Multi-kinase inhibitor E7080 suppresses lymph node and lung metastases of human mammary breast tumor MDA-MB-231 via inhibition of vascular endothelial growth factor-receptor (VEGF-R) 2 and VEGF-R3 kinase

PURPOSE: Vascular endothelial growth factor (VEGF)-C/VEGF-receptor 3 (VEGF-R3) signal plays a significant role in lymphangiogenesis and tumor metastasis based on its effects on lymphatic vessels. However, little is known about the effect of inhibiting VEGF-R3 on lymphangiogenesis and lymph node metastases using a small-molecule kinase inhibitor. EXPERIMENTAL DESIGN: We evaluated the effect of E7080, a potent inhibitor of both VEGF-R2 and VEGF-R3 kinase, and bevacizumab on lymphangiogenesis and angiogenesis in a mammary fat pad xenograft model of human breast cancer using MDA-MB-231 cells that express excessive amounts of VEGF-C. Lymphangiogenesis was determined by lymphatic vessel density (LVD) and angiogenesis by microvessel density (MVD). RESULTS: In contrast to MDA-MB-435 cells, which expressed a similar amount of VEGF to MDA-MB-231 cells with an undetectable amount of VEGF-C, only MDA-MB-231 exhibited lymphangiogenesis in the primary tumor. E7080 but not bevacizumab significantly decreased LVD within the MDA-MB-231 tumor. E7080 and bevacizumab decreased MVD in both the MDA-MB-231 and MDA-MB-435 models. E7080 significantly suppressed regional lymph nodes and distant lung metastases of MDA-MB-231, whereas bevacizumab significantly inhibited only lung metastases. E7080 also decreased both MVD and LVD within the metastatic nodules at lymph nodes after resection of the primary tumor. CONCLUSIONS: Inhibition of VEGF-R3 kinase with E7080 effectively decreased LVD within MDA-MB-231 tumors, which express VEGF-C. Simultaneous inhibition of both VEGF-R2 and VEGF-R3 kinases by E7080 may be a promising new strategy to control regional lymph node and distant lung metastases.     

Exogenous expression of UHRF1 promotes proliferation and metastasis of breast cancer cells

In the present study, we investigated the role of UHRF1 (ubiquitin-like protein containing PHD and RING finger domains 1) in proliferation, invasion and migration of breast cancer cells, and the potential mechanisms were also explored. Cell proliferation was examined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay; cell cycle distribution and apoptosis were evaluated using flow cytometry; protein expression was determined by western blotting; angiogenesis of xenografts was assessed by microvessel density (MVD); cell invasion was measured using transwell chamber; cell migration was determined by wound scratching assay. Our results demonstrated that UHRF1 transfection conferred serum independence to MDA-MB-231 cells, G1 phase shortage and apoptosis suppression, accompanied with an increased expression of cyclin D1 and decreased expression of Bax. Significant pro-invasion and pro-migration activity was observed, with no obvious effect on the expression of PTEN and maspin. Co-expression of the UHRF1/PTEN or UHRF1/maspin degraded the role of UHRF1 in regulating invasion and migration. UHRF1 induced growth of MDA-MB-231 cells by promoting tumor vessel formation in vivo. In conclusion, UHRF1 promoted the proliferation of breast cancer cells by apoptosis inhibition, G1 phase shortage and promotion of tumor vessel formation, and pro-invasion and pro-migration activity was also observed by interacting with PTEN and maspin. Thus, UHRF1 may serve as a new therapy target for breast cancer.     

RNAi‐mediated CD73 suppression induces apoptosis and cell‐cycle arrest in human breast cancer cells

