沉默激酶功能区受体抑制前列腺癌PC-3细胞的裸鼠体内成瘤能力

目的 研究激酶功能区受体(KDR)基因表达沉默后对前列腺癌PC-3细胞裸鼠体内成瘤能力的影响.方法 将15只5周龄BALB/c雄性裸鼠随机分为干扰组、阴性质粒组和未转染组,每组5只.分别接种构建的pSilencer 3.1-KDR siRNA表达质粒转染PC-3细胞、阴性对照质粒pSilencer3.1-NC转染PC-3细胞以及未转染的PC-3细胞于裸鼠皮下;观察各组PC-3细胞在裸鼠体内成瘤率、瘤体生长速度以及平均瘤质量等方面的变化,RT-PCR和Western blot技术检测瘤体KDR基因和蛋白表达.结果 pSilencer3.1KDR质粒转染组的裸鼠瘤体生长速度明显慢于未转染组和pSilencer3.1-NC质粒转染组;与未转染组和PSilencer3.1-NC组相比,pSilencer3.1-KDR组肿瘤的生长受到明显抑制,平均体积较小(0.28 cm3 vs 0.721 cm3,0.715 cm3,P＜0.01),平均瘤质量较轻(0.14g vs 0.648g,0.635g,P＜0.01);裸鼠肿瘤组织中KDR mRNA和蛋白的表达明显降低.结论 RNAi介导的KDR基因沉默可显著影响PC-3细胞裸鼠体内的瘤体生长速度,KDR有可能成为肿瘤治疗的新靶点.     

双氢青蒿素诱导人前列腺癌细胞系PC-3凋亡

目的探讨双氢青蒿素(dihydroartemisinin,DHA)对人前列腺癌细胞系PC-3的凋亡诱导作用,并探讨其可能机制。方法人前列腺癌PC-3细胞经不同浓度(0、25、50和100μmol/L)DHA处理48 h,用FCM法检测各组细胞凋亡率。用荧光定量PCR检测细胞中HSP70 mRNA的表达。用蛋白质印迹法检测细胞中HSP70蛋白、凋亡酶激活因子(Apaf-1)及caspase-3的表达;荧光定量PCR及蛋白质印迹法增加两组,即100μmol/L HSP70抑制剂槲皮素(quercetin)作为阳性药物对照组,以DMSO作为溶剂对照组。结果 DHA能明显诱导PC-3细胞凋亡(P0.05)。不同浓度DHA能明显下调HSP70 mRNA及蛋白表达水平(P0.05),上调Apaf-1及caspase-3蛋白表达水平(P0.05)。结论双氢青蒿素能诱导前列腺癌PC-3细胞凋亡,其作用机制可能是DHA干扰HSP70的表达,促进caspase信号通路中Apaf-1及caspase-3表达。     

Development of skeletal metastasis by human prostate cancer in athymic nude mice

The biology of skeletal metastasis is poorly understood. In order to establish an animal model of bone metastasis, cells from a human prostate cancer cell line (PC-3) were injected into the tail veins of athymic nude mice while the inferior vena cava was occluded. This technique was used in order to divert cells into the vertebral venous plexus. A control group of animals received tumor cells without caval occlusion. Bone lesions developed in 3/16 (19 per cent) experimental mice and in none of the control mice. The incidence of lung metastasis was significantly decreased in the experimental mice (5/16) as compared with non-occluded control mice (14/16). Two tumor sublines were established from explant cultures of bone lesions. Injection of these cells resulted in bone metastasis in 19/36 (53 per cent) mice (P=0.03 compared with the parent line). The incidence of lung lesions was also increased. The predominant site of bone metastasis was the lumbar vertebrae; other affected sites were the pelvis and femurs. All bone lesions resulted in extensive bone destruction. The successful development of bone metastasis using the technique of caval occlusion lends support to the hypothesis that entry of cells into the vertebral circulation is an important step in the development of these lesions. This model should be of value in understanding the pathogenesis of bone metastasis, and in studying the effects of various agents on the prevention and control of these lesions.     

