Effects of DN-9693, a new metastasis inhibitor, on murine hematogenous metastasis

1,5-Dihydro-7-(1-piperidinyl)-imidazo[2,1-b]quinazolin-2(3H)-one dihydrochloride hydrate (DN-9693), a new c-AMP: phosphodiesterase inhibitor was examined for its inhibitory effects on platelet aggregation induced by metastasizing tumor cells and on blood-borne metastases of these tumors. 1-3 microM of DN-9693 completely inhibited platelet aggregation induced by B16 melanoma subline BL6 and Lewis lung carcinoma (3LL) cells. Platelets prepared from mice intravenously or orally administered with DN-9693 failed to aggregate after the addition of BL6 cells. Intravenous injection of DN-9693 was effective in protecting the mice inoculated with 1 X 10(6) BL6 cells against acute pulmonary embolic death. Either intravenous or oral administration of DN-9693 (1-10 mg/kg) sufficiently suppressed thrombus formation and subsequent pulmonary metastasis caused by intravenously inoculated BL6 or 3LL cells. Spontaneous pulmonary metastasis of 3LL was also inhibited by DN-9693. Continuous administration of DN-9693 during and after surgical excision of the primary tumors was the most effective treatment against the development of pulmonary metastases of 3LL.     

stathmin基因SiRNA对人宫颈癌细胞的生长抑制作用

目的探讨S iRNA封闭stathm in基因表达对人宫颈癌细胞生长抑制作用。方法用Amb ion公司pS ilencer4.1CMV构建针对stathm in基因的S iRNA真核表达载体,以高表达stathm in基因的人宫颈癌Hale细胞系为靶细胞,脂质体法转染S iRNA表达载体,RT-PCR分析转染细胞stathm in基因表达;MTT试验观察stathm in S iRNA对Hale的生长作用影响;流式细胞仪分析细胞增殖周期的改变。结果stathm in S iRNA可有效封闭stathm in基因表达且明显抑制了宫颈癌Hale细胞的生长;Hale细胞在stathm in S iRNA作用下,细胞分裂阻滞在分裂期的中期,并诱导细胞发生凋亡。结论stathm in基因对宫颈癌细胞的生长可能起十分重要的作用,它有望成为宫颈癌治疗的新靶点。     

Influence of migratory blood cells on the attachment of tumor cells to vascular endothelium

The purpose of this study was to determine whether the presence of migratory blood cells in association with tumor emboli had the capacity to alter the attachment of tumor cells to vascular endothelium. Highly metastatic RT7-4bs rat hepatocarcinoma cells were labelled with [¹²⁵]UdR before being allowed to form mixed cellular spheroids incorporating 1) resident peritoneal macrophages, 2) activated peritoneal macrophages, 3) polymorphonuclear leukocytes, 4) splenic T lymphocytes, or 5) splenic B lymphocytes derived from both normal and tumor-bearing animals. The presence of polymorphonuclear leukocytes or activated macrophages led to a considerable increase in the number of tumor cells attaching to endothelial cell monolayers in vitro. The presence of T or B lymphocytes from either normal or tumor-bearing rats was without effect on tumor attachment to endothelium. Increased tumor cell retention in the lungs was evident for mixed spheroids containing tumor cells and polymorphonuclear leukocytes compared to homotypic tumor spheroids composed of tumor cells alone. Furthermore, preinjection of polymorphonuclear leukocytes intravascularly or inoculation of tumor cells as heterotypic spheroids containing polymorphonuclear leukocytes increased lung colony formation over that obtained after inoculation of tumor cells alone. Several simple sugars were tested for their ability to block tumor cell, polymorphonuclear leukocyte or activated macrophage binding to endothelium in vitro. The results indicate that the glycosylated cell surface components mediating tumor cell attachment to endothelium are not identical with those mediating attachment of either polymorphonuclear leukocytes or activated macrophages. Medium conditioned during mixed spheroid formation was without effect on tumor cell attachment to endothelium. We conclude that the presence of some, but not all classes of leukocytes can modulate tumor cell attachment to vascular endothelium, an effect most likely mediated by a mechanism involving direct contact between the leukocytes and the endothelial cell monolayer.     

