Serotherapy of primary rat mammary carcinoma: inhibition by ethylenedinitrilotetraacetic acid but not by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid

We reported inhibition of growth of primary rat mammary carcinomas after infusions of tumor-bearer plasma absorbed with Protein A-Sepharose or inactivated CNBr Sepharose. Absorbed plasmas were depleted of the third component of complement (C3) (other complement components defined similarly) and C5 but not C1, C4, or C2. These results suggested that activation of the alternative pathway of complement might be involved in the observed antitumor effects. To test this concept sera were treated with ethylenedinitrilotetraacetic acid or [ethylenebis(oxyethylenenitrilo)]tetraacetic acid before absorption with Protein A-Sepharose. Ethylenedinitrilotetraacetic acid, by chelating calcium and magnesium, prevents activation of both the alternative and classical complement pathways. [Ethylenebis(oxyethylenenitrilo)]tetraacetic acid, by chelating calcium but not magnesium, permits activation of the alternative pathway but inhibits activation of the classical complement pathway. Sera in the presence or absence of chelating agent were absorbed with Protein A-Sepharose twice at room temperature. After absorption calcium was added to the sera. Rats were treated by i.v. injection of sera twice a week for 2 weeks. Measurements of tumor size were made weekly for 5-7 weeks and then tumor weight was determined. Groups were compared both for size of index and total tumors. The results can be summarized as follows: tumor-bearer sera before absorption did not inhibit the growth of rat primary mammary carcinomas; tumor-bearer sera after absorption with Protein A-Sepharose showed significant consumption of C3 and did inhibit tumor growth; tumor-bearer sera absorbed in the presence of ethylenedinitrilotetraacetic acid did not show a decrease in C3 functional activity and did not inhibit tumor growth; tumor-bearer sera absorbed in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid did show a decrease in C3 functional activity and did inhibit tumor growth; sera from normal adult female rats after absorption with Protein A-Sepharose did inhibit tumor growth. The results are consistent with a role for the alternative pathway of complement in the inhibition of growth of rat primary mammary carcinomas observed after treatment with absorbed sera.     

Detection of antibodies against the cellular protein p53 in sera from patients with breast cancer

Antibodies reacting with the host protein p53 were found in the sera of patients with primary or secondary carcinoma of the breast. Fourteen out of the 155 sera from breast cancer patients tested were positive for anti-p53 antibodies (9%) and no positives were detected among 164 control sera from normal women tested. The locations of the first metastasis in patients with positive sera were unusual, with more lung metastases and fewer bone metastases than expected. The detection of anti-p53 antibodies indicates that p53 is altered in amount, type or presentation in breast tumors so that it becomes immunogenic.     

Image-guided tumor resection using real-time near-infrared fluorescence in a syngeneic rat model of primary breast cancer

Tumor involvement of resection margins is found in a large proportion of patients who undergo breast-conserving surgery. Near-infrared (NIR) fluorescence imaging is an experimental technique to visualize cancer cells during surgery. To determine the accuracy of real-time NIR fluorescence imaging in obtaining tumor-free resection margins, a protease-activatable NIR fluorescence probe and an intraoperative camera system were used in the EMR86 orthotopic syngeneic breast cancer rat model. Influence of concentration, timing and number of tumor cells were tested in the MCR86 rat breast cancer cell line. These variables were significantly associated with NIR fluorescence probe activation. Dosing and tumor size were also significantly associated with fluorescence intensity in the EMR86 rat model, whereas time of imaging was not. Real-time NIR fluorescence guidance of tumor resection resulted in a complete resection of 17 out of 17 tumors with minimal excision of normal healthy tissue (mean minimum and a mean maximum tumor-free margin of 0.2 ± 0.2 mm and 1.3 ± 0.6 mm, respectively). Moreover, the technique enabled identification of remnant tumor tissue in the surgical cavity. Histological analysis revealed that the NIR fluorescence signal was highest at the invasive tumor border and in the stromal compartment of the tumor. In conclusion, NIR fluorescence detection of breast tumor margins was successful in a rat model. This study suggests that clinical introduction of intraoperative NIR fluorescence imaging has the potential to increase the number of complete tumor resections in breast cancer patients undergoing breast-conserving surgery.     

