Tamoxifen induction of angiogenic factor expression in endometrium

Tamoxifen is the current therapy of choice for patients with oestrogen receptor positive breast cancer, and it is currently under evaluation as a prophylactic for women at high risk of developing the disease. However, tamoxifen is also known to induce proliferative changes in the endometrium increasing the risk of developing endometrial hyperplasia, polyps and carcinoma. Angiogenesis is an intimate part of this process. For this reason, we have examined the expression of several well characterized angiogenic factors, namely, acidic and basic fibroblast growth factor, thymidine phosphorylase, vascular endothelial growth factor and adrenomedullin in both normal and tamoxifen exposed pre- and postmenopausal endometrium. Vascular density and endothelial proliferation index were also quantified. We found increased expression of acidic and basic fibroblast growth factor and adrenomedullin after treatment with tamoxifen mainly in premenopausal tissue. Vascular density was significantly increased in pre- but not post-menopausal endometrium (P=0.0018) following tamoxifen treatment. These results support the notion that angiogenesis is integral to the response to tamoxifen exposure, and is a potential target with which to block these side effects of tamoxifen.     

Macrophage migration inhibitory factor: roles in regulating tumor cell migration and expression of angiogenic factors in hepatocellular carcinoma

Macrophage migration inhibitory factor (MIF) may contribute to multiple aspects of tumor progression, including control of cell proliferation, differentiation, cell survival and angiogenesis. However, the potential roles of MIF in regulating hepatocellular carcinoma (HCC) tumor cell migration and the expression of angiogenic factors by HCC tumor cells have not been studied yet. In our study, we reported that intracellular MIF mRNA and protein were overexpressed in HCC tissues compared to nontumor tissues by using in situ hybridization and immunohistochemic staining. HCC tumor cell lines also secreted large amounts of MIF into the supernatants of tumor cell culture. To assess the role of MIF in HCC, we employed the transwell invasion chamber to study the effect of MIF on tumor cell migration. Our results showed that recombinant MIF and the supernatants of tumor cell line culture could enhance the invasion and migration of HCC cells. This effect can be inhibited by the addition of a neutralizing anti-MIF antibody. We observed that increased MIF serum levels correlated with higher levels of interleukin-8 (IL-8) in the sera of patients with HCC than in normal volunteers. We therefore hypothesized that MIF may regulate the production of angiogenic factors by HCC cells. To test this hypothesis, we examined the effect of MIF treatment on vascular endothelial growth factor (VEGF) and IL-8 expression by HCC cell lines. MIF induced a significant dose-dependent increase in IL-8 and VEGF production. Taken together, our results indicated that MIF may act as an autocrine-acting factor that stimulates angiogenesis and metastasis in HCC by promoting expression of angiogenic factors and migration of tumor cells. A more detailed understanding of the MIF regulatory mechanisms involved may provide insight into new direction in the treatment of HCC.     

An in vitro model to optimize dose scheduling of multimodal radioimmunotherapy and chemotherapy: effects of p53 expression