Ecto‐5′‐nucleotidase (CD73), a cell surface protein that hydrolyzes extracellular AMP into adenosine and phosphate, is overexpressed in many solid tumors. In this study, we tested the hypothesis that increased CD73 may promote tumor progression by examining the effect of CD73 suppression via RNA interference and CD73 overexpression on tumor growth in vivo and in vitro. Using digitized whole‐body images, plate clone forming assay and TUNEL assay in frozen tissue sections, we found that the cell growth rate was significantly lower in vivo and in vitro after CD73 suppression and late apoptosis was much higher in xenograft tumors developed from the CD73‐siRNA transfected MB‐MDA‐231 clone (P1). By flow cytometry, the P1 cell cycle was arrested in the G0/G1 phase. Moreover, Bcl‐2 was downregulated, while Bax and caspase‐3 were upregulated with CD73 suppression. CD73 inhibitor α,β‐methylene adenosine‐5′‐disphosphate (APCP) functioned similarly with RNAi‐mediated CD73 suppression. In addition, in transfected MCF‐7 cells, we found that CD73 overexpression increased cell viability and promoted cell cycle progression, depending on its enzyme activity. More intriguingly, CD73 overexpression in MCF‐7 breast cancer cells produces a tumorigenic phenotype. We conclude that CD73 plays an important role in breast cancer growth by affecting cell cycle progression and apoptosis. (Cancer Sci 2010; 101: 2561–2569)     

The expression and prognostic value of ROCK I and ROCK II and their role in human breast cancer

The role and expression of ROCKI and ROCKII in human breast cancer was investigated and the effect on clinical outcome assessed. ROCK knockdown cells (MDA-MB-231DeltaROCKI and MDA-MB-231DeltaROCKII were tested for their in vitro invasiveness, motility and in vivo tumour growth. Samples of fresh frozen breast tumour tissue (n=113) and normal background tissue (n=30) were processed for immunohistochemical and quantitative RT-PCR analyses. MDA-MB-231DeltaROCKI and MDA-MB-231DeltaROCKII cells showed significantly decreased invasiveness compared with control cells (mean +/- SEM 4.33+/-0.84 MDA-MB-231DeltaROCKI vs. 18.4+/-1.4 control, p<0.001; 6.8+/-1.2 MDA-MB-231DeltaROCKII vs. 18.4+/-1.4 control, p<0.001). Similarly, both exhibited reduced motility compared with control cells (p<0.001) and lost their response to HGF. Importantly, no significant difference existed between knockdown and control cells in in vivo tumour growth. ROCKI was significantly higher in human mammary tumours than normal background tissue (2.9+/-1.1 vs. 0.29+/-0.13, p=0.023), although expression of ROCKII was fairly consistent in both (2050+/-646 vs. 2303+/-2079). ROCKI expression was greater in patients who died from breast cancer than in patients who remained disease free (11.6+/-7.1 vs. 1.95+/-0.95). Higher levels of ROCKI were associated with increased grade (0.95+/-0.73 grade-1; 2.11+/-1.72 grade-2; and 4.06+/-1.99 grade-3). Levels of ROCKI, but not ROCKII, were significantly correlated with overall survival of patients (p=0.004, Univariate analysis, median follow-up 120 month). These results show that ROCKI and possibly ROCKII are key factors in regulation of motility/invasion of breast cancer cells. This, together with significant correlation between ROCKI and poor outcome in clinical breast cancer, indicates that it is a potential target in human breast cancer.     

1,1-Bis(3′-indolyl)-1-(<Emphasis Type="Italic">p</Emphasis>-substituted phenyl)methanes inhibit proliferation of estrogen receptor-negative breast cancer cells by activation of multiple pathways

1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes containing para-trifluoromethyl (DIM-C-pPhCF(3)), t-butyl (DIM-C-pPhtBu), and phenyl (DIM-C-pPhC(6)H(5)) groups are methylene-substituted diindolylmetyhanes (C-DIMs) that activate peroxisome proliferator-activated receptor gamma (PPARgamma) in estrogen receptor alpha-negative MDA-MB-231 and MDA-MB-453 breast cancer cells. C-DIMs inhibit breast cancer cell proliferation; however, inhibition of G(0)/G(1) to S phase progression and cyclin D1 downregulation was observed in MDA-MB-231 but not MDA-MB-453 cells. Nonsteroidal anti-inflammatory drug-activated gene 1 (NAG-1), a transforming growth factor beta-like peptide, was also induced by these compounds, and the response was dependent on cell-context dependent activation of kinase pathways. However, inhibition of cell growth, induction of NAG-1 and activation of kinases by C-DIMs were not inhibited by PPARgamma antagonists. Despite the induction of NAG-1 and downregulation of the antiapoptotic protein survivin by C-DIMs in both MDA-MB-231 and MDA-MB-453 cells, apoptotic cell death was not observed. Nevertheless, the cytotoxicity of C-DIMs in vitro was complemented by inhibition of tumor growth in athymic nude mice bearing MDA-MB-231 cells as xenografts and treated with DIM-C-pPhC(6)H(5) (40 mg/kg/day). The growth inhibition of tumors derived from highly aggressive MDA-MB-231 cells suggests a potential role for the C-DIM compounds in the clinical treatment of ER-negative breast cancer.     