Stathmin基因反义核酸对人肝癌细胞系的生长抑制作用

目的 :探讨Stathmin基因反义核酸 (AS ODN)对人肝癌细胞系的生长抑制作用。方法 :以高表达Stathmin基因的人肝癌细胞系SMMC 772 1为靶细胞、反义Stathmin (AS ODN)为阻断剂 ,通过RT PCR观察AS ODN对肝癌细胞Stathmin基因表达的抑制 ,MTT试验观察AS ODN对肝癌细胞的生长抑制作用 ,流式细胞仪分析细胞增殖周期的影响。结果 :AS ODN明显抑制了肝癌细胞SMMC 772 1的生长及Stathmin基因的表达 ,P <0 0 5。SMMC 772 1在AS ODN作用下 ,细胞分裂阻滞在分裂期的中期 ,并诱导细胞发生凋亡。结论 :Stathmin基因的反义核酸 (AS ODN)对肝癌细胞的生长可能起十分重要的作用 ,它有望成为肝癌治疗的新靶点     

Laminin receptor complementary DNA-deduced synthetic peptide inhibits cancer cell attachment to endothelium.

Stable attachment of cancer cells to the endothelium is a key step in the formation of metastasis. In this study, we have investigated the possibility that interaction between laminin and its Mr 67,000 high-affinity receptor (67 LR) could play a major role in this process. Scatchard analysis of laminin-binding studies showed that bovine aortic endothelial cells exhibit 46,000 high-affinity receptors that mediate, at least in part, the attachment of highly invasive melanoma cells. This endothelial cell-melanoma cell interaction was significantly inhibited by soluble laminin and by anti-laminin antibodies. Peptide G, an eicosapeptide derived from the complementary DNA sequence of the 67 LR precursor (IPCNNKGAHSVGLMWWMLAR) that specifically binds to laminin and presumably contains the active ligand-binding site of the receptor, specifically prevented attachment of the melanoma cells to both the bovine aortic endothelial cell monolayer and human umbilical vein endothelium. Thus, peptide G may selectively interfere with the metastatic cascade by inhibiting tumor cell attachment to endothelium via the laminin-67 LR pathway and is a potential new antimetastatic agent.     

Intravascular metastatic cancer cell homotypic aggregation at the sites of primary attachment to the endothelium

The two major theories of cancer metastasis, the seed and soil hypothesis and the mechanical trapping theory, view tumor cell adhesion to blood vessel endothelia and cancer cell aggregation as corresponding key components of the metastatic process. Here, we demonstrate in vitro, ex vivo, and in vivo that metastatic breast and prostate carcinoma cells form multicellular homotypic aggregates at the sites of their primary attachment to the endothelium. Our results suggest that metastatic cell heterotypic adhesion to the microvascular endothelium and homotypic aggregation represent two coordinated subsequent steps of the metastatic cascade mediated largely by similar molecular mechanisms, specifically by interactions of tumor-associated Thomsen-Friedenreich glycoantigen with the beta-galactoside-binding protein, galectin-3. In addition to inhibiting neoplastic cell adhesion to the endothelium and homotypic aggregation, disrupting this line of intercellular communication using synthetic Thomsen-Friedenreich antigen masking and Thomsen-Friedenreich antigen mimicking compounds greatly affects cancer cell clonogenic survival and growth as well. Thus, beta-galactoside-mediated intravascular heterotypic and homotypic tumor cell adhesive interactions at the sites of a primary attachment to the microvascular endothelium could play an important role during early stages of hematogenous cancer metastasis.     

紫杉醇脂质体对卵巢癌细胞生长抑制作用的实验研究

目的分析紫杉醇脂质体对卵巢癌SKOV-3细胞的生长抑制作用,观察药物作用的时间-浓度关系,探究药物作用机制。方法应用不同剂量紫杉醇脂质体对SKOV-3细胞进行处理,通过MTT检测细胞存活率,倒置显微镜观察细胞形态,流式细胞术检测细胞凋亡作用及其对细胞周期的阻滞状态,Western blot分析Bcl-2基因蛋白表达情况。利用SPSS 13.0对数据进行统计学分析。结果紫杉醇脂质体对SKOV-3细胞有明显的剂量-时间依赖效应。紫杉醇与紫杉醇脂质体均可将SKOV-3细胞阻滞在G2/M期,且随药物浓度增加,凋亡细胞所占比例逐渐增加。通过Western blot方法发现,紫杉醇脂质体作用后SKOV-3细胞Bcl-2蛋白表达明显下降,Bax蛋白表达明显上调,Bax/Bal-2比例明显上调。结论紫杉醇以脂质体为转运载体后,并未改变其对卵巢癌SKOV-3细胞的抑制作用及作用周期,具有较好的临床应用前景。     

