The effects of estrogen on human breast carcinomas serially transplanted into nude mice

The effect and mechanisms of 17 beta-estradiol (E2) on breast cancer cells were studied in vivo and in vitro, using 5 human breast carcinomas serially transplanted into nude mice. These carcinoma strains consisted of 4 estrogen receptor (ER) positive tumors and 1 ER negative tumor. Mice bearing these tumors were treated with an intramuscular injection of E2 at a dosage of 50 mg/kg and the tumor doubling time (Td) was calculated in days. The tumor growth was significantly stimulated by E2 in 3 out of the 4 ER positive tumors, the Td of the E2 treated groups being 17.6 days for MCF-7 (control: -17.8 days), 12.8 days for R-27 (control: -12.5 days approximately 14.5 days) and 10.4 days for Br-10 (control: 14.5 days), however, in the T-61 tumor, the growth was inhibited by E2 in a dose dependent manner. In the case of the ER-negative MX-1 tumor, the tumor cell growth was not affected by E2. Discrepancies between the effects of E2 on ER-positive tumors were further analyzed by examining the steroid hormone receptor status and conducting in vitro growth studies. In vitro clonogenic cell assay reproduced the antitumor activity of E2, indicating that E2 directly inhibits part of the cell growth of T-61 tumors. The above results suggest that this experimental system provides a useful tool for analyzing the mechanism of estrogen in breast cancer and that the clonogenic assay using ER positive specimens can help to identify breast cancers sensitive to estrogen therapy.     

Changes in the hormone receptors of human breast carcinoma xenografts in nude mice by treatment with cytotoxic agents

We examined the effect of chemotherapeutic agents on the estrogen receptors (ER) of breast carcinomasin vivo using human breast carcinoma strains (Br-10, T-61) serially transplanted into nude mice. When the tumor size reached approximately 1×1×1 cm, mitomycin C (MMC) at doses of 1, 2 and 4.5 mg/kg and cyclophosphamide (CPA) at a dose of 120 mg/kg, were administered once intraperitoneally, and the ERs of the tumors were measured sequentially by the dextran-coated charcoal method. Four days after the MMC administration at above doses, the binding sites of ER in Br-10 were not reduced and binding affinity was not affected. When the changes in ER content with time after the treatment with 4.5 mg/kg MMC and 120 mg/kg CPA were investigated, the ER content was found to be stable until 4 days after the treatment with both drugs, although the growth of T-61 had been significantly inhibited by the drugs. From these findings, it seems reasonable to initiate chemotherapy before endocrine therapy, since the chemotherapeutic agents did not reduce the ER content of the breast cancer strains.     

Human breast carcinoma (MCF-7) serially transplanted into nude mice

The tumor cells (0.5 ml, 1×10⁷) of MCF-7 line were inoculated into the subcutaneous tissue or intraperitoneum of female BALB/c nude mice. Primarily tansplanted mice were treated with 17β-estradiol dipropionate (E₂) in a dose of 5 mg/kg and 17α-hydroxy progesterone caproate (Pg) in a dose of 250 mg/kg once a week. After the transferable strain was established, tumors were transplanted into female and male mice treated with E₂, Pg, and E₂+Pg. The tumors treated with E₂ or E₂+Pg grew exponentially while tumors in the other group regressed. Pg was assumed to play some role in the growth of MCF-7, in the presence of estrogen. Although cytosol estrogen receptors (ERc), nuclear estrogen receptors (ERn), and progesterone receptors (PgR) were detected by dextran coatedcharcoal method and exchange assay in the growing tumors, ERn and PgR of regressing tumors was usually negative. This MCF-7 strain in nude mice may be a promising animal model for studying chemo-hormone therapy for human breast carcinomas.     

Crosstalk Between IGF1R and Estrogen Receptor Signaling in Breast Cancer

After the discovery that depriving certain breast tumors of estrogen promoted tumor regression, therapeutic strategies aimed at depriving tumors of this hormone were developed. The tumorigenic properties of estrogen are regulated through the estrogen receptor-α (ER), making understanding the mechanisms that activate this receptor highly relevant. In addition to estrogen activating the ER, other growth factor pathways, such as the insulin-like growth factors (IGFs), can activate the ER. This review will examine the interaction between these two pathways. Estrogen can activate the growth stimulatory properties of the IGF pathway via ER’s genomic and non-genomic functions. Further, blockade of ER function can inhibit IGF-mediated mitogenesis and blocking IGF action can inhibit estrogen stimulation of breast cancer cells. Collectively, these observations suggest that the two growth regulatory pathways are tightly linked and a more thorough understanding of the mechanism of this crosstalk could lead to improved therapeutic strategies in breast cancer.     

Changes in the hormone receptors of human breast carcinoma xenografts in nude mice by treatment with cytotoxic agents

We examined the effect of chemotherapeutic agents on the estrogen receptors (ER) of breast carcinomas in vivo using human breast carcinoma strains (Br-10, T-61) serially transplanted into nude mice. When the tumor size reached approximately 1 X 1 X 1 cm, mitomycin C (MMC) at doses of 1, 2 and 4.5 mg/kg and cyclophosphamide (CPA) at a dose of 120 mg/kg, were administered once intraperitoneally, and the ERs of the tumors were measured sequentially by the dextran-coated charcoal method. Four days after the MMC administration at above doses, the binding sites of ER in Br-10 were not reduced and binding affinity was not affected. When the changes in ER content with time after the treatment with 4.5 mg/kg MMC and 120 mg/kg CPA were investigated, the ER content was found to be stable until 4 days after the treatment with both drugs, although the growth of T-61 had been significantly inhibited by the drugs. From these findings, it seems reasonable to initiate chemotherapy before endocrine therapy, since the chemotherapeutic agents did not reduce the ER content of the breast cancer strains.     