The in vitro and in vivo effects of human umbilical cord mesenchymal stem cells on the growth of breast cancer cells

The purpose of the study was to detect the effect and possible mechanism of human umbilical cord mesenchymal stem cells (hUCMSCs) on the in vitro and in vivo growth of stem cells isolated from primary human breast cancer cells and cell lines MDA-MB-231 and MCF-7. Primary human breast cancer cells and MDA-MB-231 and MCF-7 cells were sorted in vitro using flow cytometry, and the ESA+, CD44+, CD24-/low cells were isolated as breast cancer stem cells (CSCs). The inhibitory effect of hUCMSCs on CSCs was examined using the Cell Counting Kit-8 cell proliferation and soft agar colony formation assay. In vivo tumor inhibition was studied using a severe combined immunodeficient xenograft mouse model transplanted with MDA-MB-231 breast CSCs. The expression of phosphoinositide 3-kinase (PI3K) and AKT was examined in the xenograft tumors using immunohistochemistry. The number of colonies formed by breast CSCs co-cultured with hUCMSCs at the bottom of soft agar was significantly lower than those formed by the control group (P < 0.01). Compared with the control group, the CSCs co-cultured with hUCMSCs showed a higher number of cells in the G2-M phase (P < 0.05) and an increased number of apoptotic cells (P < 0.01). The mice in the medium- and high-concentration hUCMSC treatment groups exhibited clearly reduced tumor volume and tumor weight, compared with the control group (P < 0.01). Compared with the saline group, the xenograft tumor tissues from the mice treated with different concentrations of hUCMSCs showed significantly reduced levels of PI3K and AKT proteins (P < 0.001). In conclusion, hUCMSC significantly inhibited the growth of breast CSCs in vitro and in vivo. The underlying mechanism is likely related to cell cycle arrest, induction of tumor cell apoptosis, and suppressed activities of PI3K and AKT protein kinases.     

化疗对三阴性乳腺癌CD44+CD24-/low细胞亚群的影响

目的:观察化疗对三阴性乳腺癌CD44+CD24-/low细胞亚群的影响。方法:用0.2、1、5倍药物血浆峰浓度(peak plasma concentration,PPC)的紫杉醇、多西紫杉醇、氟尿嘧啶、表柔比星、长春瑞滨和吉西他滨分别作用人三阴性乳腺癌MDA-MB-231细胞24、48和72h后,MTT法检测细胞生长情况;FCM法检测1倍PPC的各药物作用48h以及细胞复苏后CD44+CD24-/low细胞含量的变化;另外,FCM法检测10例转移性三阴性乳腺癌患者在化疗前后,外周血中CD44+CD24-/low细胞的含量变化。结果:6种药物均可抑制MDA-MB-231细胞增殖。1倍PPC的各药作用后,CD44+CD24-/low细胞含量未明显升高,以氟尿嘧啶组含量降低最为明显(P<0.05)。转移性三阴性乳腺癌患者在化疗后,外周血中CD44+CD24-/low细胞含量降低(P<0.05)。结论:化疗药物紫杉醇、多西紫杉醇、氟尿嘧啶和表柔比星可降低MDA-MB-231细胞株中CD44+CD24-/low细胞亚群的含量。化疗可使转移性三阴性乳腺癌患者外周血中CD44+CD24-/low细胞含量降低。     

Antiestrogenic action of toremifene on hormone-dependent, -independent, and heterogeneous breast tumor growth in the athymic mouse