Early effects of low dose-rate radiation on cultured tumor cells

Low-dose hyperradiosensitivity (HRS) has been found for several cell types after exposure to low doses, < 0.5 Gy, of high dose-rate (typically 50-150 Gy/h) low-LET radiation. HRS precedes the occurrence of a relative resistance for doses above 0.5-1 Gy. A critical question is whether HRS is of importance in radionuclide therapy where the dose-rate is low but the total dose might be high. An indication that cells exposed to low dose-rate can be kept hyperradiosensitive has recently been published. We have in the present study applied cells without (glioma U373MG) and with (glioma U118MG and colon carcinoma HT29) HRS and studied early effects, up to one week, during low dose-rate (LDR), 0.05-0.09 Gy/hours, exposure (total dose after one week: 11.8 +/- 1.5 Gy). The cells were grown on thin foils above a (32)P source placed in a cell culture chamber. Cell number reductions, cell-cycle disturbances, and changed numbers of apoptotic cells were analyzed after continuous LDR exposures. There seemed to be no relation with HRS when the cell number reduction was considered. The U373MG cells, lacking HRS, had the strongest cell number reduction due to a combination of a G(2) block and increased apoptosis. The U118MG and HT29 cells, both having HRS, had surprisingly low cell number reductions. U118MG had only a G(2) block but no increase in apoptosis. HT29 had both a G(2) block and an increase in apoptosis but the apoptosis change was somewhat smaller than for U373MG. Thus, there seemed to be no obvious relation between HRS and early cellular effects when the cells were analyzed after continuous LDR exposure.     

Inhibitory effect of epigallocatechin-gallate on brain tumor cell lines in vitro

We investigated the effect of epigallocatechin-gallate (EGCG), the main constituent of green tea polyphenols, on human glioblastoma cell lines U-373 MG and U-87 MG, rat glioma cell line C6, and rat nonfunctioning pituitary adenoma cell line MtT/E. Cell viability was determined by assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and the extent of apoptosis was studied by flow cytometric analysis. Apoptosis was also characterized by morphology using fluorescent microscopy. The role of insulin-like growth factor-I (IGF-I) was studied by assay with MTT, immunohistochemistry, and immunoradiometric assay. After 72-h exposure, a statistically significant loss of viability (P = < 0.0001) was observed at concentrations of 12.5, 25, 50, and 100 microg/ml in U-373 MG cells and U-87 MG cells. EGCG at concentrations of 50 microg/ml and higher significantly reduced the viability of C6 cells. EGCG inhibited viability of MtT/E cells only at a concentration of 100 microg/ml. Quantitative study by flow cytometry demonstrated that lower doses of EGCG (12.5, 25, 50 microg/ml) induced apoptosis in U-373 MG, U-87 MG, and C6 cells; however, only the highest dose (100 microg/ml) induced apoptosis in MtT/E cells. Compared with other cell lines, MtT/E cells showed stronger IGF-I immunoreactivity. Neutralization of IGF-I with an antihuman IGF-I antibody reduced viability of the cell lines. It can be concluded that EGCG has an inhibitory effect on malignant brain tumors, and IGF-I may be involved in the effects of EGCG.     

