Inhibitory Effects of Mild Hyperthermia plus Docetaxel Therapy on ER(+/–) Breast Cancer Cells and Action Mechanisms

Summary： The purpose of this study was to verify that a combination of mild hyperthermia and do- cetaxel chemotherapy produces synergistic antitumor effects and to explore the action mechanisms of this treatment approach. The effects of docetaxel on the proliferation of cells from the estrogen receptor （ER）-positive human breast cancer cell line MCF-7 and the ER-negative human breast cancer cell line MDA-MB-453 were examined by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay, and effective experimental concentrations of docetaxel were determined. The effects of mild hy- perthermia plus docetaxel therapy on apoptosis rate in the MCF-7 and MDA-MB-453 human breast cancer cell lines were analyzed by using flow cytometry with Annexin-V fluorescein isothiocyanate （FITC）/propidium iodide （PI） staining. The effects of these combined treatments on cell cycle progres- sion in the MCF-7 and MDA-MB-453 human breast cancer cell lines were examined by using flow cy- tometry. The effects of these combined treatments on the expression of apoptosis-related proteins and proteins in the mitogen-activated protein kinase （MAPK） pathways were analyzed by using Western blotting. The effects of these combined treatments on the expression of the heat shock protein 70 （HSP70） and the multi-drug resistance （MDR） gene product P-glycoprotein （Pgp） were examined by using Western blotting. The results showed that the half-maximal inhibitory concentration （IC50） of do- cetaxel for MCF-7 and MDA-MB-453 cells was 19.57±1.12 and 21.64±2.31 gmol/L respectively. Mild hyperthermia with docetaxel therapy could increase apoptosis rate in the MCF-7 and MDA-MB-453 cells. Apoptosis rate in MCF-7 and MDA-MB-453 cells was increased from （23.66±3.59）% and （18.51±3.17）% in docetaxel treatment group to （47.12±6.73）% and （55.16±7.42）% in mild hyperthermia plus docetaxel group, indicating that the mild hyperthermia and docetaxel therapeutic approaches exhib- ited significant synergistic antitumor effects. Treatments of mild hyperthermia plus docetaxel induced G2/M cell cycle arrest in the MCF-7 and MDA-MB-453 cells. Western blotting demonstrated that pro- teins in the MAPK pathway were expressed at higher levels in docetaxel-treated cells following mild hypothermia than those in cells treated with docetaxel alone. As compared with blank control group, cells from the mild hyperthermia plus docetaxel group exhibited significantly decreased B-cell lym- phoma 2 （Bcl-2） protein expression but slightly increased Bcl-2-associated X protein （Bax） expression. Western blotting results revealed that HSP70 and Pgp expression levels were significantly increased following mild hypothermia. It was concluded that treatments of mild hyperthermia plus docetaxel in- hibited the proliferation of human breast cancer cells, promoted apoptosis of breast cancer cells, and produced synergistic antitumor effects.

Vaccinia-related kinase 2 variants differentially affect breast cancer growth by regulating kinase activity

Genetic information is transcribed from genomic DNA to mRNA,which is then translated into threedimensional proteins.mRNAs can undergo various post-transcriptional modifications,including RNA editing that alters mRNA sequences,ultimately affecting protein function.In this study,RNA editing was identified at the 499th base(c.499)of human vaccinia-related kinase 2(VRK2).This RNA editing changes the amino acid in the catalytic domain of VRK2 from isoleucine(with adenine base)to valine(with guanine base).Isoleucine-containing VRK2 has higher kinase activity than the valine-containing VRK2,which leads to an increase in tumor cell proliferation.Earlier we reported that VRK2 directly interacts with dystrobrevin-binding protein(dysbindin)and results in reducing its stability.Herein,we demonstrate that isoleucine-containing VRK2 decreases the level of dysbindin than valinecontaining VRK2.Dysbindin interacts with cyclin D and thereby regulates its expression and function.The reduction in the level of dysbindin by isoleucine-containing VRK2 further enhances the cyclin D expression,resulting in increased tumor growth and reduction in survival rates.It has also been observed that in patient samples,VRK2 level was elevated in breast cancer tissue compared to normal breast tissue.Additionally,the isoleucine form of VRK2 exhibited a greater increase in breast cancer tissue.Therefore,it is concluded that VRK2,especially dependent on the 167th variant amino acid,can be one of the indexes of tumor progression and proliferation.

