地锦草水提液对H22荷瘤小鼠生长抑制及其机制探讨

目的：研究地锦草对H22荷瘤小鼠的抑瘤作用并探讨其对肿瘤组织血管内皮生长因子（vascular endothelial growth factor,VEGF）和基质金属蛋白酶-3（matrix metalloproteinases-3,MMP-3）蛋白表达的影响。方法：建立H22荷瘤小鼠模型,随机分为模型对照组、地锦草264mg／（kg·d）高剂量组、地锦草132mg／（kg·d）中剂量组、地锦草66mg／（kg·d）低剂量组和环磷酰胺组50mg／（kg·d）。灌胃给药14d后处死小鼠,剥离瘤组织,测量肿瘤体积大小。采用HE染色其形态学变化;免疫组化法观察肿瘤组织VEGF和MMP-3蛋白表达情况。结果：地锦草高剂量组的肿瘤体积为（3．125±1．711）mm2,模型对照组为（5．081±1．936）mm2;地锦草高剂量组肿瘤质量为（1．784±1．765）g,模型对照组为（4．418±2．720）g。与模型组比较,地锦草高剂量组肿瘤体积明显缩小,t=2．184,P=0．037;且其瘤质量减轻,t=2．145,P=0．041。地锦草和环磷酰胺组小鼠瘤体组织切片可见癌细胞聚积成巢,肿瘤细胞数量减少,多处组织坏死,瘤体与周围组织界限清楚,且未侵犯周围组织。免疫组化结果显示,模型组VEGF和MMP=3蛋白表达增强。地锦草高剂量组VEGF蛋白表达（0．160±0．004）下降,与模型对照组（0．228±0．020）比较差异有统计学意义,t=5．011,P〈0．001。地锦草高、中、低剂量组MMP=3蛋白表达分别为0．316±0．062、0．303±0．057和0．302±0．058,与模型组比较差异均有统计学意义,t值分别为6．322、6．845和6．534,P值均〈0．001。各组肿瘤组织的VEGF与MMP3蛋白表达无直接相关性,r=0．069,P=0．709。结论：地锦草可抑制H22荷瘤小鼠肿瘤的生长,其抗肿瘤机制可能与抑制H22荷瘤小鼠的肿瘤组织VEGF和MMP-3蛋白的表达有关。     

Antitumor activity and minimal toxicity of concentrated thymidine infused in nude mice

To avoid infusing large volumes of fluid while treating patients with the standard thymidine solution (30 g/liter), it may be possible to administer this drug in more concentrated form. At 25 degrees C, thymidine is saturating at a concentration of 52 g/liter of 0.6% NaCl solution, and the thymidine concentration at saturation increases with temperature. Nude mice were infused at 29 degrees C with thymidine (60 or 72 g/liter) in cycles consisting of 4 to 5 days infusion followed by 9 days rest. Therapeutically effective doses of concentrated thymidine did not cause significant mortality in mice, and weight loss attributable to treatment was small and reversible. Significant growth inhibition of CA 1 human melanoma heterotransplants was observed after 3 treatment cycles. After 4 or 5 cycles, tumor responses were obtained in 7 mice (6 complete responses) of 12 inoculated with this tumor. These results show that concentrated thymidine solutions are highly effective against human tumor heterotransplants in nude mice and suggest that clinical use of concentrated thymidine may allow practical administration of maximum tolerated doses of this drug.     

Antitumor activity and bone marrow toxicity of aminoglucose mustard anticancer agents in mice