Virus carrier state suppresses tumorigenicity of tumor cells in athymic (nude) mice.

Nude mice injected subcutaneously with normal uninfected BHK 21 cells or HeLa cells regularly develop large, rapidly-growing tumours at the subcutaneous site of inoculation. However, these same tumour cell lines when persistently infected with VSV or other enveloped RNA viruses are either rejected or form small nodules in nude mice. This rejection phenomenon probably involves some type of immunocyte since heavily-irradiated nude mice (500 rads) cannot reject persistently infected cells but develop large, rapidly-growing tumours which shed virus and defective interfering virus (DI) and which do not exhibit the lymphocytic infiltration observed in the nodules of unirradiated mice given persistently infected cells. Finally, it was possible to select a subline of BHK 21-VSV carrier cells which regularly produces large rapidly-growing tumours in normal unirradiated nude mice, although all these carrier cells express virus antigen and shed large amounts of mature infectious virus and DI both in vivo and in vitro.     

Suppression of tumorigenicity in the human prostate cancer cell line M12 via microcell-mediated restoration of chromosome 19

Previously we immortalized human, nontransformed prostate epithelial cells with SV40 large T-antigen (SV40TAg) and derived increasingly aggressive sublines from the immortalized line. The progression of the tumorigenic sublines to metastatic capacity was accompanied by the formation of an unbalanced translocation between chromosomes 16 and 19, resulting in loss of 19p and proximal 19q. To test whether the tumorigenic and/or metastatic phenotype was causally related to this genetic alteration, we restored a neo-tagged human chromosome 19 to M12 cells by microcell-mediated transfer and assessed their growth. In vitro, the resultant hybrids grew more slowly in monolayer culture and showed a significant reduction in anchorage-independent growth as compared to M12neo controls. In vivo, all mice (13/13) injected subcutaneously (SC) with control M12neo cells developed tumors after 9-15 days. In contrast, 9/15 mice injected SC with microcell-transferred chromosome 19 hybrid cells failed to form tumors, with 6/15 producing very small tumors after 120 days. Analysis of three of these six tumors showed consistent, new chromosomal changes. Furthermore, in one of the tumors, loss of a chromosome 19 was noted in 40% of the cells. After intraprostatic injections of the hybrid cells, only 2/7 mice developed microscopic tumors, with no metastases. These data suggest the presence of a gene or genes on chromosome 19 that function to suppress growth.     

Chromosome 18 suppresses the tumorigenicity of prostate cancer cells

Microcell-mediated chromosome transfer allows for the introduction of normal chromosomes into tumor cells in an effort to identify putative tumor suppressor genes. We have used this approach to introduce an intact copy of chromosome 18 into the prostate cancer cell line DU145, and independently to introduce human chromosomes 8 and 18 into the prostate cancer cell line TSU-PR1. Introduction of an extra copy of human chromosome 8 had no effect on the growth properties in vitro or the tumorigenicity in vivo of TSU-PR1 cells. However, microcell hybrids containing an introduced copy of human chromosome 18 exhibited a longer population doubling time, retarded growth in soft agar, and slowed tumor growth in athymic nude mice. These experiments provide functional evidence for the presence of one or more tumor suppressor genes on human chromosome 18 that are involved in prostate cancer.     