Inhibitory effects of heparin plus cortisone acetate on endothelial cell growth both in cultures and in tumor masses

A combination of heparin and cortisone acetate significantly inhibited both embryonic angiogenesis and the tumor growth of Lewis lung carcinoma (3LL) transplanted into C57BL/6 mice, although each of these agents used alone affected neither angiogenesis nor tumor growth. On the other hand, this combination neither decreased the number of metastatic foci in the lung nor prolonged the survival time of mice with 3LL. All tumor-bearing mice died of hemothorax due to pulmonary metastases. Cortisone acetate by itself increased metastasis, and addition of heparin did not affect accelerated metastasis. Because an antiangiogenic activity appears independent of metastasis acceleration by cortisone acetate, the use of steroids other than cortisone acetate having no metastasis-promotion effect should be required for an antiangiogenic tumor therapy in the presence of heparin. Heparin plus cortisone acetate prevented the DNA synthesis of cultured vascular endothelial cells but not that of cultured 3LL cells. Additionally, oral administration of this combination decreased the [3H]thymidine labeling of endothelial cells of tumor blood vessels prior to the suppression of tumor growth. The specific inhibition of the growth of endothelial cells by heparin plus cortisone acetate was revealed in both the in vitro and the in vivo tests.     

PTEN基因对子宫内膜癌细胞生长的抑制作用

目的 探讨PTEN基因在子宫内膜癌基因治疗方面的可行性。方法 通过腺病毒载体(Ad-PTEN)将人野生型PrENcDNA导入Ishikawa细胞,观察外源性PTEN蛋白的表达情况,以及PTEN蛋白对Ishikawa细胞生长的影响,同时还观察了PTEN蛋白是否影响胰岛素样生长因子Ⅱ(IGF-Ⅱ)对Ishikawa细胞的作用。结果 Ad-PTEN感染IsHikaWa细胞后1d,即可见PTEN蛋白表达;第3天时,表达明显增强,并能持续表达10d。感染后的IshikaWa细胞生长受到明显抑制,并且PTEN还能明显抑制IGF-Ⅱ诱导的Ishikawa细胞生长。结论 腺病毒介导的PTEN基因导入能诱导Ishikawa细胞生长受到明显抑制,提示野生型PTEN基因可能是治疗子宫内膜癌的一种新途径。     

Intratumoral T cell subset ratios and Fas ligand expression on brain tumor endothelium

Summary Introduction: T cell presence in TIL, and the ratio of CD8⁺ and CD4⁺ T cell subsets in particular, can correlate with tumor prognosis in some tumors, although the significance of such infiltration into glioma is controversial. However, gliomas represent a lower extreme in their extent of T cell infiltration, and are thus useful in assessing factors that can decrease T cell presence within tumor tissue. Fas ligand, a pro-apoptotic cell surface protein, may play a key role in reduction of T cells in tumor tissue. Objective: To assess the level of FasL expression on brain tumor endothelium and to correlate this with relative levels of CD4⁺ and CD8⁺ T cell subsets in TIL from brain tumors. Methods: CD3⁺, CD4⁺, and CD8⁺ cells were quantified in fresh TIL by flow cytometry. Paraffin embedded sections of tumors, including meningiomas and gliomas as well as extracranial malignancies, underwent immunohistochemical staining for FasL and Von-Willebrand’s factor (Factor VIII) to determine expression levels of endothelial FasL. Results: FasL expression was high in aggressive intracranial malignancies compared to more indolent neoplasms, and correlated inversely with CD8⁺/CD4⁺ TIL ratios in all tumor classes combined (ANOVA,p<0.05). Conclusion: Low levels of T cells within TIL, as well as low CD8⁺/CD4⁺ TIL ratios appear to be a property of parenchymal tumor presence. Together with the inverse correlation seen between FasL expression and CD8⁺/CD4⁺ TIL ratios, the high levels of endothelial FasL expression in gliomas suggests that FasL decreases T cell presence in brain tumors in a subset-selective manner, thus contributing to glioma immune privilege.     

Inhibitory effect of antimetastatic fusion polypeptide of human fibronectin on tumor cell adhesion to extracellular matrices

We investigated the inhibitory mechanism of liver metastasis by using recombinant fragments with cell- and/or heparin-binding domains (C-274, H-271 or the fusion fragment CH-271). Intravenous co-injection of L5178Y-ML25 cells with CH-271 was more effective for the inhibition of liver metastasis than C-274, H-271 or C-274 + H-271. Reduction of the arrest and retention of the radiolabeled tumor cells in the liver of mice was found when CH-271 was co-injected with tumor cells. L5178Y-ML25 cells adhered both concentration- and time-dependently to the substrates precoated with fibronectin, laminin and reconstituted basement membrane, Matrigel. The tumor cell adhesions to the substrates were inhibited in the presence of CH-271. The tumor cell interaction with CH-271-substrate was inhibited by heparin, and monoclonal antibodies (IST-1 or IST-2) against the heparin-binding domain of fibronectin. However, monoclonal antibodies against the cell-binding domain failed to block the interaction. Similarly, CH-271-mediated antimetastatic activity was also inhibited by the treatment of CH-271 with IST-1 before the co-injection with tumor cells, whereas monoclonal antibody against the cell-binding domain had no effect. Thus, the antimetastatic effect of CH-271 fusion fragment on liver metastasis of L5178Y-ML25 cells may be partly due to interference with the adhesive interaction of tumor cells with extracellular matrix or basement membrane components by a heparin-binding domain-dependent mechanism.     