Several reports have appeared on the use of combined radioimmunotherapy (RAIT) and chemotherapy. The choice of drug to use with RAIT and how to space the two treatments has not been completely addressed. Because every patient's cancer presents with a specific molecular phenotype, we hypothesized that it may be necessary to tailor therapy based on specific gene expression. We addressed how the form of expression of a single gene, the p53 tumor suppressor, would impact the choice of agents, as well as sequence and spacing of agents. p53 regulates cell cycle arrest to allow for DNA repair after therapy-induced small DNA damage or induction of apoptosis if damage is great and has been shown to affect chemo- and radiosensitivity of cancer cells. We established 3 stable p53 transfectants of the SKOV-3 p53null parental line (p53(wt), p53(143mut) or p53(273mut)). p53 expression was confirmed using flow cytometry, using the DO1 pan-p53 Ab and the PAb240 anti-p53mut Ab. The colorimetric MTT assay was then used to measure dose-dependent growth inhibition from single modality chemotherapy (doxorubicin, carboplatin, paclitaxel or topotecan) or radioimmunotherapy (90Y-RS-7 IgG anti-EGP1). The % survival vs. log [drug] were plotted to obtain the IC50. We then used a matrix design in which we varied the sequence of the first and second modality of treatment and the spacing between the 2 treatments to determine the most synergistic and antagonistic combinations for the parental SKOV-3 and each of the 3 transfectants. The IC50 for each therapeutic agent varied as a function of the form of p53 expressed. For example, of the 4 lines, the p53wt transfectant was the most resistant to topotecan and the 143mut was the most resistant to carboplatin. The 273mut was quite sensitive to both doxorubicin and paclitaxel, whereas the p53null and wt were not. For multimodal treatments, most combinations of RAIT and chemotherapy resulted in a 30-40% growth inhibition (GI) and were either additive or moderately antagonistic. The 3 best (>60% GI) and 3 worst (<25% GI) combinations were identified and were unique to the parental p53null and to the 3 transfectants. Certain combinations showed clear synergy and others were antagonistic, with the first treatment modality blocking the growth inhibitory effects of the second treatment modality. The form of p53 expressed affects chemosensitivity and radiosensitivity and will influence optimal multimodal therapy with RAIT and chemotherapy and the dose-schedule (sequential with RAIT first or with drug first) when more than 1 agent is used.     

化疗后残余胃癌细胞发生上皮间质转化的体外研究

  目的  观察体外化疗能否诱导胃癌细胞发生上皮间质转化（epithelial mesenchymal transition,EMT）。   方法  使用5-Fu（30 μg/mL）对胃癌细胞株SGC7901进行4个疗程的体外化疗,残余细胞继续培养,得到能稳定传代的细胞SGC7901/Fu。比较SGC7901及SGC7901/Fu在细胞形态、EMT标记物、化疗耐药性、侵袭能力、肿瘤干细胞特性等方面的差异。   结果   与SGC7901比较,SGC7901/Fu呈间质细胞形态、上皮表型标记物表达下调、间质表型标记物表达上调。在SGC7901细胞及SGC7901/Fu细胞中,5-Fu的中效浓度（IC₅₀）分别为（43.8±7.2）μg/mL及（64.6±5.5）μg/mL,穿过Transwell小室基底膜的细胞数分别为（51.4±8.7）个及（93.2±9.5）个,克隆形成率分别为5.2%±1.0%及13.2%±2.2%,CD44+/CD24-细胞亚群所占比例分别为4.13%±0.81%及7.97%± 0.50%,两者间的差异均具有统计学意义（P<0.05）。   结论  体外化疗后残余的胃癌细胞发生EMT,同时细胞侵袭能力增强、化疗耐药性升高,并获得了肿瘤干细胞特性。      

Variant generation and selection: An In vitro model of tumor progression

Evidence for a new in vitro model of tumor progression was sought on the basis of the variant generation and selection hypothesis. The stability of a cloned murine tumor was examined during growth in standard tissue culture or in media containing the tumor promoter 12–0-tetradecanoylphorbol-13-acetate (TPA). Analysis of subclones from the appropriate tumor populations revealed that growth of the L5178Y-F9 clone in 100 ng/ml TPA and 0.1% dimethyl sulphoxide (DMSO) for 2 days yielded a tumor which exhibited increased cellular heterogeneity for susceptibility to both syngeneic and allogeneic natural antibodies (NAb). Subsequent exposure of TPA- and DMSO-treated cells to two cycles of syngeneic NAb-mediated cytolysis resulted in tumor populations which expressed a reduced sensitivity to syngeneic NAb. Thus the elements of tumor variant generation and selection were demonstrated by means of this approach, and repeated cycles of the TPA treatment and NAb cytolysis produced tumor cells with a reduced susceptibility not only to NAb in vitro but also to anti-tumor natural resistance (NR) measured in a tumor elimination assay in vivo. These observations extend the support for the notion that tumor progression can proceed through variant generation and selection. Furthermore, the association of tumor variant generation with exposure to the combination of TPA and DMSO, both non-mutagens, offers a model for studying non-mutagenic mechanisms of tumor development.     