The antiestrogen toremifene has been used to study the growth control of hormone-dependent (MCF-7), -independent (MDA-MB-231), or mixed tumor cell populations in athymic mice. Maximal MCF-7 tumor growth was produced in ovariectomized athymic mice by circulating estradiol levels of approximately 200 pg/ml (produced by 0.5-cm silastic capsules implanted s.c.). The antiestrogen toremifene (77 +/- 4 micrograms/day from a 2-cm silastic capsule) inhibited estradiol (0.5-cm capsule)-stimulated growth by more than 70%. No tumor growth was observed in mice treated with toremifene alone, although toremifene acted as a weak partial agonist on the mouse uterus. The growth of hormone-independent MDA-MB-231 breast tumors implanted in athymic mice was not influenced by either estradiol (0.5-cm capsule) or toremifene (2-cm capsule) when administered alone or in combination. Furthermore, even very large doses of toremifene (5 mg/day p.o.) did not alter the rate of MDA-MB-231 tumor growth. Mixtures of MCF-7 and MDA-MB-231 cells in 9:1 and 99:1 ratios inoculated into athymic mice produced tumors which grew in the absence of estradiol but responded to estradiol supplementation (0.5-cm capsule) with a more rapid rate of tumor growth. Tumors grown from inoculated MCF-7:MDA-MB-231 cells (99:1 ratio) in the presence of estradiol had estrogen receptor levels of 33.2 +/- 9.2 fmol/mg of protein at Day 44 compared to 84.8 +/- 4.8 fmol/mg of protein in pure MCF-7 tumors. Toremifene (2-cm capsule) treatment inhibited the estrogen stimulation of these mixed tumors (99:1 starting ratio) to that of toremifene alone. However, toremifene-alone treatment produced a more rapid rate of tumor growth than control or tumors grown from irradiated MCF-7 cells mixed with viable MDA-MB-231 cells. Increasing the ratio of MCF-7:MDA-MB-231 cells (999:1) initially inoculated resulted in tumors which developed less rapidly than the lower ratio (99:1). Toremifene (2-cm capsule) again produced partial inhibition of 17 beta-estradiol-stimulated tumor growth while increasing tumor growth above control when the antiestrogen was administered alone. These results demonstrate that toremifene is effective in inhibiting estrogen stimulation of hormone-dependent tumors and partially successful at controlling mixed hormone-dependent/independent tumors; however, the antiestrogen cannot control the growth of a hormone-independent tumor in this model.     

Reduction of TSG101 protein has a negative impact on tumor cell growth

TSG101 was defined originally as a tumor-suppressor gene, raising the expectation that absence of the encoded protein should lead to increased tumor cell growth and, perhaps, increased tumor cell aggressiveness. We have used the RNA interference (RNAi) technique to downregulate TSG101 in PC3 (prostate cancer) and MDA-MB-231 (breast cancer) cells. An approximately 85% selective downregulation at the protein level was achieved in both cell lines over a period of 12 days as detected by Western blotting. This treatment resulted in inhibition of tumor cell growth, with a decreased level of TSG101 causing partial cell cycle arrest at the G(1)/S boundary and a reduction in the rate at which cells passed from G(2) through mitosis and back into G(1). In both cell lines, the percentage of cells in S-phase was reduced significantly at day 4 after the TSG101 siRNA transfection (27% vs. 41% in MDA-MB-231 cells; 22% vs. 39% in PC3 cells). Additionally, RNAi-mediated downregulation of TSG101 reduced the colony formation capacities of both cancer cell lines. Rather more surprisingly, TSG101 downregulation affected the migratory activity of the MDA-MB-231 cells, independent of any effect on proliferation. Thus, in a Transwell assay, after 4-hr incubation, 36.0% of control MDA-MB-231 cells had migrated to the lower chamber vs. 7.3% of TSG101-downregulated cells (p < 0.001; scrambled control, 36.5%). These results show that the TSG101 gene does not comply with the usual characteristics of a tumor-suppressor gene; rather, its expression may be necessary for activities associated with aspects of tumor progression.     