LA-D-B1,a novel Abemaciclib derivative,exerts anti-breast cancer effects through CDK4/6

Background:Regulatory proteins involved in human cellular division and proliferation,cyclin-dependent kinases 4 and 6(CDK4/6)are overexpressed in numerous cancers,including triple-negative breast cancer(TNBC).TNBC is a common pathological subtype of breast cancer that is prone to recurrence and metastasis,and has a single treatment method.As one of the CDK4/6 inhibitors,abemaciclib can effectively inhibit the growth of breast tumors.In this study,we synthesized LA-D-B1,a derivative of Abemaciclib,and investigated its anti-tumor effects in breast cancer.Methods:Cellular viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay.Cell cloning and migration abilities were determined by colony formation assay and wound healing assay.Cell invasion abilities and adhesion were determined by cell invasion assay and cell adhesion assay.The impact of compound LA-D-B1 on cell proliferation and the cell cycle was analyzed through Western blotting,which quantified the levels of proteins associated with the cyclin-dependent kinase(CDK)4/6-cyclin D-Rb-E2F pathway.The in vivo anti-tumor activity of compound LA-D-B1 was investigated using a chick chorioallantoic membrane(CAM)model.Results:The study demonstrated that LA-D-B1 effectively suppressed breast cancer cell proliferation,induced apoptosis,and caused cell cycle arrest.Furthermore,LA-D-B1 reduced the expression of key proteins in the CDK4/6-cyclin D-Rb-E2F pathway,including CDK4,CDK6 and E2F1.The results also indicated significant antitumor activity of LA-D-B1 in a transplanted tumor model.Conclusion:In this study,LA-D-B1 demonstrated a potent anti-tumor effect by effectively suppressing cell proliferation and inhibiting cell cycle progression in breast cancer.These findings highlight the potential of LA-D-B1 as a valuable compound for enhancing therapeutic outcomes and controlling the progression of breast cancer.

Activation of Rac1-PI3K/Akt is required for epidermal growth factorinduced PAK1 activation and cell migration in MDA-MB-231 breast cancer cells

Epidermal growth factor （EGF） may increase cell motility, an event implicated in cancer cell invasion and metastasis. However, the underlying mechanisms for EGF-induced cell motility remain elusive. In this study, we found that EGF treatment could activate Ras-related C3 botulinum toxin substrate 1 （Racl）, PI3K/Akt and p21- actived kinase （PAK1） along with cell migration. Ectopic expression of PAK1 K299R, a dominant negative PAK1 mutant, could largely abolish EGF-induced cell migration. Blocking PI3K/Akt signalling with LY294002 or Akt siRNA remarkably inhibited both EGF-induced PAK1 activation and cell migration. Furthermore, expression of dominant-negative Racl （T17N） could largely block EGF-induced PI3K/Akt-PAK1 activation and cell migration. Interestingly, EGF could induce a significant production of ROS, and N-acetyl-L-cysteine, a scavenger of ROS which abolished the EGF-induced ROS generation, cell migration, as well as activation of PI3K/Akt and PAK, but not Racl. Our study demonstrated that EGF-induced cell migration involves a cascade of signalling events, including activation of Racl, generation of ROS and subsequent activation of PI3K/Akt and PAK1.

Cytotoxic Activity of <i>Thelesperma megapotamicum</i>Organic Fractions against MCF-7 Human Breast Cancer Cell Line

Thelesperma megapotamicum (Asteraceae) is commonly used in Argentine to treat various diseases (renal, digestive affections, and as anaesthesia). The present study showed the mechanisms involved “in vitro” cytotoxicity of T. megapotamicum Fractions. Five Fractions (F1 - F5) were separated by column chromatography (Silica gel) using hexane:diethyl ether as eluents. Viability was evaluated in Human breast carcinoma cell line (MCF-7) by staining with crystal violet. With respect to F1 Fraction treatment, the cell survival was 49.14% ± 8.87%, while the F2 and F3 ones exhibited a strong reduction of cell viability to only 26.35% ± 1.63% and 23.3%1 ± 0.53% of the control cell at 50 μg/ml, respectively. Apoptotic effect of these Fractions was detected using FITC-labeled Annexin V and propidium iodide binding assays and was confirmed by a higher proportion of apoptotic cells due to F2 and F3 treatments. T. megapotamicum active Fractions could facilitate the tumoral cells death by decreasing the activity of the enzyme Gamma-glutamyltranspeptidase and causing alteration in cell membrane sialoglycoconjugates and others involved anticancer mechanisms including apoptosis.

