correlation between positive ER expression and response to any endocrine therapy (p = 0.011) or tamoxifen only (p = 0.030) occurred when the cutoff score was set at 10%. When the evaluation was based on Allred's score (TS), a cutoff point of TS>or=4 showed a more significant association between positive ER expression and response to all kinds of endocrine therapy (p = 0.020) or tamoxifen only (p = 0.047). When evaluated at a cutoff point of 1% positive cells, there were fifteen patients with both ER- and PgR-negative tumors, and three patients (20.0%) responded to the therapy. Patients with 1% or more ER or PgR positive cells had better survival after relapse (p = 0.0005 and p = 0.0008, respectively). CONCLUSIONS: The proportion score alone might be enough to predict hormone responsiveness and post-relapse survival in metastatic breast cancer. The cutoff might be set low, for example 1%, especially for metastatic disease.     

Relation of serum levels of estrogen and dehydroepiandrosterone sulfate to hormone receptor status among postmenopausal women with breast cancer

Background: It is hypothesized that breast cancer may consist of heterogeneous diseases with different hormonal environments classified by hormone receptor status. Epidemiologic studies evaluating risk factors for breast cancer by hormone receptor status have supported the hypothesis. However, there are inconsistencies in the risk factor profiles by estrogen receptor (ER) and progesterone receptor (PR) across the studies. To clarify the heterogeneity of the disease, it is necessary to understand not only risk factor profiles but also the biologic characteristics such as the relationships among endogenous sex hormone levels and hormone receptors. Methods: We measured serum levels of estrone (E1), estradiol (E2), dehydroepiandrosterone sulfate (DHEAS), and sex hormone-binding globulin (SHBG) in 142 postmenopausal women aged 50 and over with primary breast cancer who had undergone surgical treatment, and investigated the heterogeneity in the relations of endogenous sex hormone levels to hormone receptor status, using the case-series study method. Subjects were categorized into 3 classes based on tertiles of each hormone level in receptor-negative subjects, and odds ratios (ORs) for receptor-positive status compared with receptor-negative status were computed, taking the lowest category as a reference category. Results: There were clear trends toward higher serum levels of E1, E2, and DHEAS in women with PR+ cancer. The case-series approach revealed that PR+ status might be strongly associated with serum sex hormone levels. In particular, the OR of PR+ was large for a high DHEAS level (OR for the highest category = 4.28). No significant association between serum hormone levels and ER status was observed. Conclusion: The association of serum sex hormone levels with hormone receptor status may differ by PR status, but not by ER status. This finding suggests that PR status may be related to the heterogeneity in hormonal environments associated with breast cancer risk.     

Intake of fruits, and vegetables in relation to breast cancer risk by hormone receptor status

Summary The inconsistent associations between fruit and vegetable intake and breast cancer risk may be due to heterogeneity of associations by estrogen (ER) and progesterone receptor (PR) status of the tumors. We evaluated this hypothesis in a large (2,386 cases and 2,503 controls) population-based case-control study in Poland, conducted between 2000 and 2003. We observed significant associations between reduced overall risk of breast cancer and increasing levels of total fruit intake (odds ratio (OR) for highest versus lowest quartile = 0.76, 95%CI = 0.63–0.91; p-trend = 0.01), but not for total vegetable intake (1.13 (0.93–1.37), p-trend = 0.25), after controlling for age, energy intake and known risk factors for breast cancer. The inverse association with total fruit intake was stronger for risk of ER+ (0.69 (0.54–0.88), p-trend = 0.01) than ER− tumors (0.89 (0.67–1.19), p-trend = 0.57) (p-heterogeneity = 0.02). In conclusion, this study suggests that fruit intake might have differential associations for breast tumor subtypes defined by ER status.     

