Inhibition of angiogenesis by the cancer chemopreventive agent conjugated linoleic acid

Dietary conjugated linoleic acid (CLA) has been shown previously to inhibit rat mammary carcinogenesis. In addition to direct effects on mammary epithelial cells,including decreased proliferation and induction of apoptosis, CLA may exert its effects indirectly by inhibiting the differentiation of mammary stromal cells to an endothelial cell type. Specifically, CLA was found to decrease the ability of mammary stromal cells to form complex anastomosing microcapillary networks in vitro on Engelbreth-Holm-Swarm-derived reconstituted basement membrane. This suggested that CLA might inhibit angiogenesis in vivo. To test this possibility, CD2/F(1) mice were placed on synthetic diets containing 0, 1, or 2% CLA for 6 weeks, before angiogenic challenge by s.c. injection with an angiogenic gel substrate (Matrigel pellet assay). After 7 days, the pellets from animals fed the control diet were infiltrated by abundant branching networks of blood vessels with patent lumen-containing RBCs. In contrast, pellets from the CLA-fed animals contained fewer infiltrating cells, which formed limited branching cellular networks, the majority of which had collapsed lumen and no RBCs. Both levels of dietary CLA showed similar effects, with the number of RBC-containing vessels per 20x field decreased to a third of that seen in control. Dietary CLA decreased serum levels of vascular endothelial growth factor (VEGF) and whole mammary gland levels of VEGF and its receptor Flk-1. Both cis-9, trans-11 and trans-10, cis-12 CLA isomers were effective in inhibiting angiogenesis in vitro in a dose-dependent fashion. The ability of CLA to inhibit angiogenesis may contribute to its efficacy as a chemopreventive agent.     

Fatty acid synthase inhibitors are chemopreventive for mammary cancer in neu-N transgenic mice

High levels of fatty acid synthase (FAS) have been found in cancer precursor lesions of the colon, stomach, esophagus, oral cavity, prostate, and breast. Inhibition of FAS with C75 has led to a significant antitumor effect in both human breast and prostate cancer xenografts. Recently, HER2/neu, which has also been identified in preneoplastic breast lesions, has been shown to regulate FAS expression through the PI3K/Akt signal transduction pathway rendering them susceptible to FAS inhibition. Utilizing the neu-N transgenic mouse model of mammary cancer, weekly treatment of the neu-N mice with C75 (30 mg/kg) for 10 weeks significantly delayed tumor progression. Only 20% of the C75-treated transgenic mice developed mammary carcinoma by 220 days, compared to 50% in the vehicle control animals. Two C75-treated animals never developed mammary cancer. Analysis of mammary tissue following 10 weeks of C75 treatment revealed a significant delay in mammary maturation as manifested by a reduction of the number and caliber of mammary ducts and budding epithelial structures. Apoptotic changes were increased, DNA synthesis was decreased, and the expressions of FAS, neu, Akt, phospho-Akt, and p21(waf1) were all decreased when compared to vehicle controls and FVB/N mice. Importantly, these effects were restricted to the breast epithelial cells that overexpressed neu, not involving other normal duct structures in the skin, liver, or kidney. C247, an FAS inhibitor chemically distinct from C75, significantly delayed mammary maturation similar to C75. Thus, pharmacological inhibition of FAS affects the expression of key oncogenes involved in both cancer development and maintenance of the malignant phenotype. Moreover, these data identify FAS as a potential novel drug target for breast cancer chemoprevention.     

Selection of cancer chemopreventive agents based on inhibition of topoisomerase II activity

The present study was undertaken to determine if in vitro inhibition of one or both of the two most dominant mammalian DNA topoisomerases (topos) is common among chemopreventive agents. To determine if an agent was a topo I inhibitor, we employed the DNA relaxation and nicking assays. For potential topo II inhibitors, we used the DNA unknotting and linearisation assays. 14 of 30 agents (47%) were ineffective in all four assays (IC(50) >100 microgram/ml), and 11 (37%) inhibited topo II catalytic activity. The sensitivity of the topo II assay was 63%, selectivity 93%, positive predictive value 91%, and total accuracy 77%. For chemopreventive efficacy, the positive predictive value of the unknotting assay was 92%, and the total accuracy was 60%. These data suggest that reduced topo II activity is a desirable property of many known chemopreventive agents. We conclude that the unknotting assay could be a valuable addition to the in vitro tests presently used to select chemopreventive agents.     

