Inhibitory effect of tumor necrosis serum on the metastasis of B-16 mouse melanoma cells

The inhibitory effect of tumor necrosis serum (TNS) on the artificial metastasis of B-16 melanoma cells and the spontaneous metastasis of Lewis lung carcinoma cells was investigated. The results obtained were as follows: 1) When tumor necrosis serum (TNS) was administered either 20 min or 2 days after injection of B-16 melanoma cells, a very strong inhibitory effect (99%) relative to the control was noted. 2) TNS showed a 60% inhibitory effect on spontaneous metastasis, and the weight of the original tumor regressed by 60%. 3) No histological changes in normal tissues were observed microscopically following TNS injection. The above results confirm that TNS is extremely effective in preventing metastasis.     

Combination therapy with pions and schizophyllan (spg) for murine B-16 melanoma

The B-16 melanoma transplanted into C57BL/6 mice was used to investigate the antitumor effect of Schizophyllan (SPG) when applied alone and in combination with local irradiation using pions with 4 dose fractions of 2 Gy or 6 Gy each. SPG was given intramuscularly in a daily dose of 10 mg/kg body weight for 25 consecutive days from day 4 after the initiation of irradiation, and thereafter three times a week up to day 45. The antitumor effect was evaluated by the changes in tumor volume. survival, the size of metastatic lymph nodes grossly involved, and the number of pulmonary metastatic nodules on the surface of the lungs. After 24 Gy, significant differences were found between the group treated with combined pions and SPG, and the group treated with pions alone in terms of tumor volume change. survival and lymph node, and pulmonary metastases. However, when SPG was applied to non-irradiated tumors or to tumors irradiated with only 8 Gy, it had neither antitumor nor life-prolonging effect. From the present study, it seems that a SPG, as a Biological Response Modifier, has some adjuvant effect only where a limited number of tumor cells remain following pion irradiation. Combination therapy using SPG may, therefore, be advantageous for patients with complete response or good partial response to pion irradiation.     

Combination therapy of single or fractionated x-rays and schizophyllan (spg) for murine B-16 melanoma

B-16 melanoma transplanted into C57BL/6 mice was used to investigate the antitumor effect of Schizophyllan (SPG) combination with local irradiation by X-rays using 4 fractions of 3 Gy or 9 Gy and single fractions of 12, 19 and 26 Gy. In the groups which received a single fraction of 26 Gy and 4 fractions of 9 Gy, significant differences were found between the groups with/without SPG in terms of tumor volume change and lymph node and pulmonary metastases. In terms of survival, significant difference was found only in the 4 fraction of the 9 Gy group. SPG had no antitumor effect nor life-prolonging effect, when it was applied to tumors irradiated with single fractions of 12 and 19 Gy or 4 fractions of 3 Gy, except for some metastasis-suppressing effect in the group given 19 Gy. It seems that SPG has some adjuvant effect only where a limited number of tumor cells remain following rather large dose of X-irradiation. Furthermore, the gain produced by host-mediated immune response augmented by SPG seems to be larger in the 4 fraction group. Therefore, combination radiotherapy using SPG may be advantageous for patients with complete response or a good response after multifractionated irradiation.     

20(S)-人参皂苷Rg3对B16黑色素瘤生长的抑制作用

目的 :探讨 2 0 (S) 人参皂苷Rg3对B16黑色素瘤生长的影响。 方法 :体内建立B16实体瘤模型及PCNA免疫组织化学染色 ,体外应用MTT法、生长曲线观察Rg3对B16黑色素瘤生长影响。 结果 :实体瘤模型中 ,Rg3各组瘤重明显低于对照组 (P <0 0 1) ,且Rg3组PCNALI与对照组比较有差异 (P <0 0 5 ) ,随着Rg3给予浓度的增加 ,PCNALI逐渐降低。体外实验观察到Rg3对B16黑色素瘤细胞生长同样有抑制作用。 结论 :Rg3可以明显抑制B16黑色素瘤的生长及增殖活性     

The response of murine B-16 melanoma to fractionated doses of pions.