单激素受体阳性乳腺癌患者的临床病理特征及预后因素分析

目的分析雌激素或孕激素受体阳性即单激素受体阳性乳腺癌患者的临床病理特征及预后因素,比较两种单激素受体阳性即ER单阳性和PR单阳性乳腺癌患者的不同之处。方法2000年9月至2002年9月在我院就诊的Ⅰ～Ⅲ。期单激素受体阳性乳腺癌患者共112例,分析其临床病理特征及预后因素。结果全组患者5年生存率（OS）为89．0％,5年无病生存率（DFS）为79．8％。COX多因素预后分析显示,腋窝淋巴结转移数目是全组患者的独立预后因素（P=0．003）,脉管瘤栓是淋巴结阴性单激素受体阳性患者DFS的独立预后因素（P=0．038）。PR单阳性组年龄≤50岁（P=0．021）以及绝经前患者（P=0．033）显著多于ER单阳性组。PR单阳性组分级3级、肿瘤直径〉2cm、脉管瘤栓者的比例略高于ER单阳性组,但无统计学意义。内分泌治疗可显著改善ER单阳性组患者的OS（P=0．04）及DFS（P=0．000）。内分泌治疗有一定程度上提高了PR单阳性组患者的OS（P=0．271）及DFS（P=0．387）。结论腋窝淋巴结转移数目是全组患者的独立预后因素。内分泌治疗可显著改善ER单阳性组患者的生存,有改善PR单阳性组患者生存的趋势。     

乳腺癌雌激素受体孕激素受体和C-erbB-2癌基因蛋白表达的临床意义

目的研究乳腺癌中雌、孕激素受体(ER,PR)与C,erbB-2癌基因的表达情况及临床意义。方法应用免疫组化S.P法,对82例原发性乳腺癌组织进行了ER,PR和C/erbB02检测,并进行统计学分析。结果ER,PR和C1erbB22的表达率分别为54.9%,40.2%,47.6%。ER的表达与C3erbB42的表达呈显著负相关(P<0.01)。C5erbB62的表达与肿瘤体积呈显著正相关(P<0.01),与年龄、组织学类型和淋巴结转移无明显相关性(P>0.05)。结论ER,PR的阳性表达是乳腺癌预后良好的指标,C7erbB82是乳腺癌预后不良的指标。ER,PR和C9erbB:2的测定对乳腺癌的诊断、治疗和预后判断具有重要的指导意义。     

Clinical impact of assay of estrogen receptor beta cx in breast cancer

Since 1996 when estrogen receptor beta(ER beta) was discovered, much effort has been devoted to the question of the value of ER beta as a prognostic and/or predictive factor in breast cancer and its potential as a novel target for pharmacological intervention. When estrogen receptors are applied on sucrose gradients and quantified by ligand binding, we found that in contrast to ER alpha, which has a narrow tissue distribution, ER beta is expressed in many tissues including both normal and malignant breast tissue. Receptor protein levels in tissues can also be measured from the intensities of bands after Western blotting and can be quantified when purified and quantified receptor is used as a standard. With this technique, we found that there were some tumors which had over 600 fmol/mg of ER beta protein but no detectable estradiol binding. In such tumors, RT-PCR analysis revealed that ER beta cx is the only ER beta isoform present. ER beta cx is a splice variant which utilizes an alternative exon 8. This change in the C-terminus results in very poor binding to estradiol (E2) and has a dominant negative effect on ER alpha function. Immunohistochemical analysis with an ER beta cx specific antibody in 115 ER alpha-positive breast cancers revealed that about half of the samples expressed ER beta cx protein. Initial analysis of samples from patients with preoperative tamoxifen treatment revealed that ER alpha-positive tumors expressing ER beta cx and lacking PR seemed to be resistant to the anti-estrogen. We conclude that, in order to better characterize breast cancers and design appropriate therapy for individual patients, assays for ER beta cx must be made available to clinicians.     

Short-term outcome of primary operated early breast cancer by hormone and HER-2 receptors