Estrogen receptor-alpha in the inhibition of cancer growth and angiogenesis

A high level of estrogen receptor-alpha (ER-alpha) is believed to be favorable in the prognosis and treatment of certain female cancers. ER-alpha expression in the ER-negative breast cancer cell lines inhibits their proliferation and invasive, metastatic potential in vitro. We stably overexpressed the ER-alpha in the human endometrial cancer cell line Ishikawa and showed that, unlike estradiol, high levels of ER-alpha significantly inhibit the growth of tumors xenografted from the Ishikawa cells. Subsequent to ER-alpha overexpression, in vivo down-regulation of vascular endothelial growth factor was observed in tumor xenografts. In addition, these tumors showed an inhibition of vascularization and of the angiogenic agent, integrin alphavbeta3. Involvement of a switch in the angiogenic pathways during tumorigenesis has been a recent focus of interest. Our results indicate that a high level of ER-alpha may be beneficial in the control of female cancers because of its inhibitory effect on such angiogenic pathways.     

Development of a transplantable mammary gland cancer in rats at different thyroid hormone levels]

In experiments on female rats of various age the authors studied the effect of thyroid hormones on the growth of the mammary gland transplantable cancer. In young animals the percentage of spontaneous tumor regression and the survival were found to increase with age, synchronously with a rise in blood thyroxine level. Injection of thyroxine in a dose of 5 mkg into 2-month old animals induces marked inhibition of the growth, and in later terms a complete tumor regression in 78% of cases. In thyroidectomized rats a rapid tumor growth was terminated by death in all animals of this series. It is the authors' opinion that thyroid hormones stimulate the transplantation immunity, correct the functioning of the hypothalamo-hypophyseal-genital system.     

Salicylate inhibition of rat mammary carcinogenesis and angiogenesis in female rat compatible with misoprostol administration

Epidemiological data suggest that non-steroidal anti-inflammatory drugs prevent colon cancer. The evidence for other types of tumour is less conclusive, though animal and in vitro studies indicate that they may be effective against mammary cancer cells. We assessed the effect of dietary acetylsalicylic and salicylic acid against dimethylbenzanthracene-induced rat tumours. Tumour angiogenesis was also investigated to explore the mechanism responsible for salicylate effect. Mammary tumours were induced in female Sprague-Dawley rats fed with different amounts of acetylsalicylic and salicylic acid. Serum vascular endothelial growth factor concentrations were measured and vascularization of basement membrane proteins injected in vivo (Matrigel) was determined by evaluation of haemoglobin content to assess the extent to which angiogenesis was inhibited. Dimethylbenzanthracene-induced carcinogenesis was inhibited by both acids and there was a log-dose/response correlation between the tumour diameter and salicylate concentration. Salicylic acid seems more effective than acetylsalicylic acid. Vascular endothelial growth factor was less concentrated in treated animals than in the controls and so was Matrigel haemoglobin. The mechanism involved, however, is still uncertain, though concomitant inhibition of tumour angiogenesis may be an important component. The documented salicylate serum VEGF modulation is interesting also for presence of the flk-1 receptor in mammary tumour cells of our model. Although misoprostol is a prostaglandin analogous its concomitant administration did not compromise the salicylate anti-tumour effect.     

Dairy intake and 1,25-dihydroxyvitamin D levels in men at high risk for prostate cancer

Objective  

Dairy food intake has been associated with prostate cancer in previous work, but the mechanism by which this occurs is unknown. Dairy calcium may suppress circulating levels of potentially cancer-protective 1,25-hydroxyvitamin D (1,25(OH)₂D). We examined the associations of dairy, milk, calcium, and vitamin D intake with plasma 1,25(OH)₂D levels among 296 men (194 black, 102 non-black) enrolled in a high risk program for prostate cancer from 10/96 to 10/07.     

Effect of reduced body weight gain on the evaluation of chemopreventive agents in the methylnitrosourea-induced mammary cancer model

These studies examined whether the small to moderate reductionsin body weight gain (     

Targeted therapies of cancer: angiogenesis inhibition seems not enough

The therapeutic potential of targeting tumor endothelium to induce tumor regression is now widely recognized. Tumors obtain their blood supply by the formation of new vasculature and the incorporation of pre-existent vessels. Since anti-angiogenic therapy prevents formation of neovasculature, vessels in more matured stages are not affected by such therapies. Therefore, additional vascular targeting therapy, which aim at regression of existent tumor vasculature, seems an attractive approach to effectively deprive tumors from blood supply. In this review we present an overview of different strategies to target tumor endothelium. In addition, we discuss the pitfalls of anti-angiogenic therapies in clinical settings.     

