Ipriflavone inhibits osteolytic bone metastasis of human breast cancer cells in a nude mouse model

Osteolytic bone metastasis is a frequent problem in the treatment of cancer. Ipriflavone, a synthetic isoflavone that inhibits osteoclastic bone resorption, has been used for the treatment of osteoporosis in some countries. Some other isoflavones also exhibit an antitumor effect in vitro and in vivo. Here, we studied the effects of ipriflavone on osteolytic bone metastasis of MDA-231 human breast cancer cells injected intracardially into athymic nude mice (ICR-nu/nu). Daily oral administration of ipriflavone at 12 mg/mouse significantly inhibited the development of new osteolytic bone metastases (p < 0.05) and the progression of established osteolytic lesions (p = 0.01), prolonging the life of tumor-bearing mice (p = 0.01 vs. control). In addition, ipriflavone reduced the number of osteoclasts at the bone-cancer interface with no severe adverse effects on the host. In vitro, ipriflavone inhibited the proliferation and DNA synthesis of MDA-231 cells and blocked the ligand-induced phosphorylation of Tyr(845) of the EGFR. Ipriflavone did not promote apoptosis of MDA-231 cells. Our results show that ipriflavone not only directly inhibits the growth of cancer cells but also reduces osteoclasts to prevent the soft tissue tumor burden and osteolytic bone metastases. These findings raise the possibility that ipriflavone may be of use as a therapeutic agent against osteolytic bone metastasis.     

Inhibition of colon cancer metastasis by a 3'- end antisense urokinase receptor mRNA in a nude mouse model

The role of urokinase-type plasminogen activator receptor (uPAR) in human colon cancer metastasis has not been tested using an antisense approach. In our study, the HCT116 cells, with high metastatic potential were transfected with expression vectors containing a 3' or 5' uPAR cDNA fragment in an antisense (AS) orientation. Transfection of 4 clones was confirmed by DNA hybridization analysis. Receptor-bound endogenous uPA activities of the clones were reduced to 16-68% of controls. The extracellular matrix degradation by the 4 clones was decreased to 33-76%. Two of the clones, 3'-AS7 and 5'-AS, were evaluated in an in vivo assay system of experimental metastasis using athymic mice. Pulmonary metastases were found in 63-78% mice injected with the parent HCT116 or control cells. In mice injected intravenously with the antisense transfected clones, 3'-AS7 and 5'-AS, however, pulmonary metastases were found in only 19% and 9% respectively (p < 0.05). These results provide direct evidence that both 3' and 5'-AS uPAR can inhibit colon cancer invasion and metastasis and may offer the prospect of defining specific targets for gene therapy.     

An orthotopic mouse model of remetastasis of human colon cancer liver metastasis.

Whether liver metastases from colon cancer are capable of metastasizing to other sites is an important question in surgical oncology. To answer this question, we have developed a highly metastatic orthotopic transplant model of a liver metastasis from a human colon cancer patient in nude mice that targets the liver and lymph nodes. The metastatic human tumor was transplanted in athymic nude mice by surgical orthotopic implantation (SOI) of a liver metastasis from a colon cancer patient. The human colon tumor was then subsequently implanted in the colon by SOI or, in an additional series of nude mice, in the liver by surgical hepatic implantation (SHI). The mice were then explored over time for lymph node involvement beginning 10 days after implantation. After SOI, 100% of the animals had liver metastasis within 10 days, and subsequently, 19 days after SOI, all lymph nodes draining the liver were involved with metastasis without any retroperitoneal or lung tissue involvement. After SHI, all sites of lymphatic drainage of the liver, including portal, celiac, and mediastinal lymph nodes, were massively involved by metastasis in 100% of the animals as early as 10 days after tumor implantation on the liver. The results of this study demonstrate that liver metastases from colon cancer are capable of remetastasizing to other sites. This study thus suggests that in colon cancer patients with liver metastasis, mediastinal, celiac, and portal lymph node metastases originate from the liver metastasis and not, as previously thought, from primary colon cancer.     