In previous structure-activity studies, we have demonstrated that attachment of a glucose molecule to the chloroethylnitrosourea cytotoxic group produces a compound with reduced murine bone marrow toxicity and retention of full antitumor activity. To further define this protective role conferred by the glucose moiety in bone marrow cells, we have replaced the nitrosourea cytotoxic group with another class of alkylating agent, a bifunctional nitrogen mustard. In a detailed structure-activity analysis, we have now characterized four analogues, with the mustard cytotoxic group positioned at carbon 2 [1,3,4,6-tetra-O-acetyl-2-(di-2-chloroethyl)amino-2-deoxy-D-glucopyranos e (TGM)], carbon 6, or carbon 1 (D- and L-isomers) of the aminoglucose molecule. On a molar basis, TGM was most toxic to normal BALB/c X DBA/2 F1 mice, with a 10% lethal dose (LD10) of 3.8 mumol/kg. The D- and L-isomers of 2,3,4,6-tetra-O-acetyl-N,N-bis(2-chloroethyl)glucopyranosylamine (C-1) were the least toxic, with an LD10 of 73 mumol/kg for both. Optimal antitumor activity against the murine P388 leukemia (single i.p. administration of the LD10) did not differ significantly among the four analogues, with increased life span ranging from 83-86%. P388 antitumor activity for nitrogen mustard (HN2) was significantly less, 60% increased life span (P = 0.01), while p-di(2-chloroethyl)amino-L-phenylalanine produced an increased life span of greater than 101%. An LD10 of 6-bis-(2-chloroethyl) amino-6-deoxy-D-glucose (C-6) or TGM produced significantly less depression of WBC counts than did an equitoxic dose of the C-1 isomers, HN2, or p-di(2-chloroethyl)amino-L-phenylalanine. The mean nadir WBC count for C-6 equaled 86% of control, and for TGM, 80% of control. Consistent with this sparing effect on the peripheral WBC, C-6 and TGM produced significantly less in vivo murine bone marrow DNA synthesis depression, 77 and 64% of control, respectively, as compared to the depression nadir produced by HN2 (27% of control), the D-isomer of C-1 (17%), the L-isomer of C-1 (18%), and p-di(2-chloroethyl)amino-L-phenylalanine (2%). These structure-activity studies demonstrate that conjugation of the mustard cytotoxic group to carbon 6 or carbon 2 of glucose produces an analogue that retains P388 antitumor activity significantly greater than that of HN2, with a concomitant reduction in murine bone marrow toxicity.     

Synthesis of a nickel tetracarboranylphenylporphyrin for boron neutron-capture therapy: Biodistribution and toxicity in tumor-bearing mice

Nickel-2,3,7,8,12,13,17,18-octaacetic acid-5,10,15,20-tetra-[3-carboranyl-methoxyphenyl]-porphyrin octamethylester (NiTCP) was given in a Cremophor EL, a polyethoxylated castor oil, and propylene glycol emulsion to BALB/c mice bearing transplanted s.c. KHJJ mammary carcinomas. A total dose of 244 μg NiTCP/gram body weight (gbw) (54 μg B/gbw) was given in 6 i.p. injections over a 32 hr period. Observations of behavior and changes in body weight and chemical and hematological blood tests indicated little or no toxicity from NiTCP over a period of 6–90 hr after injections. Boron concentrations near tumor margins were 160–180 μg B/g at 41–90 hr after the last injection. Tumor:normal brain boron concentration ratios reached approx. 10:1 and tumor:blood ratios reached approx. 250:1 after 4 days. There was no evidence of thrombocytopenia or other potentially important toxicities. Our findings place NiTCP among the leading candidates for pre-clinical experiments aimed toward improvement upon the compounds being tested for boron neutron-capture therapy of glioblastoma multiforme. © 1996 Wiley-Liss, Inc.     

Plasminogen activator in normal and tumor-bearing mice

The plasminogen activator levels of a series of murine tumors commonly used in cancer drug screening were determined and compared to the levels found in normal mouse tissues. Tumors high in plasminogen activator included the B16 and colon 26. The Lewis lung and M5076 carcinoma showed an intermediate level of activity, while the plasminogen activator activity in the L1210 leukemia and colon 38 was barely elevated above background. The specific activity of the enzyme (per μg protein) in extracts of B16 and colon 26 was three or four times higher than in the most active normal organs surveyed (kidney, lung, brain and intestine). The high level of plasminogen activator activity measured in extracts of the B16 tumor was reflected in a substantial elevation of the levels of fibrin degradation products in the serum of mice carrying this tumor. This result suggests that the tumor-associated plasminogen activator activity is less subject to inhibitory controls in vivo than is the plasminogen activator of most normal tissues, since the total mass of the tumor was far less than the total mass of the fibrinolytically active tissues, and yet the bulk of the fibrin degradation products were caused by the tumor. We conclude that the high levels of plasminogen activator activity which are observed in many human tumors are found only in some of the transplantable murine tumors. Since this enzyme is active at an increased level in vivo in mice carrying the B16 tumor, plasminogen activator may be a suitable target for selective, enzyme-activated chemotherapy with this tumor test system.     