Immunotherapy of Cancer—A Historical Note

We examined the possibility that the anti-estrogens, tamoxifen (TX) and toremifen (TO) interacted?with the immune system. Indeed, both TX and TO stimulated cells mediated cytotoxicity reactions by various killer cells: killer T (TK), natural killer (NK), lymphokine activated killer (LAK) cells. Both TX and TO inhibited the growth of tumors that express estrogen receptors. Thus these antiestrogens inhibited tumor growth and stimulated killer cells for cytotoxicty on such tumors. Therefore these agents were presumed to stimulate tumor immunity. We tested the P815 mouse mastcytoma with TK, LK, and TX or TO. A therapeutic effect was observed in both experiments. The SL2-5 murine lymphoma was tested with NK and TX cells or TO cells and successful immunotherapy was observed.?We digested human breast carcinomas and lung tumors with collagenase. The killer cells were separated from tumor cells on Ficoll gradients. TX and TO enhanced the cytotoxic effect of autologous killer cells on the corresponding tumor cells. This experiment indicates that the results obtained in animals are also valid for human malignant disease.

Studies on mechanism of cis9,trans11-CLA and trans10,cis12-CLA inducing apoptosis of human breast cancer cell line MCF-7

Objective： The aim of the study was to explore the activities of cis9, trans11-CLA （C9, t11-CLA） and transl0, cis12-CLA （t10, c12-CLA） inhibiting tumor, and investigate their relationships with PPARy and apoptotic proteins, and mechanism of anti-cancer. Methods： The inhibitory rate, cell growth curve and apoptotic morphological observation of MCF-7 cells were obtained by MTT assay, trypan blue staining and Hoechst33342 fluorescence staining. The apoptotic rate and cell cycle were detected with flow cytometry. Transcriptional level of genes was detected with RT-PCR semi-quantitative method, and Western blot was performed to detect proteins levels. Results： The two CLA isomers could reduce cell proliferation （P 〈 0.05）, increase apoptotic rate （P 〈 0.05）, and increase obviously the transcriptional and protein levels of PPARy （P 〈 0.01）. The synchronism and correlation between the effects of CLA to PPARy and apoptotic proteins Bax, Bcl-2, Caspase 3 changes were found with the dose- and time-dependent manners. There was cooperative relation between the levels of PPARy and the rates of Bax/Bcl-2, Caspase 3 （small fragment） by experiments of PPARy inhibitor GW9662 and ligand Rosiglitazone. Conclusion： The apoptotic pathway of PPARy-Bcl-2-Caspase 3 signaling was found. The C9, t11-CLA and tl0, c12-CLA could inhibit MCF-7 cell proliferation and promote apoptosis via activating PPARy-Bcl-2-Caspase 3 pathway. CLA may be a kind of activator of PPARv.

PROGNOSTIC SIGNIFICANCE OF MIBl PROLIFERATION MARKER EXPRESSION ON DUCTAL CARCINOMA IN SITU AND INVASIVE DUCTAL CARCINOMA OF THE BREAST

Objective To determine the prognostic significance of MIBl proliferation marker expression on ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) of the breast. Methods By means of histological and immunohis tochemical techniques, MIB1 labelling index (LI) was determined in 31 pathologic specimens (DIal 6, IDC 22 and 3 benign breast lesions as control ). Results Significantly higher (P< 0. 05) MIB1 expression wus noted in breast carcinomas in contrast with benign breast lesions. MIBl LI increased with increasing tumor invasion from DCIS to IDC (P< 0. 05). Increased tendency of MIB1 LI had been also noted in according with the increased nuclear grabing (NG) of IDC. Conclusion MIBI ex- pression can faithfully reflect the proliferative activity of the breast lesions, where the breast cancers with both high MIBl LI and NG seem to have a poor prognosis.