雌激素受体2对前列腺癌细胞系PC-3M增殖的影响

目的： 构建带有人雌激素受体2（ESR2）全长cDNA的重组质粒pcDNA3.1-hERβ,转染人前列腺癌PC-3M细胞株,观察ESR2对细胞增殖能力的影响。 方法：RT-PCR方法从人正常卵巢组织中获取ESR2全长cDNA,利用基因重组技术与真核表达载体pcDNA3.1连接,构建重组质粒pcDNA3.1-hERβ;瞬时转染前列腺癌PC-3M细胞株,应用细胞计数、MTT法及流式细胞术检测细胞增殖能力的改变;半定量RT-PCR法及Western blotting法分别检测增殖相关基因cyclinD1和 P21Cip1 mRNA及蛋白表达。 结果：DNA测序结果显示扩增的ESR2序列与GenBank（NM_001437）所公布的序列完全一致。重组质粒pcDNA3.1-hERβ瞬时转染人PC-3M细胞48 h后,与质粒对照组相比：RT-PCR法及Western blotting法显示,ESR2基因在mRNA水平和蛋白水平的表达均明显增加;细胞计数及MTT结果发现,细胞数目减少,细胞增殖活性下降（P<0.01）;流式细胞实验显示,G0/G1期细胞比例增加（P<0.05）,S期及G2/M期细胞比例减少（P<0.05）;RT-PCR及Western blotting结果还发现cyclinD1的表达减弱,而P21Cip1的表达增强。 结论：成功构建pcDNA3.1-hERβ重组质粒;带有ESR2全长基因的重组质粒转染PC-3M细胞后,细胞的增殖活性受到抑制;RSR2可能通过影响细胞增殖相关基因cyclinD1和 P21Cip1的表达抑制细胞的增殖。     

Dietary stearate reduces human breast cancer metastasis burden in athymic nude mice

Stearate is an 18-carbon saturated fatty acid found in many foods in the western diet, including beef and chocolate. Stearate has been shown to have anti-cancer properties during early stages of neoplastic progression. However, previous studies have not investigated the effect of dietary stearate on breast cancer metastasis. In this study, we present evidence that exogenously supplied dietary stearate dramatically reduces the size of tumors that formed from injected human breast cancer cells within the mammary fat pads of athymic nude mice by approximately 50% and partially inhibits breast cancer cell metastasis burden in the lungs in this mouse model system. This metastatic inhibition appears to be independent of primary tumor size, as stearate fed animals that had primary tumors comparable in size to littermates fed either a safflower oil enriched diet or a low fat diet had reduced lung metastasis. Also stearate fed mice sub-groups had different primary tumor sizes but no difference in metastasis. This anti-metastasis effect may be due, at least in part, to the ability of stearate to induce apoptosis in these human breast cancer cells. Overall, this study suggests the possibility of dietary manipulation with selected long-chain saturated fatty acids such as stearate as a potential adjuvant therapeutic strategy for breast cancer patients wishing to maximize the suppression of metastatic disease.     

Suppression of tumorigenicity in human ovarian carcinoma cell line SKOV-3 by microcell-mediated transfer of chromosome 11

To determine the pathogenic role of chromosomes 11 and 17 in the carcinogenesis of human ovarian cancers, neo(R)-tagged chromosome 11 or 17 was transferred from cell lines A9H11 or A9H17, respectively, into the ovarian carcinoma cell line SKOV-3 using microcell-mediated chromosome transfer. The chromosome transfer was verified by polymerase chain reaction detection of the neo(R) gene, fluorescence in situ hybridization detection of an extra chromosome 11, and microsatellite polymorphism detection of an exogeneous chromosome 11. Five SKOV-3/A9H11 hybrids and five SKOV-3/A9H17 hybrid clones were generated. For the chromosome 11 transfer, complete suppression of tumorigenicity was observed in four clones, (11)9-8 and 11(H)7-2, 11(H)8-3, and 11(H)7-2, 100 days post implantation. For the chromosome 17 transfer, no complete suppression of tumorigenicity was observed. However, an increased latency period ranging from 25 to 49 days in contrast to 7 days for the SKOV-3 parental line, and a significant reduction in tumor size was observed. There was no correlation between the in vitro growth rate and the tumorigenicity or length of latency period. Our results demonstrate functionally that chromosome 11 may carry a tumor suppressor gene(s) while chromosome 17 may carry a tumor growth-inhibitor gene(s) for the ovarian carcinoma cell line, SKOV-3.     