Lymphatic vessel density as prognostic factor in breast carcinoma: relation to clinicopathologic parameters

Angiogenesis and lymphangiogenesis are essential for breast cancer growth and progression. This study aimed at investigating lymphatic microvessel density (LVD) and microvessel density (MVD) as prognostic markers in breast carcinoma. Forty breast carcinomas were immunostained for D2-40, CD31 and VEGF. Median lymphatic and blood microvessel densities, as well as VEGF expression, were related to each other and to clinicopathologic parameters including lymph node (LN) status. The efficacy of haematoxylin and eosin (H&E) in detecting lymphatic vessel invasion (LVI) compared to D2-40 immunostaining was also investigated. D2-40 stained normal lymphatic endothelium and myoepithelial cells, but with different staining patterns. D2-40 LVD related significantly to CD31 counts (r=0.470; p=0.002), and LN metastasis (Mann-Whitney U=101.500; p=0.043); however, it did not relate to age, tumor grade, tumor size or LVI. D2-40 identified LVI in 3 more cases (7.5%) than those detected by H&E. VEGF was expressed in 85%of cases, and was significantly related to CD31 and D2-40 counts (p=0.033 and 0.007, respectively). In conclusion, D2-40 LVD showed a significant association with LN metastasis, and can be considered to segregate patients with positive from those with negative LNs. D2-40 enhances the detection of LVI relative to H&E staining reflecting a potential for lymphatic metastatic spread and possible poor prognosis.     

Chalkley Microvessel but not Lymphatic Vessel DensityCorrelates with Axillary Lymph Node Metastasis in PrimaryBreast Cancers

This study aimed to investigate tumor microvessel density (MVD) and lymphatic vessel density (LVD) usingthe Chalkley method as predictive markers for the risk of axillary lymph node metastasis and their relationshipto other clinicopathological parameters in primary breast cancer cases. Forty two node-positive and eightynode-negative breast cancers were immunostained for CD34 and D2-40. MVD and LVD were counted by theChalkley method at x400 magnification. There was a positive significant correlation of the MVD with the tumorsize, coexisting ductal carcinoma in situ (DCIS) and lymph node metastases (P<0.05). In multivariate analysis,the MVD (2.86-4: OR 5.87 95%CI 1.05-32; >4: OR 20.03 95%CI 3.47-115.55), lymphovascular invasion (OR3.46, 95% CI 1.13-10.58), and associated DCIS (OR 3.1, 95%CI 1.04-9.23) independently predicted axillarylymph node metastasis. There was no significant relationship between LVD and axillary lymph node metastasis.However, D2-40 was a good lymphatic vessel marker to enhance the detection of lymphatic invasion compared toH and E staining. In conclusion, MVD by the Chalkley method, lymphovascular invasion and associated DCIScan be additional predictive factors for axillary lymph node metastases in breast cancer. No relationship wasidentified between LVD and clinicopathological variables, including axillary lymph node metastasis.     

Growth inhibition and differentiation of human breast cancer cells by the PAFR antagonist WEB-2086

WEB-2086 -- an antagonist of platelet-activating factor receptor (PAFR) with known anti-inflammatory, antiangiogenic and antileukaemic properties -- also proved to inhibit the proliferation in human solid tumour cell lines of different histology, and with much higher efficacy than in normal fibroblasts. A detailed analysis of WEB-2086 anticancer activity was then performed focusing on breast adenocarcinoma MCF-7 and MDA-MB-231 cells. WEB-2086-treated cells, either expressing (MCF-7) or unexpressing (MDA-MB-231) the oestrogen receptor (ER)alpha, underwent a dose-dependent growth arrest (IC(50)=0.65+/-0.09 and 0.41+/-0.07 mM, respectively) and accumulation in G(0)-G(1) phase. WEB-2086 also induced morphological and functional changes typical of mature mammary phenotype including (i) cell enlargement and massive neutral lipid deposition (best accomplished in MCF-7 cells); (ii) decrease in motility and active cathepsin D levels (mainly observed in highly invasive MDA-MB-231 cells). The expression of ERalpha was neither increased nor reactivated in treated MCF-7 or MDA-MB-231 cells, respectively. WEB-2086-induced differentiation in breast cancer cells involved the upregulation of PTEN, a key tumour suppressor protein opposing tumorigenesis, and was apparently independent of p53, PAFR, peripheral benzodiazepine receptor and ERalpha status. Overall, WEB-2086 can be proposed as an effective antiproliferative and differentiative agent with interesting translational opportunities to treat breast cancers in support to conventional chemotherapy.     