Thymoquinone anticancer activity is enhanced when combined with royal jelly in human breast cancer

BACKGROUND Breast cancer is the most common cause of the majority of cancer-related deaths in women,among which triple-negative breast cancer is the most aggressive type of breast cancer diagnosed with limited treatment options.Thymoquinone(TQ),the main bioactive constituent of Nigella sativa,has been extensively studied as a potent anticancer molecule against various types of cancers.Honeybee products such as the royal jelly(RJ),the nutritive secretion fed to honeybee queens,exhibit a variety of biological activities besides its anticancer effect.However,the anticancer activity of the combination of TQ and RJ against breast cancer is still unknown.AIM To investigate cytotoxicity of RJ in FHs 74 Int cells and the anticancer effects of TQ,RJ,and their combinations in the MDA-MB-231 cell line.METHODS Cells were treated with TQ,RJ,and their combinations for 24 h.Using 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay,we determined the half-maximal inhibitory concentration of TQ.Trypan blue and 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were then performed to assess the cell viability in response to different treatment conditions.Cell death and cycle regulation were investigated using propidium iodide deoxyribonucleic acid staining followed by flow cytometry in response to a single dose of TQ,RJ,and their combination.Immunostaining for cleaved caspase 3 and Ki67 expression was used to determine apoptosis induction and changes in cell proliferation.RESULTS TQ alone inhibited cell viability in a dose-dependent manner at concentrations below and above the half-maximal inhibitory concentration.RJ exhibited relatively nontoxic effects against MDA-MB-231 cells and FHs 74 Int small intestinal cells at concentrations below 5μg/mL.High doses of RJ(200μg/mL)had greater toxicity against MDA-MB-231 cells.Interestingly,the inhibition of cell viability was most pronounced in response to 15μmol/L TQ and 5μg/mL RJ.A dose of 15μmol/L TQ caused a significant increase in the PreG1 population,while a more pronounced effect on cell viability inhibition and PreG1 increase was observed in response to TQ and RJ combinations.TQ was the main inducer of caspase 3-dependent apoptosis when applied alone and in combination with RJ.In contrast,no significant regulation of Ki67 expression was observed,indicating that the decrease in cell viability was due to apoptosis induction rather than to inhibition of cell proliferation.CONCLUSION This study is the first to report enhanced anticancer effects of TQ and RJ combination against MDA-MB-231 breast cancer cells,which could confer an advantage for cancer therapy.

XIAP、Smac表达与乳腺癌新辅助化疗后腋窝阳性淋巴结病理完全缓解及预后的相关性

目的 探究抑制X染色体连锁的凋亡抑制蛋白(XIAP)、第二个线粒体衍生的半胱氨酸蛋白酶激活剂(Smac)表达与乳腺癌新辅助化疗(NACT)后腋窝淋巴结(ALN)阳性病理完全缓解(pCR)及预后的相关性。方法 选取2019年6月至2021年6月期间唐山市妇幼保健院收治的195例乳腺癌ALN阳性患者纳入乳腺癌组,所有患者均进行NACT,NACT前所有患者均进行活检穿刺取病灶组织。另同期选取195例经进行乳腺活检穿刺,且病理证实为良性乳腺结节的患者,纳入对照组。采用免疫组化法检测患者XIAP、Smac表达水平。根据乳腺癌患者NACT疗效分为pCR组及非pCR组,收集患者临床资料,采用单因素和多因素Logistic回归模型分析NACT后ALN阳性患者非pCR的影响因素,采用Kaplan Meier法分析不同XIAP、Smac表达情况患者的生存预后情况。结果 乳腺癌组病灶组织XIAP低表达占比低于对照组,Smac低表达高于对照组(P<0.05);195例患中72例患者NACT后pCR,pCR发生率为36.92%;非pCR组AJCCⅢ期、原发病灶直径>3.5 cm、ALN数量≥4个、ALN pN1~pN2期、XIAP高表达及Smac低表达占比均高于pCR组(P<0.05);Logistic回归分析模型结果显示,AJCCⅢ期、ALN数量≥4个、ALN pN1~pN2期、XIAP高表达及Smac低表达是乳腺癌患者NACT后ALN阳性非pCR的独立危险因素(P<0.05,OR>1);乳腺癌患者随访时间12~36个月,中位随访时间26个月,Kaplan Meier生存分析显示,XIAP低表达者3年累积生存率为94.9%,高于XIAP高表达者的90.4%(Log rank χ^(2)=4.931,P=0.026);Smac低表达者3年累积生存率为89.8%,低于Smac高表达的94.0%(Log rank χ^(2)=4.534,P=0.033)。结论 乳腺癌ALN阳性患者组织中XIAP异常高表达,Smac异常低表达,XIAP高表达及Smac低表达是乳腺癌患者NACT后ALN阳性非pCR的独立危险因素,XIAP低表达、Smac高表达患者生存率更高,预后更好。