Antibodies reactive with the mouse mammary tumor virus in sera of breast cancer patients

IgG binding to purified mouse mammary tumor virus (MuMTV) was quantitated by an enzyme-linked immunoassay (ELISA) using sera from patients with breast cancer or benign breast disease, or from healthy age-matched controls. Significantly greater binding (p<0.01) was found in breast-cancer-derived sera than in the other categories. In addition to IgG reactivity, three breast cancer sera also possessed IgA and IgM reactive with MuMTV by the ELISA assay. Only IgG was reactive in the majority of sera while two sera possessed MuMTV binding activity only in the IgM fraction. Both IgG binding and virolysis of MuMTV were greatly reduced by preincubation of sera with MuMTV. The specificity for MuMTV was further explored with IgG of serum from one breast cancer patient. Human antibody reactive with MuMTV was progressively diminished by preincubating the human serum with increasing concentrations of MuMTV but not by incubation with the type-C AKR murine leukemia virus. Preincubation of MuMTV with a breast cancer serum partially blocked the reactivity of gp52 antiserum with the virus. The results suggest that an antigen related to an MuMTV envelope component is expressed in breast cancer.     

Methylation silencing of angiopoietin-like 4 in rat and human mammary carcinomas

Aberrant DNA methylation is deeply involved in the development and progression of human breast cancers, but its inducers and molecular mechanisms are still unclear. To reveal such inducers and clarify the molecular mechanisms, animal models are indispensable. Here, to identify genes silenced by promoter DNA methylation in rat mammary carcinomas, we took a combined approach of methylated DNA immunoprecipitation (MeDIP)-CpG island (CGI) microarray analysis and expression microarray analysis after treatment with epigenetic drugs. MeDIP-CGI microarray revealed that among 5031 genes with promoter CGI, 465 were methylated in a carcinoma cell line induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), but not in normal mammary epithelial cells. By treatment of the cell line with 5-aza-2'-deoxycytidine and trichostatin A, 29 of the 465 genes were shown to be re-expressed. In primary mammary carcinomas, five (Angptl4, Coro1a, RGD1304982, Tmem37 and Ndn) of the 29 genes were methylated in one or more of 25 samples. Quantitative expression analysis revealed that Angptl4 had high expression in normal mammary glands, but low expression in primary carcinomas. Also in humans, ANGPTL4 was unmethylated and expressed in normal mammary epithelial cells, but was methylated in 11 of 91 (12%) primary breast cancers. This is the first study to identify genes aberrantly methylated in rat mammary carcinomas, and Angptl4 is a novel methylation-silenced gene both in rat and human mammary carcinomas. The combination of the MeDIP-CGI microarray analysis and expression microarray analysis after treatment with epigenetic drugs was effective in reducing the number of methylated genes that are not methylation silenced.     

Comparison of protein profiles between acetonitrile- and non-acetonitrile-treated sera from patients with nasopharyngeal carcinomas

Serum proteins may be abnormally increased or decreased during the occurrence and development of nasopharyngeal carcinoma (NPC). However, currently there are no simple or effective methods to collect and differentiate these abnormally secreted proteins from abundant serum proteins. In this study, acetonitrile was used to remove the majority of high-abundance proteins from serum samples obtained from patients with NPC. The samples were subjected to surface-enhanced laser desorption/ionization time-of-flight mass spectrometry with a CM10 (weak cation exchange) ProteinChip, and the resulting protein profiles were compared with those of non-acetonitrile-treated serum samples. The results showed that the protein profiles differed between the acetonitrile- and non-acetonitrile-treated sera from patients with NPC. A large proportion of the non-acetonitrile-treated NPC serum protein peaks were <6000 kDa, while the detection rate of protein peaks >6000 kDa was relatively higher in the acetonitrile-treated NPC sera, accounting for more than half of all protein peaks (26.2+37.5%). Few differentially upregulated proteins were lost, and the peak value density increased after acetonitrile treatment. In conclusion, acetonitrile treatment of serum samples is effective in removing high-abundance macromolecular proteins. Therefore, acetonitrile treatment can be applied for the investigation of serum proteomics and may aid in the identification of differentially expressed proteins.     