E2F陷阱DNA对前列腺癌PC-3M细胞凋亡的诱导

目的：为观察转录因子E2F陷阱DNA 对雄激素非依赖性前列腺癌细胞PC-3M增殖和凋亡的影响。方法：采用脂质体lipofectamine将E2F decoy DNA、ARE decoy DNA和control decoy DNA分别转染PC-3M细胞，MTT检测其对细胞增生的影响，倒置相差显微镜观察细胞形态变化，流式细胞术检测细胞凋亡率，并进行染色体DNA断裂的测定；通过RT-PCR检测转染的PC-3M细胞中c-Myc mRNA、cyclin D1 mRNA表达水平的变化，通过Western blotting检测细胞中c-Myc蛋白、cyclin D1蛋白表达水平的变化。结果：E2F decoy DNA转染后的PC-3M细胞的生长受到明显抑制;转染后的细胞形态变化符合凋亡的典型改变，染色体断裂明显,细胞凋亡率为26.35%；c-Myc、cyclin D1的表达受到抑制。结论：E2F decoy DNA可诱导雄激素非依赖性前列腺癌PC-3M凋亡和抑制细胞增殖,其机制可能涉及到c-Myc mRNA、cyclin D1的表达变化。     

Rho GTPases in PC-3 prostate cancer cell morphology,invasion and tumor cell diapedesis

BACKGROUND: The Rho GTPases comprise one of the eight subfamilies of the Ras superfamily of monomeric GTP-binding proteins and are involved in cytoskeletal organization. Previously, using a dominant negative construct, we demonstrated a role for RhoC GTPase in conferring invasive capabilities to PC-3 human prostate cancer cells. Further, we demonstrated that inactivation of RhoC led to morphological changes commensurate with epithelial to mesenchymal transition (EMT) and was accompanied by increased random, linear motility and decreased directed migration and invasion. EMT was related positively to sustained expression and activity of Rac GTPase. In the current study we analyze the individual roles of RhoA, RhoC and Rac1 GTPases in PC-3 cell directed migration, invasion and tumor cell diapedesis across a human bone marrow endothelial cell layer in vitro. RESULTS: Use of specific shRNA directed against RhoA, RhoC or Rac1 GTPases demonstrated a role for each protein in maintaining cell morphology. Furthermore, we demonstrate that RhoC expression and activation is required for directed migration and invasion, while Rac1 expression and activation is required for tumor cell diapedesis. Inhibition of RhoA expression produced a slight increase in invasion and tumor cell diapedesis. CONCLUSIONS: Individual Rho GTPases are required for critical aspects of migration, invasion and tumor cell diapedesis. These data suggest that coordinated activation of individual Rho proteins is required for cells to successfully complete the extravasation process; a key step in distant metastasis.     

肿瘤坏死因子相关凋亡诱导配体在前列腺癌细胞株PC-3M中对核因子kappa B活化的影响

目的：研究TRAIL诱导激素非依赖前列腺癌细胞株PC-3M过程中核因子kappa B（NF-κB）的活化和失活现象。方法： 当不同浓度的TRAIL和LPS作用于细胞后，我们通过细胞免疫组化染色和凝胶电泳迁移试验（EMSA）来检测NF-κB核转位的情况。并通过RT-PCR的方法粗略评定二硫代氨基甲酸吡咯烷（PDTC）对抑制蛋白IκB的影响。结果： EMSA和免疫组化分析显示PC-3M细胞中NF-κB的核转位可被TRAIL或LPS明显地激活。以PDTC预处理可以上调抑制蛋白IκB的表达，阻断NF-κB的核转位。结论： TRAIL的作用于激素非依赖前列腺癌细胞时主要的负作用在于其可以显著地刺激NF-κB的活化。另一方面，在PC-3M细胞凋亡过程中NF-κB的核转位可以被PDTC有力地抑制，同时IκB的表达升高和降解减少（PDTC引起的）是抑制NF-κB活化的潜在因素，为我们提出了增强TRAIL疗效的可能性方案。     