Genetic manipulation of stromal cell-derived factor-1 attests the pivotal role of the autocrine SDF-1-CXCR4 pathway in the aggressiveness of breast cancer cells

Stromal cell-derived factor-1 (SDF-1), via its receptor CXCR4, has been implicated in metastasis of cancer, including breast cancer. Exogenous SDF-1 is known to regulate locomotion, chemotaxis and adhesion. The knowledge regarding the effect of autocrine SDF-1 on breast cancer cells is not available. The current study evaluated the effects of SDF-1 on the biological behaviour of breast cancer cells by genetically modifying the expression of SDF-1 in breast cancer cells. Two human breast cancer cell lines (MDA-MB-231 and MDA-MB-435s) and a human fetal lung fibroblast cell line (MRC5) were used. The expression of SDF-1 and the SDF-1 receptor, CXCR4 in the cell lines were studied. Expression cassettes of human SDF-1 and hammerhead ribozyme transgenes specifically targeting human SDF-1 were constructed and used to over-express SDF-1 or to knockout the expression of SDF-1 in cancer cells, respectively. Invasiveness, migration and growth of the genetically modified cells were assessed. SDF-1 was expressed in wild-type human breast cancer cell line MDA-MB-435s and fibroblast cell line MRC5, but not in MDA-MB-231 cell line. In contrast, CXCR4 expression was observed in all three cell lines tested. The ability of invasion and migration was significantly reduced in SDF-1 knockout MDA-MB-435s cells, compared with wild-type and vector control cells (p<0.01). On the other hand, SDF-1 transfected MDA-MB-231epsilonSDF1+/+ cells that stably expressed SDF-1 showed a different behaviour from MDA-MB-231SDF1+/- (plasmid control) and wild-type MDA-MB-231 cells, both being SDF-1 negative. MDA-MB-231epsilonSDF1+/+ cells displayed a higher degree of invasiveness and migration, compared with wild-type and MDA-MB-231SDF+/- cells (p<0.01). Furthermore, SDF1-knockout MDA-MB-435s cells showed a slower growth rate over a 7-day period compared with the respective control and wild-type MDA-MB-435s cells. In contrast, the growth of the SDF-1 transfected MDA-MB-231SDF1+/+ cells was markedly enhanced when compared with wild-type and vector control cells. Breast cancer cell lines, when equipped with the autocrine SDF-1-CXCR4 signal pathway, display aggressive behaviour, including an increase in invasiveness, migration together with faster growth. SDF-1, together with its receptor CXCR4 may provide important information for predicting the aggressive nature and constitute important therapeutic targets in human breast cancer.     

乳腺癌干细胞富集与鉴定的初步研究

目的:通过从MCF-7、ZR-75-1、MDA-MB-231乳腺癌细胞系中培养富集及鉴定乳腺癌干细胞(breast cancer stem cell,BCSC),寻找培养与富集乳腺癌干细胞的方法。方法:贴壁培养MCF-7、ZR-75-1、MDA-MB-231细胞系,倒置显微镜观察各细胞形态;流式细胞仪分别分选收集CD44-CD24-、CD44-CD24+、CD44+CD24-及 CD44+CD24+ 细胞,其中CD44+CD24-为乳腺癌干细胞,其余三类为对照组;MTT法计数细胞,绘制MCF-7、ZR-75-1、MDA-MB-231细胞系生长曲线;MCF-7细胞系进行无血清悬浮培养1个周期,流式细胞仪检测分子表面标记物CD44+CD24-含量,贴壁培养的CD44+CD24-乳腺癌干细胞为对照组;将分选的MCF-7（CD44+CD24-）和分选的其余MCF-7细胞（非CD44+CD24-）进行干性成球实验,鉴定CD44+CD24-干性表达。结果:MCF-7、MDA-MB-231细胞系富含表面标志物CD44-CD24-的乳腺癌细胞;ZR-75-1细胞系富含分子表面标志物CD44+CD24+的乳腺癌细胞;生长曲线显示MCF-7、ZR-75-1、MDA-MB-231均呈持续增长,MDA-MB-231细胞生长较MCF-7、ZR-75-1细胞快;通过无血清悬浮培养CD44+CD24-乳腺癌干细胞由19.4%富集到88.9%;成球实验中CD44+CD24-表型细胞成球数量较分选的其余MCF-7细胞（非CD44+CD24-表型）明显增多,成球率分别为（36.5±1.7）%,（1.1±0.5）%。结论:流式细胞仪可成功分选出分子表面标志物为CD44+CD24-的乳腺癌干细胞;CD44+CD24-可能不是乳腺癌干细胞唯一的表面标志物;MDA-MB-231细胞系较MCF-7、ZR-75-1细胞系生长快;无血清悬浮培养法可简便、高效地富集乳腺癌干细胞;CD44+CD24-乳腺癌干细胞干性表达较强。     