槲皮素对乳腺癌细胞的影响及作用机制实验研究

目的:探讨槲皮素对乳腺癌细胞的影响及其作用机制。方法:将人乳腺癌MDA-MB-231细胞分为空白对照组(无药物干预)、低、中及高剂量槲皮素组(分别加入10、20、40μmoL/L槲皮素共培养)和顺铂组(加入3 mg/L顺铂共培养)。显微镜下观察各组细胞形态。Transwell实验检测各组细胞侵袭能力。划痕实验检测各组细胞迁移能力。流式细胞仪检测各组细胞凋亡率。免疫印记实验检测细胞因子信号转导抑制蛋白1(SOCS1)、信号传导及转录激活蛋白3(STAT3)表达。结果:空白对照组MDA-MB-231细胞呈不规则状生长,分布较紧密。低、中剂量槲皮素组MDA-MB-231细胞间隙变大,数目减少,细胞质变浑浊,而高剂量槲皮素组及顺铂组MDA-MB-231细胞形态呈片状球形,并出现肿胀破裂且数目减少。与空白对照组比较,低、中及高剂量槲皮素组MDA-MB-231细胞侵袭数目、划痕距离、STAT3水平降低,凋亡率、SOCS1水平升高(均P<0.05)。低、中及高剂量槲皮素组MDA-MB-231细胞侵袭数目、划痕距离、STAT3水平依次降低,凋亡率、SOCS1水平依次升高(均P<0.05)。高剂量槲皮素组与顺铂组细胞侵袭数目、划痕距离、凋亡率及STAT3、SOCS1水平比较差异无统计学意义(均P>0.05)。结论:槲皮素能够抑制乳腺癌细胞侵袭和转移,促进细胞凋亡,其机制可能与上调SOCS1、抑制STAT3表达有关。

信号转导和转录激活因子3/CC趋化因子配体2信号通路对乳腺癌细胞活力、迁移和侵袭的影响实验研究

目的:探讨信号转导和转录激活因子3(STAT3)/CC趋化因子配体2(CCL2)信号通路对乳腺癌细胞活力、迁移和侵袭的影响。方法:体外培养人乳腺癌细胞株(HCC1937、MCF-7、MDA-MB-231、ZR-75-1)和人正常乳腺上皮细胞(MCF-10A)。采用蛋白印迹法检测HCC1937、MCF-7、MDA-MB-231、ZR-75-1和MCF-10A细胞磷酸化STAT3(p-STAT3)、STAT3、CCL2蛋白表达。选取p-STAT3/STAT3蛋白比值、CCL2蛋白表达最高的人乳腺癌细胞进行后续实验。将MCF-7细胞分为对照组、STAT3抑制剂(WP1066)Ⅰ组(2μmol/L WP1066)、Ⅱ组(4μmol/L WP1066)、Ⅲ组(8μmol/L WP1066)。分别采用细胞计数试剂盒-8、划痕实验、Transwell实验检测MCF-7细胞活力、迁移率及侵袭能力。采用蛋白印迹法检测MCF-7细胞p-STAT3、STAT3、CCL2、基质金属蛋白酶(MMP)-2、MMP-9蛋白表达。结果:与MCF-10A细胞比较,HCC1937、MCF-7、MDA-MB-231、ZR-75-1细胞p-STAT3/STAT3蛋白比值、CCL2蛋白表达升高(均P<0.05)。其中MCF-7细胞p-STAT3/STAT3蛋白比值、CCL2蛋白表达最高,因此选用MCF-7细胞进行后续实验。与对照组比较,STAT3抑制剂Ⅰ、Ⅱ、Ⅲ组MCF-7细胞活力、迁移率、侵袭数目依次降低(均P<0.05)。与对照组比较,STAT3抑制剂Ⅰ、Ⅱ、Ⅲ组MCF-7细胞p-STAT3/STAT3蛋白比值、CCL2、MMP-2及MMP-9蛋白表达依次降低(均P<0.05)。结论:乳腺癌细胞中STAT3/CCL2呈高表达,抑制STAT3/CCL2信号通路能够降低乳腺癌细胞活力,减少迁移和侵袭。