Development of a metastatic human colon cancer xenograft model in the nude mouse

The objective of our experimental protocols was to develop a metastatic model for a human colon carcinoma xenograft in congenitally athymic nude mice. This model would be useful in evaluating the efficacy of radiolabeled monoclonal antibodies for detection and treatment of regional and distant metastases. The LS-174T human colon carcinoma line was used to establish primary subcutaneous tumors in nude mice. Mice were killed at varying time intervals to establish the incidence of spontaneous metastases. Only lung micrometastases were observed during the 2-month observation period. To increase the metastatic rate, the site of primary implantation was varied and/or surgical manipulations were performed. Excision of small primary tumors resulted in a low incidence of local recurrence and no distant metastases. However, with excision of large primary tumors, a high local recurrence rate was noted and over 30% of mice had gross metastases. Mice bearing hind footpad tumors underwent excision when tumors were at least 1 cm in size. There were no local recurrences, but by 8 weeks over 40% had large pulmonary metastases. The LS-174T tumor was also established as a primary implant in the spleen from which 10 to 15% of the mice developed liver or lung metastases. The LS-174T tumor can metastasize in the nude mice and the latter two models may prove very useful in imaging and therapy studies.     

Mitogen-activated protein kinase antisense oligonucleotide inhibits the growth of human lung cancer cells

Mitogen-activated protein kinase (MAPK) pathway is proposed to be a therapeutic target for cancer cells. In order to find the potential therapeutic usefulness of MAPK for cancer cells, the effect of EAS1, an antisense oligonucleotide for an MAPK, on cancer-cell-growth were investigated in vitro. EAS1 effectively inhibited the growth of several human lung cancer cell lines such as PC-14 cells upon exposure to 10-0-10-1 microM of EAS1 determined dye-formation (MTT) assay. The ED50 values were comparable to those obtained for the inhibition of MAPK activity, DNA synthesis. EAS1 arrested the PC-14 cells at the G2/M phase of cell cycle followed by apoptosis in a dose-dependent manner. In order to determine the factors which influence the cellular sensitivity against MAPK inhibition, the effect of EAS1 on H-ras-transformed murine fibroblast cells were compared with that on parental cells. The NIH3T3 cells transformed by the H-ras gene (PT22-3) showed higher sensitivity against the effects of EAS1. Because MAPK activity was activated by H-ras gene transfection in PT22-3, the status of the MAPK cascade in cells was the determining factor for the efficacy of EAS1. In addition, cell permeabilization by digitonin enhanced the growth inhibitory effect of EAS1. Penetration of the cell membrane by EAS1 is also crucial for the growth inhibitory effect of EAS1. In conclusion, MAPK is an important target for cancer treatment and MAPK antisense oligonucleotide is a potentially significant antitumor oligonucleotide.     

The camptothecin derivative CPT-11 inhibits angiogenesis in a dual-color imageable orthotopic metastatic nude mouse model of human colon cancer

Recent studies have shown the expression of a stem cell marker protein, nestin, in nascent blood vessels in nestin-driven green fluorescent protein (ND-GFP) transgenic nude mice. In the present study, we visualized tumor angiogenesis and evaluated the antiangiogenic efficacy of CPT-11 in ND-GFP nude mice using dual-color fluorescence imaging. We orthotopically implanted ND-GFP nude mice with the human cancer cell line HCT-116 expressing red fluorescent protein (RFP). The mice were treated with CPT-11 at 40 mg/kg on days 7, 10, 14. Tumor angiogenesis was imaged and visualized by dual-color fluorescence imaging on day 17, three days after the last CPT-11 treatment. Tumor volume and the mean nascent blood vessel density were determined and compared to the control mice. The growing tumor had high expressions of nestin in the nascent blood vessels. The nascent blood vessels showed co-localization of the endothelial-cell-specific marker CD-31 under immunohistochemical staining. The nascent blood vessels were highly visible and their density was determined. ND-GFP nude mice that were administered CPT-11 showed significant reduction in the mean nascent blood vessel density and tumor volume. The dual-color model of ND-GFP transgenic nude mice orthotopically implanted with HCT-116 expressing RFP proved to be effective in visualizing and quantitating tumor growth and tumor angiogenesis. The results showed that CPT-11 is an effective inhibitor of angiogenesis and provided strong implications for wider clinical application of CPT-11 for colon cancer.     