沉默CD44表达对乳腺癌细胞MDA-MB-231增殖与侵袭的影响

目的:探索乳腺癌细胞MDA-MB-231及MCF-7中CD44分子的表达水平差异及沉默CD44对乳腺癌细胞MDA-MB-231增殖、侵袭和迁移的影响。方法:利用qRT-PCR及Western blot技术检测细胞中CD44基因表达水平;设计并合成CD44的siRNA片段(CD44-siRNA)转染乳腺癌细胞,利用qRT-PCR、Western blot技术检测细胞中CD44基因表达水平的变化;MTT检测MDA-MB-231细胞增殖;Transwell侵袭实验检测MDA-MB-231细胞的迁移与侵袭能力变化。结果:CD44在侵袭性乳腺癌细胞MDA-MB-231中的表达高于非侵袭性乳腺癌细胞MCF-7,CD44-siRNA下调了 MDA-MB-231细胞中CD44 mRNA与蛋白水平的表达,并抑制了细胞的增殖和侵袭转移能力。结论:CD44-siRNA能够下调CD44的表达,并有效抑制乳腺癌细胞MDA-MB-231的增殖及其侵袭迁移力。     

Expression of CD34 in gastric cancer and its correlation with histology, stage, proliferation activity, p53 expression and apoptotic index

The formation of new blood vessels is essential for tumor growth and progression. Until today there are only few studies of the immunohistochemical assessment of angiogenesis in gastric cancer by the evaluation of the expression of CD34 antigen. The aim of this study was to analyze the relationship between microvessel density (MVD) expressed as the mean count of CD34 immunostained vessels and clinicopathologic features of gastric tumors (the histological type according to the Lauren classification, tumor grade G; presence of lymph node metastases N; depth of tumor invasion; stage of disease (UICC-AJCC 1988 1992), p53 expression, tumor cell proliferative activity described as the Ki67 labelling index and apoptotic index of tumor cells TUNEL method). We assessed formalin-fixed, paraffin-embedded tissue samples obtained during potentially radical gastrectomy from 58 patients with primary gastric adenocarcinoma. The representative tissue blocks from each tumor were used for the immunohistochemical assay and examined by two pathologists independently. MVD was counted in five tumor areas of the most intensive neovascularization (x 200 field by light microscopy) and the mean counts were recorded. The mean MVD (CD34 expression value+/-SD) in this study was 43,15+/-19,8 per x 200 field. The study demonstrated the statistically significant correlation between MVD and two main histological parameters: tumor grading (p < 0.001) and tumor histological type according to Lauren s classification (p<0.05). In well and moderately differentiated tumors (G1/2) MVD was significantly lower in comparison to the group of poorly differentiated cancer G3 (mean value: 31,62 vs. 49,89). MVD was higher in diffuse type of gastric cancer comparing to intestinal type (50.05+/-19,03 vs. 39.17+/-20,09). However, the authors failed to find a significant correlation between MVD and other investigated histopathological features in malignant gastric tumors. The close relationship between CD34 immunostaining, gastric cancer tumor vascularity and main histological parameters was shown in this study. It can be stated that analysis of expression of angiogenesis in gastric cancer may be helpful for better estimation of hematogenous recurrence and the selection of the group of patients for adjuvant antiangiogenic treatment.     