基于自噬抑制剂3-MA介导的大叶蛇葡萄总黄酮提取物对人乳腺癌细胞增殖、凋亡的影响

目的探讨抑制自噬时,大叶蛇葡萄总黄酮提取物(total flavonoid extract,TFE)对人乳腺癌细胞增殖、凋亡的影响及其作用机制。方法针对人宫颈癌细胞Hela、人肺癌细胞A549、人肝癌细胞SMMC-7721、人乳腺癌细胞MCF-7、MDA-MB-231及人正常肝细胞L-02,采用MTT法优选敏感细胞株;通过TFE与自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)联合运用,采用MTT法检测其对敏感细胞增殖的抑制作用;透射电子显微镜及Hoechst 33258单染法观察细胞的形态学变化;Annexin V-FITC/PI双染法检测细胞凋亡率的变化;Western blot检测凋亡相关蛋白和通路蛋白(死亡受体途径、线粒体途径、内质网应激途径)的表达水平;免疫荧光法检测线粒体途径关键蛋白Cyt-c表达,并选择自噬激动剂雷帕霉素(rapamycin,RA)进行反向验证。结果TFE可浓度依赖性抑制人乳腺癌细胞增殖,MCF-7细胞为敏感细胞株,与TFE组相比,TFE+3-MA组在24、48、72 h对MCF-7细胞的抑制率均明显增加(P<0.01),细胞数量减少、间隙增大,凋亡小体增多,凋亡率升高(P<0.01),Bax/Bcl-2(P<0.01)、cleaved caspase-3(P<0.01)、Cyt-c(P<0.05)、FADD、cleaved caspase-12的表达量均升高,核内凋亡蛋白Cyt-c表达增加;TFE+RA组荧光减弱,逆转了TFE诱导的线粒体途径凋亡。结论TFE能明显抑制人乳腺癌细胞的增殖,抑制自噬时可能通过线粒体途径促进MCF-7细胞凋亡,激活自噬可逆转细胞凋亡。

活化白细胞黏附分子在乳腺癌中的研究进展

乳腺癌是全球女性最常见的肿瘤,也是导致女性死亡最主要的疾病之一。肿瘤相关标志物及治疗靶点的研究已成为肿瘤领域的一个热点话题。研究发现活化白细胞黏附分子(Activated leukocyte cell adhesion molecule,ALCAM)在乳腺癌的发生发展过程中发挥重要的作用,并且与乳腺癌患者的预后具有相关性。本文针对ALCAM的分子结构特征、ALCAM与乳腺癌临床指征及预后的相关性,以及ALCAM作为乳腺癌临床诊断及治疗靶点的研究和应用进展进行综述。