The relative biological effectiveness (RBE) of pions has been studied in mouse B-16 melanoma transplanted into C57BL/6 mice. To determine the RBE at both high and low doses per fraction, a range of fractionation schedules was used, with 1, 4 and 10 fractions. The reference 250 kV X ray dose rate was 1.5 Gy/min which was much higher than the dose rate of pions (0.25 Gy/min). The RBE varied depending on the number of fractions and, within the same fractionation schedule, also on the dose per fraction. The RBE ranged from 1.15 for single fractions at 12.5 days of growth delay, to 1.80 for 10 fractions at 5 days of growth delay, which was determined by the time taken for the tumors to reach 5 times the average of their original volume. RBEs at the iso-effect level of 10 days growth delay were 1.20, 1.29 and 1.62 for single, 4 fractions and 10 fractions, respectively. RBE values were influenced by both the number of fractions and the dose per fraction, that is, the larger the number of fractions and the smaller the dose per fraction, the larger the value of RBE. In comparison with RBE of normal mouse skin, it was suggested that pion therapy may provide advantage over conventional photontherapy for radioresistant tumors such as this melanoma with the maximum therapeutic gain factor of 1.2. alpha/beta ratios for B-16 melanoma were also obtained from the 10 day growth delay iso-effect curve, and were 10.5 Gy and 32.6 Gy for X ray and pions, respectively.     

姜黄素水溶性制剂对小鼠黑素瘤自发性转移的抑制作用

目的: 观察姜黄素水溶性制剂对小鼠黑素瘤转移的抑制作用。方法：以羟丙基β环糊精（hydroxypropylβ cyclodextrin, HPβCD）为包合剂制备姜黄素水溶性制剂——姜黄素羟丙基β环糊精包合物（CHPβCD），采用小鼠黑素瘤细胞B16J10建立小鼠移植瘤伴自发性转移模型，于移植瘤细胞的第2日开始以腹腔注射药物进行治疗，共分低剂量姜黄素组(30 mg/kg)、高剂量姜黄素组（60 mg/kg)、阴性对照组及阳性对照TNP470组 (30 mg/kg)4组，隔日1次，连给7次。观察各组小鼠成瘤及腹腔淋巴结转移情况，以免疫组化法检测移植组织的微血管密度（MVD）。结果: 制备的CHPβCD易溶于水，不加任何有机溶剂即可用于腹腔注射给药。两个CHPβCD治疗组的肿瘤移植瘤均明显小于阴性对照组（P<0.05），移植瘤组织MVD均明显低于阴性对照组（P<0.05）。高剂量姜黄素组的腹腔淋巴结转移程度显著低于阴性对照组（P<005），但低剂量姜黄素组则与阴性对照组无显著差异（P>0.05）。结论:姜黄素水溶性制剂经腹腔注射给药时可显著抑制小鼠黑素移植瘤转移，且其抗转移作用与抗血管生成作用密切相关。     

Inhibition of growth of B16 murine malignant melanoma by exogenous interferon

We previously reported that polyinosinic-polycytidylic acid, a potent interferon inducer, inhibits the growth of B16 malignant melanoma in the C57BL/6 mouse. Two experiments were done to evaluate the effectiveness of interferon in tumor inhibition in vivo. In the first, mice were implanted with melanoma and divided into four groups, according to treatment: interferon preparation; interferon control preparation ("breakthrough fraction"); phosphate-buffered saline control; and murine serum albumin control. Daily, each mouse was given i.p. injections of 200,000 NIH reference units (hereafter called units) of interferon or of one of the control substances. The second experiment was similar to the first, except that bovine serum albumin was an additional control. In both experiments, the average tumor volume in interferon-treated mice was statistically significantly smaller than that of each control group. Mouse interferon preparations also inhibited the multiplication of B16 malignant melanoma cells in culture. This inhibition was statistically significant from interferon levels as low as 5 to as high as 5000 units/ml. The degree of inhibition markedly increased from 5 up to 500 units, the inhibition reaching its maximum at this concentration. The inhibitory effect of interferon was abrogated by anti-murine interferon serum produced in a rabbit. These findings suggest that the in vivo inhibition of the growth of B16 melanoma demonstrated with polyinosinicpolycytidylic acid and with exogenous interferon probably results, at least in part, from a direct effect of interferon on the tumor cells themselves.     