Truncated galectin-3 inhibits tumor growth and metastasis in orthotopic nude mouse model of human breast cancer.

PURPOSE: The goal of this research was to evaluate a potential therapeutic agent for breast cancer based on galectin-3 that has been implicated in tumorigenicity and metastasis of breast cancer. The hypothesis was that therapy with NH(2)-terminally truncated form of galectin-3 (galectin-3C) will be efficacious for reduction in tumor growth and for inhibition of metastases. EXPERIMENTAL DESIGN: Recombinant human galectin-3 was produced in Escherichia coli from which galectin-3C was derived by collagenase enzyme digestion. Toxicity, pharmacokinetic, and organ biodistribution studies were performed in nude mice. For efficacy studies, nude mice bearing orthotopically implanted tumors derived from breast cancer cell line MDA-MB-435 were treated with galectin-3C or a vehicle control i.m. twice daily for 90 days. RESULTS: The maximum tolerated dose of galectin-3C in nude mice was determined to be >125 mg/kg without overt adverse effects. The elimination half-life when administered i.m. was found to be 3.0 h in the serum and 4.3 h in the cellular fraction of the blood. Organ biodistribution studies revealed that galectin-3C localized in the liver, kidneys, and spleen but not in the heart or lungs. We found that the mean tumor volumes and weights were statistically significantly less in mice treated with galectin-3C compared with control mice, and that fewer numbers of mice exhibited lymph node metastases in the treated group compared with the control group. CONCLUSIONS: Galectin-3C is not overtly toxic, and is efficacious in reducing metastases and tumor volumes and weights in primary tumors in an orthotopic nude mouse model of human breast cancer.     

Experimental nude mouse model of human colorectal cancer liver metastases

A nude mouse model (BALB/c) was established for investigating the production of hepatic metastases by human colorectal carcinoma (HCC) cells. The malignant potentials of 3 different HCCs derived from a primary tumor (HCC-P4733), a lymph node metastasis (HCC-M14328), and a hepatic metastasis (HCC-M1410) were investigated following implantation into the spleens of athymic nude mice. Cells of the HCC-M1410 line produced extensive liver tumors in all mice by 30 days after injection. Cells of the HCC-M14328 and HCC-P4733 lines produced few liver tumors in the inoculated mice and then only after a 90-day period. Isozyme and karyotype analyses ascertained the human origin of all the tumors. Studies with [125I]IdUrd-labeled HT-29 carcinoma cells suggested that tumor cells reached the liver shortly after injection into the spleen. Thus the production of HCC tumors in livers of nude mice was determined by the ability of HCC cells to proliferate in the liver parenchyma rather than by the ability of the cells to reach the liver. The results suggest that the intrasplenic injection of HCC cells can provide a valuable model for the study of the biology and therapy of the liver metastases.     

双调蛋白反义核酸表达对裸鼠乳腺癌血管生成的抑制作用

目的 研究双调蛋白（AR）反义RNA表达对乳腺癌血管生成的抑制作用。方法 乳腺癌NS2T2A1细胞经AR反义cDNA质粒转染后,经筛选获得表达AR反义RNA的AR-AS1及AR-AS3克隆,转染空载体获得NS2T2A1 V对照细胞,接种裸鼠皮下形成肿瘤。研究条件培养液对人微血管内皮细胞（HMEC）增殖的影响,以EL1SA法测定细胞血管内皮生长因子（VEGF）分泌量。以定量RT-PCR分析肿瘤组织的VEGF表达水平。以免疫组化法标记CD31研究肿瘤内血管数量。结果 HMEC在AR-AS1和AR-AS3细胞条件培养液中的增殖比例明显降低。AR-AS1和AR-AS3细胞VEGF分泌量亦降低。AR-AS1和AR-AS3肿瘤的血管数量仅有对照组的50％左右,且VEGF表达显著降低。结论 AR反义RNA表达可有效抑制乳腺癌的血管生成,应作为新治疗靶点进一步研究．     