氧化苦参碱对肿瘤诱导血管内皮细胞增殖的抑制作用

目的 探讨氧化苦参碱（Ｏｘｙ）对肺癌和胃癌细胞诱导血管内皮细胞（ＶＥＣ）增殖的抑制作用。方法 用ＭＴＴ法检测不同浓度Ｏｘｙ对ＶＥＣ、人肺腺癌ＳＰＣ－Ａ－１细胞、人低分化胃癌ＭＫＮ－４５细胞增殖及ＳＰＣ－Ａ－１和ＭＫＮ－４５细胞诱导ＶＥＣ增殖的抑制。结果 Ｏｘｙ浓度为２．５～１０ｍｇ／ｍｌ时,对ＶＥＣ增殖的抑制率为４．６％～３６．４％;Ｏｘｙ浓度为０．１５６～１０ｍｇ／ｍｌ时,对肺癌ＳＰＣ－Ａ－１和     

Adhesion molecules and tumor cell interaction with endothelium and subendothelial matrix

Cancer metastasis poses the greatest challenge to the eradication of malignancy. The majority of clinical and experimental evidence indicates that metastasis is a non-random, organ-specific process. Tumor cell interaction with endothelium and subendothelial matrix constitutes the most crucial factor in determining the organ preference of metastasis. A plethora of cell surface adhesion molecules, which encompass four major families (i.e., integrins, cadherins, immunoglobulins and selectins) and many other unclassified molecules, mediate tumor-host interactions. Adhesion molecules and adhesion processes are involved in most, if not all, of the intermediate steps of the metastatic cascade. Decreased E-cadherin expression and increased CD44 expression are clearly correlated with the acquisition of the invasive capacity of primary tumor cells. Similarly, altered expression pattern of many other adhesion molecules such as upregulated expression of the laminin receptors and depressed expression of fibronectin receptors (51) appears to be involved in tumor cell invasion into the subendothelial matrix. Tumor cell-endothelium interactions involve several well-defined sequential steps that can be analyzed by the Docking and Locking hypothesis at the molecular level. Tumor cell-matrix interactions are determined by the repertoire of adhesion receptors of tumor cells and the unique composition of organ-specific matrices. Our experimental data, together with others', suggest that the integrin IIb3 is one of the major players in these tumor-host interactions. Tumor-host interaction is a dynamic process which is constantly modulated by a host of factors including various cytokines, growth factors and arachidonate metabolites such as 12(S)-HETE. Delineation of the molecular mechanisms of tumor-host interactions may provide additional means to intervene in the metastatic process.     

Inhibitory effects of canthaxanthin on in vitro growth of murine tumor cells.

The antitumorigenic effects of carotenoids, in addition to their immuno-enhancing effects, may occur by their direct action on growing tumor cells. To test this hypothesis the direct inhibitory effect of various concentrations of canthaxanthin (CX; 4,4'-diketo-beta-carotene), a non-provitamin A carotenoid, was tested on the in vitro growth of JB/MS, B16F10 melanomas and PYB6 fibrosarcoma and murine non-transformed NIH-3T3 (ATCC CRL 1658) cells. At concentrations of 1 x 10(-8) M up to 1 x 10(-4) M, CX significantly reduced the overall number of tumor cells. The greatest inhibition was observed at a CX concentration of 1 x 10(-4) M after 72 h and 96 h of incubation. However, CX had no inhibitory effect on the growth of the non-transformed NIH-3T3 cell line; rather it significantly enhanced growth of this cell line (P less than 0.05) after 96 h of incubation. Thus, the inhibitory action of CX on growing tumor cells appears to be due to its direct actions on tumor cells and not via its conversion to vitamin A or its immuno-enhancing effects.     