外源性TGFBI抑制乳腺癌细胞生长的体内外实验

目的 研究转化生长因子β诱导基因 (transforming growth factor-β induced gene, TGFBI) 是否能抑制人乳腺癌细胞株(MDA-MB-231)的体内外增生。方法 将外源性TGFBI稳定转染到人乳腺癌细胞株(MDA-MB-231)中,测定细胞增殖率、细胞周期、软琼脂克隆形成率及P21、P53蛋白表达变化,检测乳腺癌细胞的致瘤性。结果 外源性TGFBI在人乳腺癌细胞株(MDA-MB-231)中能够稳定地高表达;外源性TGFBI可以明显地抑制乳腺癌细胞因血清生长因子刺激的增生;与转染空质粒的对照组细胞V23101比较,外源性TGFBI相对软琼脂克隆形成数减少了90.89%; TGFBI可以使人乳腺癌细胞阻滞在G1期,延缓其进入S期的时间。外源性TGFBI可以延长裸鼠肿瘤发生的潜伏期,并降低肿瘤发生率。结论 TGFBI能够抑制人乳腺癌细胞株(MDA-MB-231)的体内外增生。     

顺铂耐药乳腺癌细胞株的建立及FANCF 基因在耐药中的作用

[摘要] 目的：探讨三阴性乳腺癌顺铂（cisplatin, DDP)耐药细胞和敏感细胞中FA/BRCA通路关键基因FANCF的表达和功能,以及与DDP耐药的相关性。方法：DDP浓度递增法诱导建立乳腺癌细胞MDA-MB-231的DDP耐药细胞株MDA-MB-231/DDP;通过RNAi技术敲减MDA-MB-231 敏感细胞和DDP耐药细胞中FANCF,并在mRNA和蛋白水平进行敲减效果验证。CCK-8 法检测DDP耐药细胞株增殖活性,Western blotting 法检测该细胞中FANCF蛋白表达,流式细胞仪检测MDA-MB-231细胞周期和凋亡情况,实时定量PCR（RT-qPCR）法检测FANCF mRNA的表达。结果：MDA-MB-231细胞DDP诱导3个月建立的MDA-MB-231/DDP细胞株耐药指数为13.5,其G0/G1 期细胞增多、S期和G2/M期细胞减少。MDA-MB-231/DDP细胞中FANCF mRNA和蛋白表达水平显著升高（均P<0.01）。FANCF敲低后MDA-MB-231/DDP细胞凋亡增加,细胞对DDP的药物敏感性显著升高（均P<0.01）。结论：FANCF基因通过抗凋亡作用导致MDA-MB-231细胞对DDP的耐药性,FANCF是乳腺癌治疗的一个潜在靶点。     

Quantification of the heterogeneity in breast cancer cell lines using whole-cell impedance spectroscopy.

PURPOSE: Quantification of the heterogeneity of tumor cell populations is of interest for many diagnostic and therapeutic applications, including determining the cancerous stage of tumors. We attempted to differentiate human breast cancer cell lines from different pathologic stages and compare that with a normal human breast tissue cell line by characterizing the impedance properties of each cell line. EXPERIMENTAL DESIGN: A microelectrical impedance spectroscopy system has been developed that can trap a single cell into an analysis cavity and measure the electrical impedance of the captured cell over a frequency range from 100 Hz to 3.0 MHz. Normal human breast tissue cell line MCF-10A, early-stage breast cancer cell line MCF-7, invasive human breast cancer cell line MDA-MB-231, and metastasized human breast cancer cell line MDA-MB-435 were used. RESULTS: The whole-cell impedance signatures show a clear difference between each cell line in both magnitude and phase of the electrical impedance. The membrane capacitance calculated from the impedance data was 1.94 +/- 0.14, 1.86 +/- 0.11, 1.63 +/- 0.17, and 1.57 +/- 0.12 muF/cm(2) at 100 kHz for MCF-10A, MCF-7, MDA-MB-231, and MDA-MB-435, respectively. The calculated resistance for each cancer cell line at 100 kHz was 24.8 +/- 1.05, 24.8 +/- 0.93, 24.9 +/- 1.12, and 26.2 +/- 1.07 MOhm, respectively. The decrease in capacitances of the cancer cell lines compared with that of the normal cell line MCF-10A was 4.1%, 16.0%, and 19.1%, respectively, at 100 kHz. CONCLUSIONS: These findings suggest that microelectrical impedance spectroscopy might find application as a method for quantifying progression of cancer cells without the need for tagging or modifying the sampled cells.