Introduction Prognostic subgroup classification of operable breast cancers using cDNA clustering of breast cancer-related genes resembles the classification based on the combined immunohistochemical (IHC) expression of the hormone and HER-2 receptors. We here report the short-term disease-free interval (DFI) of operable breast cancers by their joint hormone receptor/HER-2 phenotype. Patients and methods Short-term follow-up (FU) of a prospective cohort of 1,958 breast-cancer patients primary operated at our institution between 2000 and 2005. Receptors were evaluated using IHC. Steroid receptors were considered positive for any nuclear staining; HER-2 for strong (3+) membrane staining or positive fluorescence in situ hybridization (FISH). Kaplan–Meier (KM) DFI curves were calculated for any relapse defined as a local, regional, contralateral, or distant breast cancer event for the six predefined breast cancer subgroups: ER + PR + HER-2 − (PPN), ER + PR − HER-2 − (PNN), ER + PR + HER-2 + (PPP), ER – PR − HER-2 − (NNN), ER – PR − HER-2 + (NNP), and ER + PR − HER-2 + (PNP). P-values were calculated for comparison of the six different survival curves using two possible adaptations for multiple testing. A multivariate model for the receptors predicting DFI did incorporate local and systemic adjuvant therapy. Results Median patient age was 57 years (ranges 26–96) and median FU was 3.35 years. Overall, DFI at median FU was 91%; 94% for PPN, 89% for PNN, 86% for NNN, 81% for PPP, 80% for PNP, and 76% for NNP cases. Some receptor subgroups had a significantly better DFI than others based on multiple testing, especially when the PPN group was compared against the four most frequent subtypes. The multivariate model with local and systemic adjuvant therapy confirmed the prognostic value of ER, PR, and HER-2 for short-term DFI. Conclusion It is possible to distinguish short-term prognostic breast cancer subgroups only on the basis of ER, PR, and HER-2 even when stratified for local and systemic adjuvant therapy. While gene expression profiles based on microarray data of over hundreds of genes will probably teach us much about breast cancer biology, heterogeneity, and prognosis, we emphasize the important short-term prognostic value of currently used IHC markers for ER, PR, and HER-2.     

Immunohistochemical image analysis of estrogen and progesterone receptors in breast cancer

Background  Recently objective quantification of immunohistochemical estrogen receptor (ER) and progesterone receptor (PgR) staining in breast cancer by image cytometry has been predominantly performed by measuring the area of positively stained cells. However, in sample preparations of immunostained hormone receptors, both the stained area and the intensity of staining vary. In this study, we performed quantification of the stained area by measuring, tailing intensity using image cytometry. Methods  Quantitative analysis of ER and PgR immunohistochemistry was performed using image cytometry. The obtained values were presented as % of positive staining (%PS). Comparison of %PS with values obtained by EIA and with clinicopathological features was performed. Results  The %PS values and the natural logarithm of the EIA levels of the hormone receptors showed a significant positive correlation for both ER and PgR. The concordance of the results obtained by the two methods was 96.3% for ER and 73.7% for PgR. The ER-%PS values of postmenopausal patients were significantly higher than those of premenopausal patients, whereas the PgR-%PS values of the former group were significantly lower than those of the latter group. Conclusions  The quantification of ER and PgR in immunostained preparations using %PS as a parameter was reproducible and showed a high correlation with values obtained by EIA. It was shown that only menopausal status affects hormone receptor levels when analyzing the relationship between %PS measurements and clinicopathological features.     

新辅助化疗对局部晚期乳腺癌组织中雌激素受体孕激素受体和人表皮生长因子受体2表达的影响

目的探讨新辅助化疗对晚期乳腺癌组织中雌激素受体(ER)、孕激素受体(PR)和人表皮生长因子受体2(HER-2)表达的影响。方法收集ⅡB^ⅢB期的局部晚期乳腺癌患者48例,应用免疫组化方法检测ER、PR和HER-2的表达后,给予表阿霉素(EPI)加多西紫杉醇(DOX)方案进行新辅助化疗,3个疗程后行根治手术,将术后标本中ER、PR和HER-2的表达与化疗前表达情况进行对照,分析新辅助化疗对免疫表达的影响。结果新辅助化疗前ER阳性表达率为33.3%,化疗后ER阳性表达率为52.1%;化疗前PR阳性表达率为35.4%,化疗后PR阳性表达率为50.0%。ER和PR阴性表达的患者在新辅助化疗后转为阳性表达的比例高于化疗前阳性转为阴性的比例,但差异没有统计学意义(P>0.05)。新辅助化疗前HER-2阳性表达率为62.5%,化疗后HER-2阳性表达率为52.1%,新辅助化疗后阳性转阴率略高于阴性转阳率,差异无统计学意义(P>0.05)。结论新辅助化疗能使部分乳腺癌组织中ER、PR和HER-2的表达发生改变,为乳腺癌的治疗提供了一定的依据。