乳腺癌患者不同病理特征中H3K9ac、PD-L1、VISTA表达及其联合检测对乳腺癌不良预后的预测价值

目的探讨乳腺癌患者不同病理特征中组蛋白H3第9位赖氨酸残基乙酰化(H3K9ac)、程序性死亡因子配体1(PD-L1)、T细胞激活抑制物免疫球蛋白的可变区结构域(VISTA)及其联合检测对乳腺癌不良预后的预测价值。方法选取2018年5月至2020年7月平顶山市第一人民医院收治的130例乳腺癌患者,免疫组化检测乳腺癌组织及癌旁组织中H3K9ac、PD-L1及VISTA表达情况。比较乳腺癌组织、癌旁组织中H3K9ac、PD-L1及VISTA表达情况;比较乳腺癌不同病理特征中H3K9ac、PD-L1及VISTA表达情况;随访3 a,根据是否发生转移、复发、死亡分为预后不良和预后良好;比较预后不良和预后良好乳腺癌患者H3K9ac、PD-L1及VISTA表达情况;分析H3K9ac、PD-L1、VISTA表达联合检测对乳腺癌不良预后的预测价值。结果乳腺癌组织中H3K9ac、PD-L1、VISTA高表达率均高于癌旁组织(P<0.05);不同组织分化程度、T分期、N分期、阳性淋巴细胞数目中H3K9ac、PD-L1、VISTA高表达率比较,差异有统计学意义(P<0.05);随访3 a,130例乳腺癌患者失访7例(5.38%),预后不良22例,预后良好101例。预后不良患者H3K9ac、PD-L1、VISTA高表达率高于预后良好患者(P<0.05);H3K9ac、PD-L1、VISTA联合检测预测乳腺癌患者不良预后的AUC为0.902,大于各指标单独预测的0.714、0.866、0.781(P<0.05)。结论乳腺癌患者癌组织中H3K9ac、PD-L1及VISTA均呈高表达,其高表达与分化程度、分期、淋巴细胞数目等病理特征相关,H3K9ac、PD-L1、VISTA联合检测对乳腺癌患者不良预后具有较高的预测价值。

长链非编码RNA活化T细胞核因子非编码基因和长链非编码RNA小分子NF90相关RNA与三阴性乳腺癌预后的相关性分析

目的 探究长链非编码RNA(lncRNA)活化T细胞核因子非编码基因(NRON)和lncRNA小分子NF90相关RNA(snaR)与三阴乳腺癌(TNBC)预后的相关性。方法 2018年2月~2020年2月我院收治的TNBC病人100例,术中取其肿瘤组织及癌旁组织,根据3年随访预后情况将其分为预后良好组(72例)与预后不良组(28例)。采用实时定量荧光PCR(RT-qPCR)法检测肿瘤组织与癌旁组织lncRNA NRON、lncRNA snaR表达水平。Pearson法分析TNBC病人肿瘤组织lncRNA NRON与lncRNA snaR的相关性。多因素Cox回归分析TNBC病人预后的影响因素。受试者工作特征(ROC)曲线分析TNBC病人肿瘤组织lncRNA NRON和lncRNA snaR表达水平对预后的预测价值。结果 与癌旁组织相比,TNBC病人肿瘤组织lncRNA NRON表达水平显著下降(1.01±0.10比0.65±0.08,P<0.001),lncRNA snaR表达水平显著上升(1.03±0.13比2.42±0.30,P<0.001)。lncRNA NRON、lncRNA snaR表达水平与TNM分期、肿瘤分化程度有关(P<0.05)。与预后良好组相比,预后不良组TNBC病人肿瘤组织lncRNA NRON表达水平显著下降(0.69±0.09比0.55±0.05,P<0.001),lncRNA snaR表达水平显著上升(2.28±0.26比2.78±0.40,P<0.001)。Pearson法分析显示,TNBC病人肿瘤组织lncRNA NRON与lncRNA snaR表达水平呈负相关(r=-0.617,P<0.001)。多因素Cox回归分析结果显示,lncRNA NRON是TNBC病人预后的保护因素(P<0.05),lncRNA snaR是TNBC病人预后的危险因素(P<0.05)。lncRNA NRON和lncRNA snaR表达水平联合预测TNBC病人预后优于lncRNA snaR单独预测(Z_(二者联合-lncRNA snaR)=3.200,P=0.001)。结论 TNBC病人肿瘤组织中lncRNA NRON表达下调,lncRNA snaR表达上调,二者与TNBC病人预后有关,可能是TNBC预后预测的生物标志物。

Breast cancer stem-like cells can promote metastasis by activating platelets and down-regulating antitumor activity of natural killer cells