三肽化合物酪丝缬肽对小鼠黑色素瘤细胞B16-F10侵袭和转移的抑制作用

背景与目的:三肽化合物酪丝缬肽(tyroservaltide,YSV)对实验性肝癌具有明显抑制作用,本研究观察YSV对小鼠黑色素瘤细胞B16-F10侵袭和转移的抑制作用。方法:MTT法检测YSV对B16-F10的细胞毒作用;利用基质胶Matrigel研究YSV对细胞粘附能力的影响;用Transwell小室侵袭模型研究YSV对肿瘤细胞侵袭能力的改变;经C57/BL6小鼠尾静脉注射B16-F10细胞,建立人工肺转移模型,观察YSV对B16-F10肺转移的影响;免疫组化方法观察YSV对细胞间粘附分子(intercellularadhesionmolecule-1,ICAM-1)在肺组织中表达水平的影响。结果:100μg/mlYSV作用48h对B16-F10细胞的增殖抑制率为24.36%;作用24h对B16-F10细胞在Matrigel胶上的粘附抑制率为36.51%;10μg/mlYSV作用48h对B16-F10细胞的侵袭抑制率为36.53%;640μg·(kg·d)-1YSV抑制B16-F10的肺转移,抑制率为62.21%;YSV组ICAM-1的组织量明显少于生理盐水组。结论:YSV具有抑制B16-F10生长和侵袭转移的作用。     

Control of melanogenesis by glycosylation inhibitors and the inhibitory effect of interferon on melanoma metastasis

The Glycosylation inhibitors, glucosamine or tunicamycin induced a marked loss of pigment within melanoma cells in addition to their reduced metastatic ability. Electrophoresis of tyrosinase demonstrated the disappearance of or a marked decrease in membrane-bound tyrosinase, T3 in the small and large-granule fractions. Glycoprotein synthesis in the melanogenic subcellular compartments of pigment cells seems to play an integral role in melanogenesis which is principally enhanced in their carcinogenic status. The effect of interferon (IFN) on melanoma metastasis was investigated using B16-F10 melanoma cells. The inhibitory effect was maximal when given 3 h prior to tumor cell inoculation. IFN given 12 and 24 h prior to, as well as simultaneously with, tumor cell inoculation, also reduced metastases, but to a lesser extent. When given 2 h after the inoculation, no effect was shown. The salutary effect of IFN was abolished by anti-asialo GMI, but NK activity was enhanced equally throughout 3 to 24 hrs. This indicates that the effect is substantially dependent on NK cell activity, although the implication of other factors is not excluded.     

Inhibitory effect of epigallocatechin gallate on adhesion of murine melanoma cells to laminin

We examined the effects of five kinds of green tea catechin on the adhesion of mouse melanoma B16 cells to laminin. (-)-Epigallocatechin gallate (EGCG) and (-)-epicatechin gallate in the culture medium were found to inhibit the cell adhesion. The adhesion to laminin pre-treated with EGCG was also impaired. Affinity chromatography revealed the binding affinity between laminin and EGCG. These data suggest that the inhibitory effect of EGCG on adhesion of melanoma cells to laminin is included in the mechanism(s) of previously reported metastasis inhibition elicited by EGCG and green tea infusion.     