Therapy of primary and metastatic mouse mammary carcinomas with doxorubicin encapsulated in long circulating liposomes.

The purpose of our study was to compare the therapeutic effects of doxorubicin in 3 different formulations: (1) in PBS, (2) in conventional liposomes composed of egg phosphatidylglycerol/egg phosphatidylcholine/cholesterol/dl-alpha tocopherol, and (3) in sterically stabilized, long-circulating "Stealth" liposomes composed of hydrogenated soy phosphatidylcholine/cholesterol/polyethylene glycol-distearoylphosphatidylethanolamine. The doxorubicin formulations were used to treat recently implanted and well-established, growing primary mouse mammary carcinomas, and to inhibit the development of spontaneous metastases from intra-mammary tumor implants. In the treatment of recently implanted primary tumors, the formulations were given in 3 i.v. injections over 15 days, starting 3 or 10 days after tumor implantation. In the treatment of well-established primary tumors, the mice received 4 i.v. injections over 22 days, starting an average 38 days after tumor implantation. In the preventive treatment against metastases, the formulations were given in 4 i.v. injections over 22 days, starting 22 days or 58 days after primary tumor implantation. The Stealth liposome formulation was significantly more effective than the conventional liposome formulation or the free drug in reducing the incidence of metastases from intra-mammary implants of tumor MC19 and tumor MC65, in curing mice with recent implants of tumor MC2A, tumor MC2B, and tumor MC65, and in increasing the 8-week survival of mice with well-established implants of tumor MC2B. It is concluded that the long circulation time of the Stealth liposome doxorubicin formulation accounts for its superior therapeutic effectiveness.     

Relationships between cellular superoxide dismutase and susceptibility to chemically induced cancer in the rat mammary gland

Increasing evidence suggests a role for reactive free radicaloxygen species in the multi-stage events of chemical carcino-genesis.We hypothesized that variations in the level of super-oxidedismutase (SOD), a major endogenous antioxidant enzyme, mayaccount in part for variations in susceptibility to cancer inducedby polycyclic aromatic hydrocarbons (PAH). The SOD activityof mammary epithelial cells from rats with varying susceptibilityto dimethylbenz[a]anthracene (DMBA)-induced breast cancer wasassayed. Ageing, pregnancy and previous multiple pregnanciesreduce susceptibility of Sprague Dawley female rats to DMBA.These decreases in susceptibility were correlated with increasedlevels of SOD activity. Only minor differences in SOD activitywas observed in mammary epithelium of genetic strains of ratswith differences in susceptibility to DMBA. These data suggestthat, in models where physiological differences may accountfor variations in effectiveness of PAH to induce mammary cancer,SOD activity is inversely correlated with breast cancer susceptibilityand support the hypothesis that cancer susceptibility may bepartially mediated through reactive free radical oxygen intermediates.     

Pathogenic characterization of 1-methyl-1-nitrosourea-induced mammary carcinomas in the rat

The induction of mammary carcinogenesis in the rat by 1-methyl-1- nitrosourea (MNU) is widely used in experimental breast cancer research. In the experiments reported, the Ha-ras codon 12 (ras12) mutation (GGA-->GAA) was used as a molecular marker to address issues of the clonality of carcinomas induced, pathogenetic independence among multiple carcinomas within the same animal and topographic distribution of mutant ras12 carcinomas in different mammary gland chains. In order to determine whether the frequently observed morphologically distinguishable lobules within carcinomas originate from the coalescence of independent lesions or whether cancerous cells within a carcinoma share a common origin, 44 randomly selected MNU-induced mammary carcinomas were genotyped for two to four lobules each for the ras12 mutation. A total of 43 carcinomas out of 44 (97.7%) had concordant ras12 genotypes among the multiple sites within each tumor, which is consistent with the latter possibility. Next, it was observed that as carcinoma multiplicity increased, the discordance rate of ras12 genotypes among multiple carcinomas within the same animal increased in a manner that was in excellent agreement with the expected discordance rate based on an assumption of no pathogenetic association among carcinomas. Furthermore, a significant difference was observed in the occurrence of mutant ras12 carcinomas between the cervical-thoracic and the abdominal-inguinal mammary glands in that three times as many carcinomas were mutant in the former as in the latter glands, whereas the occurrence of wild-type carcinomas was approximately the same in both regions. Taken together, the data are consistent with (i) carcinomas induced by MNU and detected by palpation are monoclonal in origin, (ii) independently-initiated cells emerge as distinct mammary carcinomas in the same animal, and (iii) the anatomical location of the gland may affect the prevalence of mammary carcinomas that harbor a mutant ras12.      