化疗对乳腺癌患者血清血管生成调节因子水平的影响

目的探讨乳腺癌患者化疗期间血清血管内皮生长因子(VEGF)和内皮细胞抑制素(ES)水平的变化及其与疗效的关系。方法收集40例转移性乳腺癌患者化疗前、化疗1个周期和5—6个周期后的系列血清标本120份,采用酶联免疫吸附试验(EUSA)检测VEGF和ES水平;同时采用该方法检测血清可溶性血管细胞黏附因子-1(VCAM-1)水平。结果(1)化疗前,乳腺癌患者血清VEGF中位水平为496.6 pg/ml,是健康对照组的4.7倍(P<0.001);ES中位水平为95.5 ng/ml,比健康对照组低18.3%(P=0.183);VCAM-1中位水平为1077.1 ng/ml,较健康对照组明显增高(P< 0.001)。化疗前VEGF与血清VCAM-1水平、疾病分期和转移部位相关(P<0.05)。(2)化疗1个周期后,乳腺癌患者血清VEGF中位水平为524.8 pg/ml,较化疗前增高(P=0.047);ES中位水平为110.5 ng/ml,与化疗前差异无统计学意义(P=0.055);VCAM-1中位水平为975.6 ng/ml,与化疗前差异亦无统计学意义(P=0.27)。(3)化疗5—6个周期后,乳腺癌患者血清VEGF中位水平为306.5 pg/ml,较化疗前、化疗1个周期后明显下降(P值分别为0.009和0.005);ES中位水平为113.3 ng/ml,比化疗前明显增高(P=0.042),但与化疗1个周期差异无统计学意义(P>0.05)。血清VEGF水平的变化与疗效相关。病情稳定或缓解的27例乳腺癌患者,VEGF均出现不同程度下降, 13例病情进展者无VEGF下降;VCAM-1水平也出现了与VEGF类似的治疗相关反应;而ES水平与疗效无关(P>0.05),提示化疗对ES水平影响可能较小。结论乳腺癌全身化疗明显影响血清VEGF水平,VEGF下降可能是疾病得到控制的一个指标;随着治疗后病情的稳定或缓解,肿瘤血管生成具有向着抗血管生成活性状态发展的趋势。     

Metastasis suppressor CC3 inhibits angiogenic properties of tumor cells in vitro

Resistance to apoptosis and ability to promote angiogenesis are integral features of the metastatic phenotype. Human gene CC3 is a metastasis suppressor for variant small cell lung carcinoma and a mouse melanoma in vivo. We have shown previously that metastasis-suppressing function of CC3 might be due at least in part to the ability of CC3 protein to predispose tumor cells to apoptosis. Here we demonstrate that CC3 has a previously unidentified effect on the ability of tumor cells to induce angiogenesis in vitro. Expression of CC3 in three different tumor cell lines significantly diminished their angiogenic character as manifested in the in vitro proliferation and migration assays with endothelial cells of both macro- and microvascular origin. Expression of CC3 induced changes in RNA levels of several angiogenic modulators consistent with the overall reduction in angiogenic properties. These results indicate that expression of CC3 has a dual effect on phenotype of tumor cells ultimately inhibiting their metastatic potential.     

基于共培养技术的肿瘤微血管细胞模型的建立

目的建立可研究肿瘤微血管的分子学特性及结构特性的细胞模型,应用于药物筛选及靶向药物输送的研究.方法通过TRANSWELL共培养肿瘤细胞MCF-7和内皮细胞HUVEC的方法,建立肿瘤微血管细胞模型,检测了该模型下的细胞形态、结构、及通透性的变化.结果在肿瘤细胞共生条件下的内皮细胞层结构疏松,微环境呈明显酸性,而且细胞间隙增大,对100纳米左右粒子的通透性有显著提高.结论这一细胞模型的建立将有助于肿瘤血管的分子、结构特性以及相关药物的筛选和输送研究.     