Clonal origin of metastasis in B16 murine melanoma: a cytogenetic study

A cell line isolated from the B16 melanoma and carried in continuous culture for 8 years (the parent line) exhibited great heterogeneity in terms of marker chromosome content. A lung metastasis from a C57BL/6 mouse inoculated im with cells of this line showed karyotypic homogeneity. Inoculation iv of cells from the parent line produced numerous tumor foci in various organs. Cytogenetic analyses of 18 such lesions led to the following conclusions: Cells from each metastatic colony exhibited relatively homogeneous karyotypic characteristics, indicating that metastases are of clonal origin; many parental cells with different marker chromosomes had metastatic potential; and some genomes maintained homogeneity longer than others.     

Immunotherapy of B-16 melanoma with peptidoglycan monomer

B-16 melanoma-bearing mice received intravenously or intratumorally one or multiple injections of peptidoglycan monomer (PGM) derived from Brevibacterium divaricatum cell wall. Multiple injections of this non-toxic, water-soluble, low-molecular-weight peptidoglycan reduced the growth rate of tumor nodule on the leg, but did not significantly prolong the survival of tumor-bearing mice. One milligram of PGM administered 3 or 7 days after tumor inoculation inhibited formation of pulmonary metastases, induced either by intravenous injection of malignant cells or seeded spontaneously from tumor nodules in the legs before amputation. The inhibition reached about 50% of control values in saline-treated mice. Addition of PGM to in vitro cultures of B-16 melanoma cells did not change their growth rate. The phagocytic activity in the lungs, but not in the spleen and liver, was significantly augmented 3 and 7 days after treatment with PGM. These data indicate that the antimetastatic potency of PGM is probably due to activation of local (pulmonary) macrophages, and not due to direct cytotoxic effects on B-16 melanoma cells or to activation of systemic antineoplastic defence.     

重组腺病毒介导小鼠内皮抑素对荷骨肉瘤MG-63细胞裸鼠肺转移的抑制

目的: 构建携小鼠内皮抑素(endostatin， ES)基因的重组腺病毒载体，观察其对荷骨肉瘤裸鼠肺转移的抑制，探讨内皮抑素表达水平与骨肉瘤肺转移的关系。方法:构建pDC315mEndo表达质粒，同源重组产生重组腺病毒AdmEndo。裸鼠右前肢皮下注射骨肉瘤MG63细胞建立移植瘤裸鼠模型；随机分为4组：小鼠内皮抑素腺病毒（AdmEndo）组，携带EGFP基因腺病毒（AdEGFP）组， PBS组，未接种肿瘤细胞裸鼠空白对照组。各组裸鼠每周分别注射相应药物200 μl，连续5次，观察各组动物移植瘤体积、瘤组织病理，ELISA法检测各组裸鼠血ES水平；7周后处死动物，观察有无肺转移及肺转移灶病理。结果：AdEGFP组肿瘤体积为（1.53±0.05） cm3,PBS组为（1.56±0.07） cm3, AdmEndo组为（0.91±0 .03） cm3，AdmEndo治疗的抑瘤率达40.7％。AdmEndo组裸鼠血内皮抑素表达水平明显高于AdEGFP组和PBS组（P<0.05）。AdmEndo组裸鼠肺部未发现肿瘤转移灶，其他两组肺部见大量散在转移灶，肺转移率分别为80%和90%。未发生肺转移裸鼠的ES水平显著高于发生肺转移的裸鼠（P<0.05）。结论：腺病毒介导的小鼠内皮抑素显著抑制了荷骨肉瘤裸鼠肺转移，内皮抑素表达水平与肺转移有着直接的关系。     