康莱特注射液对结肠癌肝转移裸鼠血浆骨桥蛋白的影响

目的　建立人结肠癌ＬｏＶｏ细胞裸鼠肝转移模型,观察康莱特注射液（ＫＬＴ）对该模型裸鼠血浆骨桥蛋白的影响。方法　将３０只裸鼠随机分为５组：对照组、环磷酰胺（ＣＴＸ）组、ＫＬＴ组、ＣＴＸ＋ＫＬＴ组和正常组。前４组将０.２ｍｌＬｏＶｏ细胞悬液（１×１０７个／ｍｌ）接种于裸鼠脾脏,建立结肠癌肝转移模型,分别经腹腔注射给予生理盐水、ＣＴＸ、ＫＬＴ和ＣＴＸ＋ＫＬＴ。正常组未接种肿瘤细胞。接种４５天后处死全部裸鼠,称取４组脾脏接种瘤和肝脏转移瘤重量,并检测血浆骨桥蛋白浓度。结果　与对照组相比,ＣＴＸ组、ＫＬＴ组、ＣＴＸ＋ＫＬＴ组的脾脏接种瘤和肝脏转移瘤重量均减轻（Ｐ＜０.０５）,其中ＫＬＴ组分别为（４０１.７±５３.１）ｍｇ和（３５３.３±３９.８）ｍｇ,ＣＴＸ组＋ＫＬＴ组分别为（２３５.０±５２.４）ｍｇ和（１８５.０±２７.４）ｍｇ。与正常组相比,其余４组裸鼠血浆骨桥蛋白浓度均升高（Ｐ＜０.０５）;对照组血浆骨桥蛋白浓度为（２.１０±０.５５）ｎｇ／ｍｌ,ＣＴＸ＋ＫＬＴ组为（０.９２±０.３１）ｎｇ／ｍｌ,两组差异有统计学意义（Ｐ＜０.０５）,而ＣＴＸ组、ＫＬＴ组与对照组比较差异无统计学意义（Ｐ＞０.０５）。结论　ＫＬＴ联合ＣＴＸ可以抑制模型裸鼠的脾脏接种瘤及肝脏转移瘤的生长,抑制其血浆骨桥蛋白的浓度。     

Extensive liver metastasis from human colon cancer in nude and scid mice after orthotopic onplantation of histologically-intact human colon carcinoma tissue.

Clinically-relevant animal models of human cancer are greatly needed for the study of human cancer biology and the development of new cancer therapeutics and diagnostics. We report here that by orthotopically transplanting histologically-intact human colon cancer to the colon of the immunodeficient nude and scid mouse mutants that extensive local growth and liver metastases occur consistently even after extensive in vivo orthotopic passage. We demonstrate that the liver metastases arise by hematogenous spread. The models described in this report for human colon cancer should prove useful for individual cancer patients as well as for basic and applied studies to develop improved treatment.     

Interaction between colon cancer cells and human liver sinusoidal endothelial cells promotes liver metastasis of tumor cells

Objective  

To investigate the effect of co-culture between colon cancer cells (SW1116) and human liver sinusoidal endothelial cells (HLSECs) on cancer cell metastasis, and to provide a novel model for studying the mechanism of colon cancer liver metastasis.     

The nude mouse as anin vivo model for human breast cancer invasion and metastasis

Summary Human breast cancer xenografts only rarely invade and metastasize in nude mice, and have therefore only had limited use as a model for studying mechanisms involved in breast cancer spreading. However, recent reports describe differences not only between various cell lines but also between strains of immune-deficient mice in terms of providing a model for studies of the invasive and metastatic capability of human breast cancer xenografts. Genetic labelling of human cancer cells with the lacZ gene is described as a specific and highly sensitive method for identification of micrometastases in such a model.     