Combination chemotherapy in a prostate tumor model: Nb rat prostatic adenocarcinoma model

This report presents the experience with single and combination chemotherapy in the Noble rat prostatic adenocarcinoma model. The best single agent in treatment of these tumors is cyclophosphamide, the best combination was triple drug therapy cyclophosphamide, cis-plati-num, and adriamycin. This report examines the effect of the various chemotherapeutic regimens on tumor volume, number of metastasis, and complete tumor regression.     

Identification of angiogenic properties of insulin-like growth factor II in in vitro angiogenesis models

Insulin-like growth factor II (IGF-II), highly expressed in a number of human tumours, has been recently known to promote neovascularization in vivo. Yet, the detailed mechanism by which IGF-II induces angiogenesis has not been well defined. In the present study, we explored an angiogenic activity of IGF-II in in vitro angiogenesis model. Human umbilical vein endothelial cells (HUVECs) treated with IGF-II rapidly aligned and formed a capillary-like network on Matrigel. In chemotaxis assay, IGF-II remarkably increased migration of HUVECs. A rapid and transient activation of p38 mitogen-activated protein kinase (p38 MAPK) and p125 focal adhesion kinase (p125FAK) phosphorylation was detected in HUVECs exposed to IGF-II. IGF-II also stimulated invasion of HUVECs through a polycarbonate filter coated with Matrigel. Quantitative gelatin-based zymography identified that matrix metalloproteinase-2 (MMP-2) activity generated from HUVECs was increased by IGF-II. This induction of MMP-2 activity was correlated with Northern blot analysis, showing in HUVECs that IGF-II increased the expression of MMP-2 mRNA, while it did not affect that of TIMP-2, a tissue inhibitor of MMP-2. These results provide the evidence that IGF-II directly induces angiogenesis by stimulating migration and morphological differentiation of endothelial cells, and suggest that IGF-II may play a crucial role in the progression of tumorigenesis by promoting the deleterious neovascularization.     

术前化疗对乳腺癌组织VEGF表达和血管生成的影响

目的研究术前化疗对乳腺癌血管内皮生长因子(VEGF)表达、肿瘤组织微血管密度的影响.方法36例术前化疗的乳腺癌和32例未做术前化疗的乳腺癌标本,进行VEGF、CD34免疫组织化学标记,并进行微血管计数和统计学处理.结果术前化疗组VEGF阳性率为47.22%(17/36),对照组53.13%(17/32),微血管计数化疗组和对照组分别为23.67±13.45,26.12±11.32,两者差异均无显著性.化疗组VEGF阳性和阴性的标本,MVC均值差异无显著性;对照组VEGF阳性和阴性的标本,MVC均值差异有显著性.结论常规术前化疗并未显示出明显的血管生成抑制作用,也未显示出明显的血管内皮生长因子表达的改变.     

Adjuvant chemotherapy after cystectomy of bladder tumor

Adjuvant chemotherapy mainly consisting of cisplatin, adriamycin and mitomycin C was administered to 17 patients after cystectomy for bladder tumor (chemotherapy group). Seventeen concurrently treated patients did not receive adjuvant chemotherapy (control group). From the comparison of survival curves of these two groups, the following results were obtained. 1) Survival curves of the patients in all stages did not differ significantly between chemotherapy and control groups. 2) Among patients in stage pT3, pT4 and/or N+, survival of the chemotherapy group seemed to have some advantage over that of the control group, but the survival curves did not differ significantly between these two groups. 3) Among patients in N+, survival of the chemotherapy group was far better than that of the usual cases. Review of the literature on adjuvant chemotherapy after cystectomy for bladder tumor revealed the necessity of randomized study to determine whether adjuvant chemotherapy is effective.