Semliki Forest virus biodistribution in tumor-free and 4T1 mammary tumor-bearing mice: a comparison of transgene delivery by recombinant virus particles and naked RNA replicon

Semliki Forest virus (SFV) vectors are promising tools for cancer gene therapy because they ensure a high level of transgene expression and a rapid and strong cytopathic effect. However, broad tissue tropism and transient expression make it more difficult to develop an optimal cancer treatment strategy. In this study, we have compared the distribution of recombinant SFV particles (recSFV) and naked viral RNA replicon (recRNA) in tumor-free and 4T1 mammary tumor-bearing mice as a consequence of different vector administration strategies. The high potential of SFV recRNA as a biosafe approach for the development of therapeutic treatment was demonstrated. Intravenous (i.v.) inoculation of recRNA provided primary brain targeting in both tumor-free and 4T1 tumor mouse models, but local intratumoral inoculation revealed a high expression level in tumors. Moreover, we observed the predominant tumor targeting of recSFV at a reduced viral dose on i.v. and intraperitoneal (i.p.) virus inoculation, whereas the dose increase led to a broad virus distribution in mice. To prolong transgene expression, we have tested several i.v. and i.p. reinoculation strategies. A detailed evaluation of vector distribution and readministration properties could have an impact on cancer gene therapy clinical trial safety and efficacy.     

Antitumor and other effects of 24R,25-dihydroxycholecalciferol in Lewis lung carcinoma causing abnormal calcium metabolism in tumor-bearing mice

Lewis lung carcinoma was found to cause hypercalcemia in tumor-bearing mice. 24R,25(OH)2D3 (K-DR, prepared by Kureha Chemical Ind.) significantly prolonged the survival time of mice with Lewis lung carcinoma. K-DR exhibited an antimetastatic effect on Lewis lung carcinoma, and also had an analgesic effect in mice with Lewis lung carcinoma.     

In vivo modulation of myelopoiesis and immune functions by maleic anhydride divinyl ether copolymer (MVE-2) in tumor-free and MBL-2 tumor-bearing mice treated with cyclophosphamide

Treatment of normal or MBL-2 tumor-bearing mice with cyclophosphamide (CY) caused severe suppression of myelopoiesis and macrophage (M phi) functions, both of which may limit further use of chemotherapy. Additional treatment with the chemically defined biological response modifier maleic anhydride divinyl ether copolymer (MVE-2) was able to ameliorate the myelosuppressive effects of CY and to restore normal bone marrow cellularity. The stimulatory effects on myelopoiesis, however, could only be obtained by administering MVE-2 at greater than or equal to 3 days after CY, which correlated with an MVE-2-induced simultaneous increase in granulocyte and/or macrophage colony-stimulating factor secretion by bone marrow cells or M phi. Injection of MBL-2 tumor-bearing mice with MVE-2, at 3 days after Cy treatment, caused a decrease in tumor burden and a significant increase in median survival time as compared to treatment with CY alone. At the same time, MVE-2 induced an increase in the number of cytotoxic M phi and a complete restoration within the myelopoietic lineage, which might prevent delayed side effects of CY, such as secondary infections, and might permit more intensive chemotherapeutic treatment. Treatment of MBL-2 tumor-bearing mice with MVE-2, at 6 days after CY, induced a significant increase in M phi cytotoxicity but did not prolong median survival time, probably due to a rapid regrowth of tumor after treatment with CY. Our studies thus show that successful combined therapy with the primary cytotoxic agent CY and the biological response modifier MVE-2 depends on precise timing of the drug regimen and is influenced by the extent and reversibility of CY-induced immunosuppression, as well as by the kinetics of recruitment of new effector cells from bone marrow and by the tumor burden present at the time of treatment.     