Novel diet-related mouse model of colon cancer parallels human colon cancer

AIM: To investigate the close parallels between our novel diet-related mouse model of colon cancer and human colon cancer. METHODS: Twenty-two wild-type female mice (ages 6-8 wk) were fed the standard control diet (AIN-93G) and an additional 22 female mice (ages 6-8 wk) were fed the control diet supplemented with 0.2% deoxycholic acid [diet + deoxycholic acid (DOC)] for 10 mo. Tumors occurred in the colons of mice fed diet + DOC and showed progression to colon cancer [adenocarcinoma (AC)]. This progression is through the stages of tubular adenoma (TA), TA with high grade dysplasia or adenoma with sessile serrated morphology, intramucosal AC, AC stage T1, and AC stage T2. The mouse tumors were compared to human tumors at the same stages by histopathological analysis. Sections of the small and large intestines of mice and humans were evaluated for glandular architecture, cellular and nuclear morphology including cellular orientation, cellular and nuclear atypia, pleomorphism, mitotic activity, frequency of goblet cells, crypt architecture, ulceration, penetration of crypts through the muscularis mucosa and presence of malignant crypts in the muscularis propria. In addition, preserved colonic tissues from genetically similar male mice, obtained from a prior experiment, were analyzed by immunohistochemistry. The male mice had been fed the control diet or diet + DOC. Four molecular markers were evaluated: 8-OH-dG, DNA repair protein ERCC1, autophagy protein beclin-1 and the nuclear location of beta-catenin in the stem cell region of crypts. Also, male mice fed diet + DOC plus 0.007% chlorogenic acid (diet + DOC + CGA) were evaluated for ERCC1, beclin-1 and nuclear location of beta-catenin. RESULTS: Humans with high levels of diet-related DOC in their colons are at a substantially increased risk of developing colon cancer. The mice fed diet + DOC had levels of DOC in their colons comparable to that of humans on a high fat diet. The 22 mice without added DOC in their diet had no colonic tumors while 20 of the 22 mice (91%) fed diet + DOC developed colonic tumors. Furthermore, the tumors in 10 of these mice (45% of mice) included an adenocarcinoma. All mice were free of cancers of the small intestine. Histopathologically, the colonic tumor types in the mice were virtually identical to those in humans. In humans, characteristic aberrant changes in molecular markers can be detected both in field defects surrounding cancers (from which cancers arise) and within cancers. In the colonic tissues of mice fed diet + DOC similar changes in biomarkers appeared to occur. Thus, 8-OH-dG was increased, DNA repair protein ERCC1 was decreased, autophagy protein beclin-1 was increased and, in the stem cell region at the base of crypts there was substantial nuclear localization of beta-catenin as well as increased cytoplasmic beta-catenin. However, in mice fed diet + DOC + CGA (with reduced frequency of cancer) and evaluated for ERCC1, beclin-1, and beta-catenin in the stem cell region of crypts, mouse tissue showed amelioration of the aberrancies, suggesting that chlorogenic acid is protective at the molecular level against colon cancer. This is the first diet-related model of colon cancer that closely parallels human progression to colon cancer, both at the histomorphological level as well as in its molecular profile. CONCLUSION: The diet-related mouse model of colon cancer parallels progression to colon cancer in humans, and should be uniquely useful in model studies of prevention and therapeutics.     