VEGF and c-Met blockade amplify angiogenesis inhibition in pancreatic islet cancer

Angiogenesis inhibitors that block VEGF receptor (VEGFR) signaling slow the growth of many types of tumors, but eventually the disease progresses. Multiple strategies are being explored to improve efficacy by concurrent inhibition of other functionally relevant receptor tyrosine kinases (RTK). XL880 (foretinib, GSK1363089) and XL184 (cabozantinib) are small-molecule inhibitors that potently block multiple RTKs, including VEGFR and the receptor of hepatocyte growth factor c-Met, which can drive tumor invasion and metastasis. This study compared the cellular effects of XL880 and XL184 with those of an RTK inhibitor (XL999) that blocks VEGFR but not c-Met. Treatment of RIP-Tag2 mice with XL999 resulted in 43% reduction in vascularity of spontaneous pancreatic islet tumors over 7 days, but treatment with XL880 or XL184 eliminated approximately 80% of the tumor vasculature, reduced pericytes and empty basement membrane sleeves, caused widespread intratumoral hypoxia and tumor cell apoptosis, and slowed regrowth of the tumor vasculature after drug withdrawal. Importantly, XL880 and XL184 also decreased invasiveness of primary tumors and reduced metastasis. Overall, these findings indicate that inhibition of c-Met and functionally related kinases amplifies the effects of VEGFR blockade and leads to rapid, robust, and progressive regression of tumor vasculature, increased intratumoral hypoxia and apoptosis, and reduced tumor invasiveness and metastasis.     

Role of chemopreventive agents in cancer therapy

Tumorigenesis or carcinogenesis is a multi-step process that is induced primarily by carcinogens leading to the development of cancer. Extensive research in the last few years has revealed that regular consumption of certain fruits and vegetables can reduce the risk of acquiring specific cancers. Phytochemicals derived from such fruits and vegetables, referred to as chemopreventive agents include genistein, resveratrol, diallyl sulfide, S-allyl cysteine, allicin, lycopene, capsaicin, curcumin, 6-gingerol, ellagic acid, ursolic acid, silymarin, anethol, catechins and eugenol. Because these agents have been shown to suppress cancer cell proliferation, inhibit growth factor signaling pathways, induce apoptosis, inhibit NF-kappaB, AP-1 and JAK-STAT activation pathways, inhibit angiogenesis, suppress the expression of anti-apoptotic proteins, inhibit cyclooxygenase-2, they may have untapped therapeutic value. These chemopreventive agents also have very recently been found to reverse chemoresistance and radioresistance in patients undergoing cancer treatment. Thus, these chemopreventive agents have potential to be used as adjuncts to current cancer therapies.     

Clinical biomarkers of angiogenesis inhibition

INTRODUCTION: An expanding understanding of the importance of angiogenesis in oncology and the development of numerous angiogenesis inhibitors are driving the search for biomarkers of angiogenesis. We review currently available candidate biomarkers and surrogate markers of anti-angiogenic agent effect. DISCUSSION: A number of invasive, minimally invasive, and non-invasive tools are described with their potential benefits and limitations. Diverse markers can evaluate tumor tissue or biological fluids, or specialized imaging modalities. CONCLUSIONS: The inclusion of these markers into clinical trials may provide insight into appropriate dosing for desired biological effects, appropriate timing of additional therapy, prediction of individual response to an agent, insight into the interaction of chemotherapy and radiation following exposure to these agents, and perhaps most importantly, a better understanding of the complex nature of angiogenesis in human tumors. While many markers have potential for clinical use, it is not yet clear which marker or combination of markers will prove most useful.     

Rg3对甲状腺癌血管生成抑制作用的临床观察

为了探讨人参皂甙Rg3对人甲状腺癌血管生成的抑制作用 ,对 18例甲状腺癌患者在术前 2周口服Rg3 ,观察手术切除癌内微血管密度 (microvesseldexsity ,MVD)的变化。结果实验组血管减少明显 ,高倍镜下可见血管内皮细胞支架塌陷、变性、坏死 ;而对照组血管无明显改变。实验组MVD值计数 10 8 69± 2 5 2 4,对照组MVD计数 160 5 2±47 45 ,两组比较差异有统计学意义 ,P <0 0 0 1。初步研究结果提示 ,Rg3具有抑制甲状腺癌血管生成的作用 ,可有效抑制其生长和转移。     