OBJECTIVE:To investigate whether cancer stem cells(CSCs) more efficiently activating platelets and evading immune surveillance than non-CSCs thus promoting metastasis.METHODS:We enriched and identified sphereforming cells(SFCs) and coincubated washed platelets with several platelet activators including collagen,4T1 and SFCs.Platelet-coating tumor cells,platelet activation and TGF-β1 release were analyzed.Then natural kell cells(NK) were incubated with supernatants of different activated platelet samples what we called sample release(SR).The degranulation assay and NKG2 D expression on NK cells were conducted by flow cytometry.Finally tissue factor(TF) expression of SFCs or 4T1 were evaluated by western blot.RESULTS:Breast cancer cell line 4T1 could form spheres in serum-free medium at low adherence.Sphere-forming cells expressed high levels of the CD24-/lowCD44 + stem cell phenotype.Both sphere-forming cells or 4T1 were coated with abundant platelets while sphere-forming cells induced significantly higher expression of platelet activating receptor CD62 p than 4T1 did(P < 0.01).And sphere-forming cells induced platelets to produce more TGF-β1 than 4T1 did(P < 0.01).Furthermore,sample releases induced by sphere-forming cells caused more vigorous inhibition of NK cells antitumor reactivity(P < 0.05) and reduced NKG2 D expression(P < 0.01).The final results showed that sphere-forming cells expressed higher levels of TF than 4T1(P < 0.05).CONCLUSION:Our findings indicate that CSCs could efficiently activate platelets,induce platelets to secrete more TGF-β1,decrease NKG2 D expression and inhibit antitumor activity of NK cell,compared with 4T1.And higher levels of TF expression of CSCs may account for this correlation of CSCs and platelets.

Effects and Potential Mechanisms of Danzhi Xiaoyao Pill(丹栀逍遥丸) on Proliferation of MCF-7 Human Breast Cancer Cells in vitro

Objective： To investigate the effects of 50% ethyl alcohol （EtOH） extracts from Danzhi Xiaoyao Pill （丹栀逍遥丸, DXP） on the proliferation of MCF-7 human breast cancer cells and potential mechanisms. Methods： ATP-Lite assay was performed to test the proliferation of the MCF-7 breast cancer cell line; and antioxidant activity was measured by the oxygen radical absorbance capacity （ORAC）. The effects of DXP on nitric oxide （NO） production were tested by lipopolysaccharide （LPS）- stimulated RAW 264.7 murine macrophages using the Griess reaction. Results： The 50% EtOH DXP extracts displayed a cytotoxic response on MCF-7 cells at 0.10, 0.25 and 0.50 mg/mL dosedependently with the proliferation inhibited by more than 85%. The ORAC value of the DXP was 820μ moL Trolox equivalent/g, about 40% of the vitamin C value. DXP extracts had significant inhibitory effect on NO production at the concentration from 0.0625 mg/mL to 0.5 mg/mL （P〈0.05, P〈0.01）. Conclusion： The extracts of DXP could significantly inhibit the proliferation of MCF-7 cells, with the effect possibly related to its antioxidant activity and the inhibition of NO production.

Effect of 1,25(OH)_2D_3 on the growth and apoptosis of breast cancer cell line MCF-7

Objective To study the effect of 1,25 dihydroxyvitamin D 3 (1,25(OH) 2D 3) on the growth and apoptosis of breast cancer cell line MCF 7 Methods Cell number was determined using the MTT method Flow cytometric analysis was performed on cell cycles, and the percentage of apoptosis was counted Apoptotic cells were quantified by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL), and bcl 2 protein expression was estimated with Western blotting Results After incubation with 1,25(OH) 2D 3 10 7 mol/L for 48 hours, MCF 7 cells exhibited significant growth in a dose and time dependent manner Flow cytometric analysis indicated that cell numbers in G 0/G 1 increased along with increasing apoptotic peak and percentage With microscope and electron microscope observation, characteristics of apoptosis such as typical apoptotic bodies were commonly found TUNEL also showed that 1,25(OH) 2D 3 10 8 mol/L and 10 7 mol/L groups had significantly high apoptosis percentage than control group with dose dependence on induction apoptosis And Western blot showed that 1,25(OH) 2D 3 10 8 mol/L could down regulate bcl 2 protein and 10 7 mol/L could almost block bcl 2 protein expression Conclusions 1,25(OH) 2D 3 can inhibit cell growth with G 0/G 1 arrest, enhance the proliferation inhibition action of adriamycin, and induce apoptosis which may result from the down regulation of the anti apoptotic bcl 2 protein