人参皂苷Rg3抑制B16黑色素瘤新生血管生成及其机制的探讨

目的:观察20(S)-人参皂苷Rg3(SPG-Rg3)对B16黑色素瘤血管生成的影响,并探讨其可能的作用机制.方法:体内采用肿瘤诱导血管生成实验,观察SPG-Rg3对B16黑色素瘤血管生成的影响;免疫细胞化学法观察SPG-Rg3对B16黑色素瘤细胞血管内皮细胞生长因子(VEGF)表达的影响;取不同浓度SPG-Rg3作用后的B16黑色素瘤细胞条件培养基(BMCM)作用于人脐静脉内皮细胞,用MTT法、PCNA免疫荧光染色以及迁移实验观察SPG-Rg3对内皮细胞增殖和迁移的作用.结果:在肿瘤诱导血管生成实验中,SPG-Rg3组肿瘤周围的血管数明显少于对照组,P＜0.01;SPG-Rg3作用组BMCM的促内皮细胞增殖和迁移作用均明显弱于无SPG-Rg3作用组,且SPG-Rg3(5 μg/mL)降低了B16黑色素瘤细胞VEGF的表达,P＜0.01.结论:SPG-Rg3可明显抑制B16黑色素瘤的血管生成,且可能通过降低肿瘤细胞分泌VEGF来抑制肿瘤细胞对血管内皮细胞增殖和迁移的促进作用,从而达到抑制肿瘤新生血管形成的作用.     

重组人血管内皮抑制素抑制内皮细胞血管生成的实验研究

目的:观察国产重组人血管内皮抑制素(Endostar,恩度)对人脐静脉内皮细胞(HUVECs)的抗血管生成作用,并对其机制进行初步探讨.方法:采用MTS/PMS系统测定不同浓度恩度对HUVECs和人肝癌细胞株HepG2的生长抑制作用;流式细胞术检测恩度在相同时间内诱导HUVECs和HepG2细胞早期凋亡和晚期凋亡情况;通过迁移/侵袭实验、体外管腔形成实验和鸡胚尿囊膜(CAM)实验观察恩度对HUVECs迁移/侵袭和体内外管腔形成及功能的影响.结果:不同浓度的恩度持续作用72h,对HUVECs具有抑制增殖,且在50～200ng/ml的剂量范围内呈现浓度依赖关系;而对HepG2细胞未见明显作用.药物处理方式的不同,恩度对HUVECs诱导凋亡和迁移/侵袭作用差异显著;对HepG2细胞的迁移/侵袭未见明显影响.高剂量的恩度对HUVECs体外管腔形成和鸡胚尿囊膜的血管发生及成熟具有显著影响.结论:重组人血管内皮抑制素(恩度)能够有效地抑制血管内皮细胞形成复杂的管腔,并显著影响血管的成熟;对人肝癌细胞系HepG2生长及迁移/侵袭未见明显影响.     

Effects of the immunomodulator LS 2616 on growth and metastasis of the murine B16-F10 melanoma

The carboxamide-quinoline LS 2616 is a novel immunomodulator augmenting natural killer (NK) cell activity and T-lymphocyte related effector functions. To investigate the possible usefulness of LS 2616 in immunotherapy of tumors, the effect of the substance on growth and metastasis of the B16-F10 melanoma in syngeneic C57BL/6 mice was investigated. Treatment with LS 2616 from the time of s.c. inoculation of B16-F10 cells significantly reduced tumor take. Continuous treatment of mice with LS 2616 initiated 4 days prior to i.v. injection of tumor cells reduced the number of pulmonary metastases by 85%. When treatment with LS 2616 was started 4 days after i.v. injection of tumor cells, a time when established tumor foci were readily detectable in the lungs, a significant reduction in the number of pulmonary metastases resulted. LS 2616 significantly reduced the number of spontaneous pulmonary metastases developing from a B16-F10 tumor growing in the footpad. When treatment with LS 2616 was initiated after the establishment of grossly visible spontaneous pulmonary metastases, no significant effect on the number of metastases was found after 2 weeks of treatment. However, combined treatment with a dose of cyclophosphamide which in itself was ineffective resulted in a statistically significant 70% reduction in the number of remaining pulmonary metastases. Injection of antibodies to asialomonoganglioside which strongly reduce NK cell activity in various organs was used as a probe for the involvement of NK cells in the effects of LS 2616 on the B16-F10 tumor. The therapeutic efficiency of LS 2616 on tumor take when given from the time of s.c. inoculation, on the number of i.v. induced pulmonary metastases when treatment was started before tumor cell injection, as well as the spontaneous development of pulmonary metastases during exposure to the substance was abrogated by simultaneous injection with antibodies to asialomonoganglioside. In contrast, the beneficial effects of LS 2616 on already established i.v. produced or spontaneous pulmonary metastases were unaltered in mice made NK cell deficient by injection of anti-asialomonoganglioside antibodies. In conclusion, LS 2616 has potent antitumor activities mediated by NK cells as well as non-NK cell related defense mechanisms.     