Intracellular 58-kd selenoprotein levels correlate with inhibition of DNA synthesis in mammary epithelial cells

Five mouse mammary epithelial cell lines (MMEC) with differentgrowth rates were used to examine the relationship between selenoproteinlevels and selenite-mediated inhibition of DNA synthesis. Theinhibition of DNA synthesis preceded and was significantly greaterthan inhibition of protein synthesis. Cycloheximide, a proteinsynthesis inhibitor, caused a coordinate inhibition of bothDNA and protein synthesis over a 50-fold dose range. Of theselenoproteins detected by one-dimensional SDS-PAGE, the 58-,26- and 23-kd proteins were the only major selenoproteins observedin common among the five MMEC lines before and during inhibitionof DNA synthesis. Other selenoproteins were present in somecell lines or after inhibition of DNA synthesis. The level ofthe 58-kd selenoprotein was most closely correlated with thedegree of inhibition of DNA synthesis (r² = 0.85), whereas the26- and 23-kd proteins most closely correlated with seleniteretention (r² = 0.78). Upon selenite withdrawal from the growthmedium, the decrease in 58-kd, but not in the 26- and 23-kdproteins correlated with resumption of DNA synthesis. Similarly,dose-response studies indicated that the 58-kd protein increased> 20-fold, whereas the 26- and 23-kd proteins increased only5-fold. At high doses of selenite, other selenoproteins of mol.wts 30–101 kd were present but these proteins seemed toappear after inhibition of DNA synthesis. The possibility existsthat these proteins may be product/ precursors of the majorselenoproteins. Since these experiments attempted to quantitateselenoproteins by densitometry of one-dimensional SDS-PAGE autoradiographs,the quality controls used for the experiments are discussed.The results are discussed in terms of the hypothesis that the58-kd selenoproteins may mediate the effects of selenite onDNA synthesis.     

Characterization of effective chemopreventive agents in mammary gland in vitro using an initiation-promotion protocol

Mouse mammary glands respond to carcinogen stimulus to form mammary lesions in organ culture. Prevention of the formation of mammary lesion has been utilized as a test to evaluate the effectiveness of a variety of classes of agents. In the present study we determined whether the effective chemopreventive agents are active against initiation or the promotion phase of lesion development. Mammary glands were subjected to 24 hours exposure to 2 mg/ml dimethylbenz (a) anthracene (DMBA) followed by a 5 day exposure to 7,12-tetradecanoyl phorbol - 13 - acetate (TPA). This treatment protocol allows one to study initiation and promotion aspects of lesion development. Chemopreventive agents effective when present prior to the carcinogen were considered as anti-initiators, whereas agents effective when present after the DMBA treatment along with TPA were considered as anti-promoters. Within the chemopreventive agents evaluated, limonene, oltipraz, aspirin, curcumin and b-sitosterol were anti-initiators. Esculatin, thiolutin, silymarin, DHEA and a few others were found to be anti-promoters. Results presented in this report can be utilized to study the efficacy of these agents in vivo.     

A combination of resveratrol and melatonin exerts chemopreventive effects in N-methyl-N-nitrosourea-induced rat mammary carcinogenesis

The neurohormone melatonin is primarily involved in the regulation of circadian rhythms, but also acts as an antioxidant and anticarcinogenic agent, especially in breast cancer. Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a widely known polyphenolic agent from red wine, which has been shown to exert antioxidant, anti-inflammatory and anticarcinogenic effects. The objective of this study was therefore to investigate the effects of melatonin in combination with resveratrol in a rat model of experimental mammary carcinogenesis. Female Sprague-Dawley rats aged 31 days were used in the experiment. Mammary carcinogenesis was induced by N-methyl-N-nitrosourea (NMU), which was administered in two intraperitoneal doses (50 mg/kg of body weight). Chemoprevention with resveratrol and melatonin started 2 weeks before the first dose of NMU and lasted until the end of the experiment. The basic parameters evaluated were: tumour incidence, latency period, tumour frequency per group and tumour volume. In addition, oestrogen receptors ERα and ER?, melatonin receptor MT1, proliferating cell nuclear antigen and vascular endothelial growth factor were determined by immunohistochemical staining. The combination of resveratrol and melatonin reduced tumour incidence by approximately 17% and significantly decreased the quantity of invasive and in-situ carcinomas. Food intake declined in the second and seventh weeks after the administration of carcinogen. Resveratrol in combination with melatonin returned food intake to the level of intact controls. Resveratrol in combination with melatonin has some protective effects on NMU-induced rodent breast cancer. Further studies are necessary to confirm these effects of this promising combination.     