Growth and metastasis of human breast cancers in athymic nude mice

To evaluate critically the merit of utilizing a wound model for growing human tumors, a series of increasingly difficult human tumor types were tested for growth at sites of trauma in athymic nude mice. In vitro tumor lines as well as fresh tumors from the breast, colon, rectum, lung, and a metastasis from an unknown primary were intraperitoneally injected into mice subjected to intra-abdominal organ injury. Successful xenografts were obtained from nine of 10 cell lines and 14 of 24 fresh tumors. The latter included five of six (83%) colon cancers, one lung tumor, metastatic tumor of unknown primary, three of four (75%) metastatic breast cancers and four of six (67%) estrogen receptor (ER)-negative breast primary tumors. Six ER-positive breast tumors tested failed to grow in mice without estrogen supplementation. Xenografts from two breast, two colon and the lung cancers formed spontaneous metastases and all xenografts tested were able to yield serial transplants in the surgical wound model. Histologically, all xenografts and their metastases were identical to their respective donor tumors. Transplantability in mice without exogenous estrogen supplementation was linked to the absence of estrogen and progesterone receptors in breast tumors. Transplantability of the cell lines was associated with the expression of cell surface receptors for fibronectin and hyaluronic acid. Receptors for other extracellular matrix components, namely, laminin, vitronectin, collagen, fibrinogen or von Willebrand factor were not associated with transplantability. These results demonstrate that a large proportion of human tumors, including the breast tumors, can be successfully xenografted into athymic mice by providing them with a healing wound environment, and that such xenografts grown at ectopic sites exhibit metastatic ability.     

Human chromosome II inhibits tumorigenicity of a murine squamous cell carcinoma cell line

Loss of heterozygosity (LOH) of mouse chromosome 7 has been consistently demonstrated in chemically induced murine squamous cell carcinomas (SCCs). The region of this chromosome presenting LOH in the mouse tumors is syntenic to human chromosome segments II p I5 and II q. To determine whether the introduction of human chromosome (Hchr) II can suppress the growth of murine SCC, we injected four clones of a chemically induced murine SCC cell line bearing an Hchr II into athymic BALB/c nude mice. All microcell hybrid clones with Hchr II (CH721Hchr II) had latency periods twice as long as those of the parental CH72 cells and control hybrids containing a Hchr 12. Tumor-derived cells from CH721Hchr II hybrids had lost centromeric and telomeric sequences from Hchr II. All repressed cell lines grew significantly m e slowly in vitro than did the controls. These results suggest that Hchr II contains a tumor-suppressor gene capable of inhibiting tumorigenicity in chemically induced SCC, confirming common pathways in the development of human neoplasias and the murine model. © 1995 Wiley-Liss, Inc.     

BCSG1-siRNA在乳腺癌裸鼠移植瘤的表达

目的 探讨小分子干扰RNA(siRNA)对人乳腺癌移植瘤组织中乳腺癌特异性基因BCSG1表达的抑制作用.方法 根据BCSG1的已知cDNA序列,设计并体外转录合成4条特异性siRNA,分别导入载体质粒中,用脂质体介导分别转染人乳腺癌细胞系MCF7细胞中,以无关序列转染和未转染的MCF7细胞为对照.将用上述4条特异性siRNA和无关序列转染后的细胞连同未转染的MCF7细胞(共6组细胞)注入裸鼠皮下,构建裸鼠乳腺癌移植瘤模型,分析肿瘤生长抑制情况.采用半定量逆转录聚合酶链反应和免疫组织化学SP法分别检测6组裸鼠移植瘤中BCSG1 mRNA和BCSG1蛋白的表达情况.3组人肿瘤组织(浸润性导管癌、癌旁乳腺导管增生组织和乳腺纤维腺瘤)同时用作对照.结果 BCSG1-siRNA转染的4组的抑瘤率均明显大于无关序列转染组及未转染对照组(P＜0.01);与无关序列转染组及未转染对照组相比,BCSG1-siRNA转染的4组肿瘤组织中BCSG1蛋白的表达明显减弱;未转染细胞对照组和无关序列转染组、人乳腺浸润性导管癌组均有大量BCSG1 mRNA阳性条带,而在BCSG1-siRNA转染的4组中,仅有少量BCSG1较弱的mRNA阳性条带,与未转染细胞对照组和无关序列转染组相比,差异均有统计学意义(P＜0.01).结论 BCSG1-siRNA能有效抑制肿瘤的生长,下调裸鼠乳腺癌组织中BCSG1蛋白及mRNA的表达.     