Neoadjuvant immunotherapy with interferon of the spontaneously metastasizing murine B16F10L melanoma.

We have previously demonstrated that administration of interferon a/b (IFN) for 4-5 days after challenge with a transplantable Moloney sarcoma virus-induced tumor completely inhibited tumor development. In the present study, we examined the therapeutic effects of IFN on mortality induced by metastatic dissemination of the B16F10L murine melanoma. IFN was administered at various times in relation to the surgical removal of primary tumor: days -5 to -1 prior to tumor excision (neo-adjuvant protocol), or for 5 days after tumor excision, beginning on days 1, 6 or 11 after excision of the primary tumor (adjuvant protocols). The neo-adjuvant protocol was superior to all other protocols, significantly increasing percentage survival (56% vs. 0%) and median survival time (greater than 84 days vs. 33 days) compared to untreated controls, as well as to all adjuvant protocols. In contrast, IFN treatment on days 1 to 5 after excision of the primary tumor decreased median survival time of cases compared to untreated controls (20 days vs. 33 days). Both IFN-induced inhibition and enhancement of metastatic dissemination were dose-dependent, with higher amounts of IFN producing greater inhibition or enhancement. The superior therapeutic efficacy of the neo-adjuvant IFN treatment was associated with increased spleen and lung-derived natural killer cell cytolytic activity (on days -4, 0 and 2) followed by a later (day 13) increase in lung-associated cytolytic T-cell responses.     

4-1BBL联合Hsp70-肿瘤抗原肽抑制小鼠黑色素瘤肺转移

背景与目的正调节性共刺激分子表达偏低是肿瘤逃避机体免疫系统攻击的重要原因之一,增加这些分子的表达是促进抗肿瘤免疫的研究热点。小鼠黑色素瘤B16-F1细胞株是弱免疫原性癌细胞株。本研究旨在观察4-1BBL联合Hsp70-抗原肽对小鼠黑色素瘤肺转移的抑制作用,并探讨其机制。方法建立小鼠黑色素瘤肺转移模型,Hsp70-B16抗原肽小鼠皮下免疫,同时从尾静脉注射4-1BBL表达质粒p4-1BBL。于接种后第17天,解剖小鼠取肺组织,解剖显微镜下计数黑色素瘤肺转移结节的数目,并检测小鼠部分免疫学指标及肝、肾功能。结果4-1BBL联合Hsp70-B16抗原肽治疗组小鼠黑色素瘤肺转移结节数降至50±8,明显少于二者单独治疗组的500±80和450±40;联合治疗组小鼠血清IL-2和IFN-γ的分泌水平分别增加了4倍和3倍,与对照组相比均存在显著性差异(P<0.01);单独应用4-1BBL基因或与Hsp70-B16抗原肽联合应用对小鼠肝、肾的功能均无明显影响。结论表达4-1BBL联合应用Hsp70-B16抗原肽主要通过增强外周T淋巴细胞功能活性而有效抑制小鼠黑色素瘤肺转移。