Targeting angiogenesis for mammary cancer prevention: factors to consider in experimental design and analysis.

An experimental model developed to investigate premalignant stages of breast cancer was used to establish a rationale for designing experiments that target angiogenesis for cancer prevention. Blood vessels were identified via CD31 immunostaining, and all vessels that occurred in a 50 microm wide region circumscribing each pathology were counted using a digital imaging technique. The blood vessel density associated with terminal end buds was unaffected by carcinogen treatment, whereas vessel density was higher in intraductal proliferations and ductal carcinoma in situ than in terminal end buds (P < 0.001) and total vascularity increased with morphologic progression. In comparison with intraductal proliferation or ductal carcinoma in situ, mammary carcinomas had higher vascular density in the tissue surrounding the cancer with a marked increase in the number of blood vessels <25 microm(2). These data suggest that antiangiogenic chemopreventive agents would inhibit cancer occurrence if initiated at any premalignant stage of the carcinogenic process. Because increased vascular density observed during premalignancy could be explained by the size expansion of the lesion and its encroachment on a preexisting blood supply, by pathology-associated vessel expansion, and/or by angiogenesis, it remains to be determined if antiangiogenic agents will reduce the prevalence of premalignant lesions or cause their accumulation by blocking conversion to carcinomas. Failure to recognize the patterns of vascularization that accompany morphologic progression could limit the success of efforts to target angiogenesis for cancer prevention and lead to misinformation about how agents that affect blood vessel formation or growth inhibit the carcinogenic process.     

Tissue selenium levels in selenium-supplemented rats and their relevance in mammary cancer protection

The present study was designed to investigate whether there is any correlation between the anticarcinogenic efficacy of selenium (Se) compounds and tissue Se retention under high levels of supplementation. With the use of the dimethylbenz[a]anthracene-induced mammary tumor model in chemoprevention experiments, our data showed that selenomethionine was not as active as selenite over a graded dose range from 1 to 5 p.p.m. Se. Tissue Se concentrations in blood, liver, kidney and skeletal muscle were always higher in rats given selenomethionine compared with those given selenite at each of the three levels tested (1, 3 and 5 p.p.m. Se). The difference was only minimal in blood, but became more pronounced in the liver and kidney, and was quite dramatic in the skeletal muscle. Thus a high tissue concentration or total body burden of Se is not necessarily an indicator of reduced susceptibility to carcinogenesis. The bioavailability of the Se pool in maintaining liver glutathione peroxidase activity during a period of Se deprivation, following excess selenite or selenomethionine loading, was also assessed. The half-life of decay of the enzyme was calculated to be 4.2 and 9.1 days respectively, in those rats that had already been exposed to 3 p.p.m. Se as either selenite or selenomethionine. From a nutritional viewpoint, selenomethionine may be superior to selenite, especially with respect to maintenance of glutathione peroxidase during periods of Se inadequacy, but the reverse seems to be true in terms of anticarcinogenic potency under high levels of Se supplementation. These results suggest that the nutritional and anticarcinogenic efficacies of a given Se compound may not be parallel to each other.     

Correlation between retinoid inhibition of N-methyl-N-nitrosourea-induced mammary carcinogenesis and levels of retinoic acid binding proteins

A correlation was made between the ability of retinoids to suppressN-methyl-N-nitrosourea (MNU)-induced mammary carcinogenesisand the levels of cytosolic retinoic acid binding proteins (cRABP)in the cytosol of MNU-induced mammary tumors. Although retinylacetate and N-(4-hydroxyphenyl)retinamide were found to be effectiveinhibitors of mammary carcinogenesis in intact hosts, both retinoidswere significantly more active in ovariectomized rats than inintact animals. Quantitative analyses of cRABP in the tumorsindicated that mammary cancers arising in animals which wereovariectomized one week after MNU administration contained significantlyincreased concentrations of cRABP compared to cancers appearingin intact rats. In addition, when animals bearing palpable mammarytumors were ovariectomized, the tumors which continued to growcontained significantly higher levels of cRABP than did tumorswhich stopped growing or regressed. These data suggest thatthe selective inhibition of ovarian hormone-independent mammarycancer by retinyl acetate and N-(4-hydroxyphenyl)retinamidemay be mediated through an increased level of cRABP in tumorcells of ovariectomized hosts.