DIM增强顺铂抑制PC-3细胞增殖及诱导凋亡的效应

目的：观察顺铂(DDP)和3，3-二吲哚基甲烷(3,3-diindolylmethane ,DIM)联合应用对人前列腺癌PC-3细胞增殖和凋亡的影响。方法:采用MTT法检测PC-3细胞增殖抑制情况，用流式细胞术及吖啶橙染色法分析细胞凋亡的变化，用RT-PCR检测抑癌基因p21的表达变化。结果:60 μmol.L-1的DIM 与0.4 mg·L^-1的DDP 联合可有效抑制PC-3细胞增殖并诱导其凋亡，其效果与单用4 mg·L-1DDP相同。DIM与DDP联合试验中细胞生长抑制率和凋亡率也明显高于单用DIM处理组（P＜0.05）。RT-PCR结果表明：单用DIM和DIM与DDP联合用药都能明显增强p21基因的表达，但联合用药的效果更明显。结论:DIM能显著增强DDP对PC-3细胞的增殖抑制和诱导凋亡的效应。     

Sublines of the Burkitt line Daudi,selected on the basis of reactivity with soybean agglutinin,differ in tumorigenicity in athymic mice

Two subpopulations were isolated on the basis of soybean agglutinin (SBA) binding, from the human Burkitt lymphoma line Daudi. The low- and high-binder sublines maintained this characteristic in continuous passages. Their surface marker profiles, antibodies, scanning electron microscope (SEM), cytochemical reactions and binding of other lectins (concanavalin A and wheatgerm agglutinin) were not different. They differed, however, in growth potential in athymic mice. The low-binder subline had lower frequency of takes, tumor weight and volume, and did not metastasize as compared to the high-binder subline. However, the reaction with F-SBA of all the tumor cells examined was strong (> 70%), indicating in vivo selection and tumor development of high binder cells.     

Chrysin reduces proliferation and induces apoptosis in the human prostate cancer cell line pc-3

INTRODUCTION:

Honey is a common household product with many medicinal uses described in traditional medicine. Only recently has its antioxidant properties and preventive effects against disease been highlighted. Chrysin is a natural flavone commonly found in honey that has been shown to be an antioxidant agent. In this study, we investigated the antiproliferative and apoptotic effects of honey and chrysin on cultured human prostate cancer cells.

METHODS:

Cells were cultured in RPMI medium and treated with different concentrations of honey and chrysin for three consecutive days. Cell viability was quantitated by the 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The percentage of apoptotic cells was determined by flow cytometry using Annexin V-fluorescein isothiocyanate.

RESULTS:

The MTT assay revealed that both compounds had an antiproliferative effect on PC-3 cells in a dose- and time-dependent manner. The IC₅₀ values for honey and chrysin against PC-3 cells were 2.5% and 24.5% after 48 h and 1.8% and 8.5% after 72 h, respectively. Chrysin induced apoptosis in PC-3 cells, as determined by flow cytometry.

CONCLUSION:

Our results suggest that honey has anti-proliferative effects on prostate cancer cells and the effects are mainly due to chrysin. Therefore, chrysin may be a potential compound for both cancer prevention and treatment. Further in vivo investigation is needed to support the use of chrysin in cancer therapy.     

高转移性人成骨肉瘤细胞系在裸小鼠体内的筛选和维持

目的：筛选人成骨肉瘤高转移细胞并探讨维持其高转移性状的方法。方法：采用低转移性成骨肉瘤细胞系在裸小鼠腹腔内连续传代和体外培养肺转移灶筛选高转移亚系,通过体内、外交替传代维持其高转移性状,并用流式细胞仪、染色体分析等方法,研究该高转移亚系的细胞周期、形态、增殖等生物学特性。结果：筛选所得腹水型高转移亚系自发性肺转移率达100％。在细胞形态、增殖速度、染色体数目等方面与母系有较大差别,经体内、外交替传代高转移性状得以维持,且出现较高比例的脑、骨、肌肉转移。结论：腹腔连续传代培养转移灶和体内、外交替传代是筛选和维持高转移细胞的可靠方法。