Differential expression of cAMP-kinase subunits is correlated with growth in rat mammary carcinomas and uterus.

The expression of the regulatory (RI and RII) and catalytic (C) subunits of cAMP-dependent protein kinase was found to depend on the growth-state in oestrogen-dependent DMBA-induced mammary adenocarcinomas as well as in uteri of the rat. Castration-induced atrophy of the oestrogen-dependent tissues was accompanied by a decrease of the concentration of regulatory subunits (RI and RII) relative to both the catalytic subunit (C) and total protein, decreasing the R/protein and R/C ratios. A hyperplastic burst caused by high-dose oestrogen-replacement treatment was associated with an increased level of RI and little change in RII and C levels. Only minor differences were noted for the expression of mRNA for the alpha and beta subtypes of RI, RII and C between rat uteri from castrated and oestrogen-treated animals, or between mammary tumours from normal and castrated animals. Expression of RI beta-mRNA was detected only in the uterus. Our findings provide an experimental correlate for the reported value of the parameter R/protein in human mammary cancer biopsies to predict prognosis and outcome of therapy. Due to the sensitivity of the R/protein ratio towards changes in extracellular protein content, we recommend the biologically more meaningful R/C ratio in further clinical evaluations of mammary tumour biopsies.     

Preferential growth stimulation of metastatic rat mammary adenocarcinoma cells by organ-derived syngeneic fibroblasts in vitro.

To determine whether organ-derived fibroblasts differentially affect the growth of cells from tumors that preferentially metastasize to specific organs, we investigated the effect of medium conditioned with primary cultured rat fibroblasts from various organs on the in vitro growth of metastatic cell lines and clones of the rat 13762NF mammary adenocarcinoma. The conditioned medium from fibroblasts derived from rat mammary fat pad differentially stimulated tumor cell growth in monolayer culture and clonogenic growth in soft agarose of the highly metastatic clone MTLn3 in a dose-dependent manner. Conditioned medium from fibroblasts derived from the lung and liver also stimulated the growth of clone MTLn3 cells but to a lesser extent than did mammary fat pad fibroblasts. In contrast, poorly metastatic cell clones (MTC, MTPa) did not respond to the growth stimulatory factor(s) from the fibroblast-conditioned medium. The factor(s) responsible for the growth stimulation were inactivated by heat and trypsin treatment and inhibited by low pH and cycloheximide. The result suggest that fibroblasts in different organs have different effects on tumor cell growth, and they may determine, in part, the organ specificity of tumor development and metastasis.     

Expression of c-erbB3 protein in primary breast carcinomas.

Expression of c-erbB3 protein was investigated in 104 primary breast carcinomas comprising nine comedo ductal carcinoma in situ (DCIS), 91 invasive ductal carcinomas and four invasive lobular carcinomas using two monoclonal antibodies, RTJ1 and RTJ2. Of the 91 invasive ductal carcinomas, seven contained the comedo DCIS component adjacent to the invasive component. An immunohistochemical technique was used to evaluate the association between expression of c-erbB3 and clinical parameters and tumour markers such as epidermal growth factor receptor (EGFR), c-erbB2, cathepsin-D and p53 in archival formalin-fixed paraffin-embedded tumour tissues. Our results indicated that RTJ1 and RTJ2 gave identical staining patterns and concordant results. It was found that the overexpression of c-erbB3 protein was observed in 67% (6/9) of comedo DCIS, 52% (44/84) of invasive ductal carcinomas, 71% (5/7) of carcinomas containing both the in situ and invasive lesions and 25% (1/4) of invasive lobular carcinomas. A significant relationship (P < 0.05) was observed between strong immunoreactivity of c-erbB3 protein and histological grade, EGFR and cathepsin-D, but not with expression of c-erbB2, p53, oestrogen receptor status, lymph node metastases or age of patient. However, we noted that a high percentage of oestrogen receptor-negative tumours (59%), lymph node-positive tumours (63%) and c-erbB2 (63%) were strongly positive for c-erbB3 protein. We have also documented that a high percentage of EGFR (67%), c-erbB2 (67%), p53 (75%) and cathepsin-D-positive DCIS (60%) were strongly positive for c-erbB3. These observations suggest that overexpression of c-erbB3 protein could play an important role in tumour progression from non-invasive to invasive and, also, that it may have the potential to be used as a marker for poor prognosis of breast cancer.     