熊果酸对小鼠H22 移植瘤的抑制作用研究

目的： 观察熊果酸 (ursolic acid,UA)对小鼠H22移植瘤的抑制作用并对其机制进行探讨。方法：将75只昆明种小鼠于左前腋下皮下接种H22肝癌细胞,24 h后随机分为5组,每组15只,模型组、环磷酰胺 (cyclophosphamide,CP)对照组[25 mg/ (kg·d)] 和UA低、中、高剂量药物组[50、100、150 mg/ (kg·d)]。UA各剂量组和CP对照组分别以相应剂量UA和CP灌胃,模型组以等体积花生油灌胃,每天灌胃1次,连续给药2周。末次给药24 h后,称小鼠体质量,摘除眼球取血后处死。无菌条件下完整剥离小鼠肿瘤及肝、肾、脾组织,计算抑瘤率及肝、肾、脾指数;HE染色观察肿瘤组织病理学改变;分离脾淋巴细胞,CCK8法测定小鼠脾淋巴细胞增殖活性;免疫组化法检测肿瘤组织中CD4+、CD8+ T细胞亚群的分布情况,计数CD4+ T细胞和CD8+ T细胞个数。并采用酶联免疫吸附法 (ELISA)检测血清中白细胞介素12 (interleukin-12,IL-12)的浓度。结果：UA各剂量组小鼠H22肿瘤质量增长均较缓慢,其中UA中、高剂量组肿瘤质量明显减小,与模型组比较,差异均具有统计学意义 (P<0.05);UA中、高剂量组抑瘤率分别为30.15%和39.80%。UA各剂量组肝、肾指数与模型组比较差异无统计学意义 (P>0.05),而脾指数则随UA剂量增加逐渐升高,其中,UA中、高剂量组脾指数与模型组比较差异具有统计学意义 (P<0.05)。HE染色病理观察结果显示,模型组肿瘤细胞生长旺盛,细胞体积较大,胞质丰富,少见坏死区域;UA干预组肿瘤组织中瘤细胞生长增殖明显受到抑制,肿瘤细胞数量减少,密度降低,排列稀疏,细胞核固缩深染或破裂,细胞间质较多,核浆比例减少,坏死区域明显增多。CCK8实验结果显示,随着UA剂量增加,脾淋巴细胞增殖活性逐渐升高,其中,UA中、高剂量组脾淋巴细胞增殖活性显著升高,与模型组间的差异具有统计学意义 (P<0.05)。免疫组化结果显示,UA中、高剂量组肿瘤组织中CD4+、CD8+ T细胞个数与模型组比较均明显增加,差异具有统计学意义 (P<0.05)。UA高、中、低剂量组血清中IL-12的含量均较模型组升高,差异均有统计学意义 (P<0.05);UA高剂量组与CP对照组相比亦显著升高 (P<0.05)。 结论：一定剂量UA可抑制H22荷瘤小鼠肿瘤生长,其作用机制可能与UA促进机体免疫器官发育和淋巴细胞增殖,增加CD4+、CD8+ T细胞向肿瘤组织浸润,并提高血清中IL-12等抗肿瘤活性细胞因子的浓度诱导细胞免疫有关。     

Polymorphic enzyme analysis of cultured human tumor cell lines

Of the total of 137 cultured cell lines from a wide variety of human neoplasms now typed at a maximum of 16 of their polymorphic enzyme loci, the phenotype combinations for 72 are described in this paper. The phenotype frequency product of each line was less than 0.05, and only 148 of the 9,316 (1.6%) pairwise comparisons between lines had phenotypes that did not differ in at least one locus. Thus the probability of any two randomly selected lines having indistinguishable genetic signatures in this sample of 137 lines was 0.016. Fifty-two of the 137 lines (38%) had phenotype combinations distinguishable from each of the other 136 lines. The frequency products of the 85 lines that could not be distinguished from at least one other line were all sufficiently high to preclude a suspicion that contamination had occurred among them. No additional cell lines with phenotype combinations indistinguishable from the combination characteristic of HeLa cells were identified among these 72 cell lines.