A human colon cancer metastasis model for radioimmunodetection

One important issue in radioimmunodetection is how well the current methods can locate and disclose small metastatic foci in visceral sites. We have developed a human colonic tumor metastasis model by surgically implanting GW-39 tumor cells in the liver of unconditioned hamsters. Tumors were produced in 71 of 73 animals and were macroscopically apparent within 1 wk. In addition, multiple nodular lung metastases of GW-39 were found in about 80% of the animals given implants of tumor in the liver, but implantation of tumor in the spleen failed to show lung metastases even after 4 wk. Hamsters bearing GW-39 liver and cheek pouch grafts or normal hamsters were given injections of a mixture of 131I-labeled anti-carcinoembryonic antigen antibody and 125I-labeled irrelevant immunoglobulin G. After 7 days, tumor was localized by external scintigraphy without subtraction techniques in both the liver and cheek pouch, but even in animals with extensive lung metastases we failed to unequivocally detect tumor in the lungs by external imaging or by comparing tissue counting data from uninvolved and tumor-bearing lungs. However, whole-body autoradiography confirmed specific localization of anti-carcinoembryonic antigen antibody in the tumors at all sites indicating that tissue counting and external imaging were not sensitive enough to reveal micrometastatic tumors. Thus, the current methods used for this model appear to be useful for further investigation of the radioimaging of tumors growing in visceral organs.     

塞来昔布抑制裸鼠乳腺癌骨转移实验研究

目的探讨塞来昔布(西乐葆)作为血管抑制剂对乳腺癌骨转移的形成及生长的影响。方法建立裸鼠乳腺癌骨转移模型。将12只乳腺癌骨转移裸鼠随机分成两组,对照组隔日腹腔注射生理盐水;西乐葆组隔日腹腔注射西乐葆30mg/kg;30天后处死。检测血清碱性磷酸酶,采用免疫组化和图像分析系统测定血管内皮生长因子(VEGF)和肿瘤微血管密度(MVD),光镜下观察各组肿瘤组织,流式细胞仪检测肿瘤细胞的凋亡。结果西乐葆组肿瘤生长明显受到抑制,肿瘤组织中的VEGF和MVD与对照组比较显著降低,抑瘤率为50.5%,凋亡指数上升,血清碱性磷酸酶下降。结论西乐葆能明显抑制裸鼠乳腺癌骨转移瘤的形成及新生血管形成。     

脾切除法建立人结肠癌细胞肝转移模型及对肝转移的评估

目的：建立类似临床大肠癌根治术后肝转移动物模型，并建立精确评估肝转移的方法。方法：在裸鼠脾内注入人结肠癌细胞５ｍｉｎ后切除脾脏。术后第３１天对裸鼠行病理检查并测定肝转移ＤＮＡ含量。结果：发现肝转移率为１００％，肝外其它脏器未见转移结节，组织学证实肝转移结节为低分化腺癌。脾内注入１０＾５癌细胞、脾切除组肝转移癌ＤＮＡ的含量比脾内注入１０＾６癌细胞、脾切除组肝转移癌ＤＮＡ的含量低６１．４％。结论：本实     

Anti-metastatic effect of capecitabine on human colon cancer xenografts in nude mouse rectum

Capecitabine (N4-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine) is a new fluoropyrimidine carbamate, which is converted to 5-fluorouracil (5-FU) by 3 sequential steps of enzyme reactions. We investigated the possibility of using capecitabine to prevent metastasis with a metastasis model of gastrointestinal cancer developed by the intrarectal injection of green fluorescent protein (GFP)-expressing colon cancer HT-29 cells (HT-29-GFP) into nude mice. Lung and lymph node metastasis in the HT-29-GFP rectal xenograft was assessed through both observation of GFP fluorescence and quantification of metastasis by amplification of a cancer-related human DNA by TaqMan PCR. Furthermore, for each organ, we examined mRNA levels of cancer-specific thymidine phosphorylase (dThdPase), which is an essential enzyme for capecitabine activation, by the quantitative RT-PCR method. Capecitabine inhibited the HT-29-GFP xenograft growth by 60.8% and 43.8% in the subcutaneous and rectal xenograft models, respectively. Furthermore, it inhibited both lung and lymph node metastasis by 99.9%. dThdPase expression in the tumor cells of both the rectal xenograft and metastatic lung tumor cells was upregulated by 10.0- and 24.3-fold that in the HT-29-GFP cells in vitro, respectively. These results indicated that capecitabine might effectively inhibit or suppress metastasis via upregulation of dThdPase expression. Capecitabine administration might be highly expected to reduce metastasis and improve survival of patients with gastrointestinal cancers.