A variable neovascularization threshold in tumor-bearing mice

The development of solid tumors is dependent on angiogenesis in such a way that a restriction in neovascularization would cause secondary tumor implants to remain in dormant state, establishing an apparent paradox. In this study an attempt has been made to demonstrate that in the tumor-host relationship a variable threshold of angiogenic response is generated which can be normal, enhanced or diminished depending on the intensity of the stimulus. The latter was determined by the number of PEM, semiallogeneic lynphocytes or irradiated tumor cells which were intradermally injected to induce the host angiogenic response. As compared to the normal controls, in tumor-bearing mice, capillary neoformation i) induced by a low angiogenic stimulus was progressively inhibited by tumor growth; ii) when induced by higher stimuli, in 9 day tumor-bearing mice the response was enhanced while in those of 12 days it was normal being completely inhibited in 15 day tumor-bearing mice; iii) when in a specific day of tumor growth (9, 12 or 15) progressively higher angiogenic stimuli were applied, the response was higher in tumor-bearing mice than in the corresponding controls. Similar results were obtained with PEM induced granulomas, suggesting the participation of a phenomenon of counter-inflammation. It can be concluded that there is an angiogenic threshold that increases as a function of tumor growth so that the response will depend on whether the stimulus attains or surpasses the threshold.     

Heart levels of adriamycin in normal and tumor-bearing mice

Adriamycin accumulates in the hearts of mice bearing Lewis lung carcinoma more than in controls. The effect is more evident at longer (25 days) than earlier times (11 days) after tumor transplantation and it is more consistent at high (15 mg/kg i.v.) than at low doses (5 mg/kg i.v.). The effect is probably related to an impaired pulmonary circulation due to the presence of lung metastases but is not related to a different capacity of the heart in control versus tumor bearing animals to take up adriamycin.The effect is observed in Lewis lung carcinoma bearing animals is present also in mice bearing a B16 melanoma, but not in (C₃H × O₂₀)F₁ mice with a spontaneous mammary carcinoma or in rats transplanted with the Walker carcinosarcoma.In most experimental conditions adriamycin accumulates less in the lungs of tumor bearing animals than in controls. Like heart and lung, other tissues show concentrations of Adriamycin much higher than in blood, but differences in AM distribution between normal and tumor bearing animals are not so evident.Daunomycin is similar to adriamycin in this respect.     

High proliferation of granulocyte-macrophage progenitors in tumor-bearing mice

Cancer may affect hemopoiesis by altering the proliferative status of hemopoietic progenitor cells. In Lewis lung carcinoma (LLC), the proliferative rate of the granulocyte-macrophage colony-forming unit (culture) (GM-CFUc) was studied using in vivo hydroxyurea techniques. The disposal of mature elements to the periphery was also monitored during tumor growth. Neutrophilia, anemia, and splenic hypertrophy developed during the course of the disease. By Day 6 post-tumor implant, myeloid hyperplasia of the marrow was evident, but the content of GM-CFUc in LLC mice was similar to that of control. However, by Day 11, the marrow of LLC mice displayed an increased concentration of GM-CFUc, which tripled by Day 19. There was an increased percentage of proliferating GM-CFUc in LLC mice by Day 6 which was highest by Day 11 and thereafter declined. The level of colony-stimulating activity was higher in the serum of tumor bearers than in that of controls. The early increase in proliferative rate of these early hemopoietic precursors can account for the later accumulation of GM-CFUc and myeloid elements in the marrow. Increased cycling of hemopoietic stem cells raises questions concerning the potential for early exhaustion of hemopoietic progenitor cells in these animals.     

Dysfunction of Ia-positive antigen-presenting cells in tumor-bearing mice

The activities of T cells and antigen-presenting cells in spleen from tumor-bearing mice were studied in vitro. The in vitro induction of trinitrophenyl (TNP)-specific proliferative T cell responses and TNP-specific cytotoxic T cell responses was markedly impaired in spleen cells from X5563 plasmacytoma-bearing C3H/He mice. The activity of TNP-hapten presentation to proliferative T cells and cytotoxic T cells was also impaired in spleen from tumor-bearing mice. Suppressor macrophage activity was not observed in spleen from tumor-bearing mice, because the addition of TNP-modified spleen cells from tumor-bearing mice to TNP-modified spleen cells from normal mice did not suppress the activity of TNP-hapten-presenting cells from normal mice and the addition of indomethacin, an inhibitor of prostaglandin synthesis, to the culture of T cells with TNP-modified spleen cells from tumor-bearing mice did not restore their activity. The population of I-region associated antigen (Ia)-positive cells in spleen macrophages decreased in tumor-bearing mice. Furthermore, the production of interleukin 1 by spleen macrophages was also impaired in tumor-bearing mice. These results suggest that one of the mechanisms leading to immunological abnormality in the tumor-bearing host is mediated by the dysfunction of Ia-positive antigen-presenting cells.