Mechanisms by which energy restriction inhibits rat mammary carcinogenesis: in vivo effects of corticosterone on cell cycle machinery in mammary carcinomas

Increased secretion of adrenal cortical steroids may account in part for its cancer inhibitory activity of energy restriction (ER). To test this hypothesis, a study was conducted to determine the effects of dietary administration of corticosterone on the post-initiation stage of mammary carcinogenesis. Eighty-four female Sprague-Dawley rats were injected with 50 mg 1-methyl-1-nitrosourea/kg body wt (i.p.) at 21 days of age. One week later, animals were randomly divided into three groups and fed control diet, or that diet to which was added 200 or 400 mg corticosterone/kg. Diets were fed for 5 weeks after which the experiment was terminated. With increasing dietary corticosterone, a dose-dependent reduction in the incidence (P=0.03), multiplicity (P=0.003) and size (P<0.003) of mammary carcinomas was observed. Dietary administration of corticosterone also reduced plasma insulin-like growth factor-1 (IGF-1) and levels of IGF-1 receptor in mammary carcinomas (P<0.01). In order to investigate molecular mechanisms underlying anticancer activity, the levels and activities of cell cycle components involved in the G1-S transition were investigated in mammary carcinomas that emerged in treated animals. Levels of cyclin D1, cyclin E, cyclin-dependent kinase (CDK)-2 and CDK-4 were reduced in carcinomas from corticosterone treated rats; whereas, levels of cyclin-dependent kinase inhibitors (CKI) Kip1/p27 and Cip1/p21 were elevated. Binding of these CKIs to both the cyclin D1-CDK-4 complex and the cyclin E-CDK-2 complex were increased and the kinase activities of these complexes were reduced with increasing dietary corticosterone. These effects were consistent with those observed in response to ER in vivo and corticosterone exposure in vitro. Whereas the effects of exogenously administered corticosterone and ER had many similarities, the lower efficacy of corticosterone versus ER in inhibiting the carcinogenic process imply that changes in cortical steroid metabolism alone are unlikely to explain the cancer inhibitory activity of ER.     

乳腺癌与乳腺增生病某些内分泌激素的差异及其临床意义

目的通过比较乳腺癌与乳腺增生病患者内分泌激素变化,对内分泌激素与免疫系统的关系进行初步分析. 方法随机选取绝经前和绝经后的乳腺癌患者各28例、乳腺增生病患者各22例,在治疗前采血样,对垂体激素6项（PRL、GH、TSH、ACTH、FSH、LH）和性类固醇激素3项（E2、P、T）进行检测,采用秩和检验,正态近似法（Wilcoxon法）对结果行统计学分析. 结果绝经前乳腺癌患者FSH 水平高于乳腺增生患者,其它垂体激素（PRL,GH,TSH,ACTH,LH ）和性类固醇激素（E2,T,P）水平无显著性差异（P〉0.05）.绝经后乳腺癌患者ACTH水平高于乳腺增生病患者,其它垂体激素（PRL,GH,TSH,FSH, LH）和性类固醇激素（E2,T, P） 比较含量无显著差异（P〉0.05）.结论绝经前乳腺癌患者血浆中的FSH和绝经后乳腺癌患者ACTH水平高于乳腺增生病患者,说明乳腺癌患者内分泌激素与免疫系统之间正常规律被打破.下丘脑-垂体-肾上腺轴（HPA）短、长反馈失调,使免疫功能受抑制;雌激素诱导癌细胞基因突变,雄激素刺激细胞的敏感性增加,加速乳腺癌的发展.