Ipriflavone, a Synthetic Phytoestrogen, Enhances Intestinal Calcium Transport In Vitro

Ipriflavone (IP), a synthetic isoflavone, prevents bone loss associated with ovarian hormone deficiency in women and animal models. This protective effect of IP may be partly due to its ability to enhance calcium absorption. The purpose of this study was to examine the effects of IP and 17β-estradiol (E₂) on in vitro intestinal calcium transport in an ovariectomized rat model using E₂ as a positive control. Forty-eight 90-day-old female Sprague-Dawley rats were divided into four groups: one sham-operated (sham) and three ovariectomized groups. The ovx groups were either control (ovx), supplemented with IP (100 mg/kg body weight daily) via gavaging (ovx+IP), or injected with E₂ (10 μg/kg body weight) (ovx+E₂). Animals were fed diets containing 0.4% calcium, 0.3% phosphorus, and 0.195 nmol vitamin D₃/g for 35 days from the date of surgery. Animals were exsanguinated, and isolated cells from the duodenum, jejunum, ileum, and colon were used to measure in vitro calcium uptake. Calcium uptake by duodenal cells was significantly greater in the IP and E₂-treated animals compared with the ovx control group. In addition, calcium uptake by the ileal and colonic cells of the E₂-treated animals was significantly greater compared with all the other groups. The results confirm our earlier findings implicating a role for estrogen in duodenal calcium uptake. The findings also indicate that IP, although less potent than estrogen, significantly enhances calcium uptake in the duodenum, the active site of calcium absorption. Received: 21 July 1999 / Accepted: 10 February 2000     

Steroid Receptor Heterogeneity in Relation to DNA Index in Breast Cancer: A Multiparameter Flow Cytometric Approach on Paraffin-Embedded Tumor Samples

Abstract: Steroid hormone (estrogen and progesterone) receptor (ER and PR) status at the time of breast carcinoma surgery is used as a marker for hormone dependency to guide adjuvant therapy. In a significant number of cases a discrepancy exists between the detected number of hormone receptors and the response to hormonal therapy. One of the explanations for this could be intratumoral heterogeneity. Our objective was to investigate the heterogeneity of steroid hormone receptor expression in breast cancer by using multiparameter flow cytometry (MP-FCM) on routinely processed formalin-fixed, paraffin-embedded tumors. A series of 232 routinely processed breast carcinomas were analyzed using a recently developed technique for the isolation of single cells from paraffin-embedded material. After dewaxing and rehydrating, 50-μm thick sections were heated for 2 hours at 80°C in a citrate solution. Single-cell suspensions were prepared by a short pepsin digestion. The obtained single-cell suspensions were immunostained simultaneously for cytokeratin and ER or PR. Finally, DNA was stained using propidium iodide, after which the samples were analyzed on a flow cytometer. The fractions of ER- and PR-positive cells were determined in the total, as well as the G₀ /G₁ fraction of the diploid, and in case of nondiploid tumors, also in the G₀ /G₁ fraction of the aneuploid cell population. Of 232 cases, 88 (38%) were diploid, 38 (16%) were tetraploid, and 106 (46%) were aneuploid. In the diploid tumors the mean fraction of ER- and PR-positive cells was 81% and 76%, respectively. The ER- and PR-positive fractions in the total cytokeratin-positive fraction decreased significantly in the tetraploid (56% and 55%, respectively) and aneuploid tumors (both 47%, p < 0.0001). When analyzing the ER- and PR-positive fractions separately in the diploid and aneuploid cell populations of the nondiploid tumors, it became apparent that the ER and PR status in the diploid fraction of the tumor was significantly higher than in the aneuploid fraction (p < 0.0001). For the tetraploid tumors the mean ER- and PR-positive fractions were 79% and 76%, respectively, in the diploid fraction, and this decreased to 45% in the aneuploid cell subpopulation. In the aneuploid tumors this decrease was even more drastic: in the diploid cell population the ER- and PR-positive fractions were 66% and 62%, while this was 38% and 39% in the aneuploid population. These findings illustrate clearly the existence of a heterogeneous distribution of ER/PR expression in breast cancer, related to the loss of a diploid DNA index. Because of its objective quantification of subfractions within the same tumor, MP-FCM can be regarded as a superior method compared to more conventional techniques such as immunohistochemistry and biochemistry.     

Endogenous NIS Expression in Triple-Negative Breast Cancers

Background  The sodium iodide symporter (NIS) mediates iodide transport into cells and has been identified in approximately 70% of breast cancers. Functional NIS expression raises the possibility of using ¹³¹I for therapeutic targeting of tumor cells. Treatment of triple-negative breast cancers [estrogen/progesterone receptor-negative and HER2-negative (ER−/PR−/HER2−)] is primarily limited to chemotherapy. Our aim was to characterize NIS expression in this subset of tumors. Methods  Archival tissue sections from 23 women with triple-negative breast cancer were analyzed for NIS expression using immunohistochemical methods and an anti-human NIS antibody. Tumors were evaluated for the presence of plasma membrane immunoreactivity. One patient with a NIS-expressing positive tumor underwent ¹²³I scintigraphic imaging with dosimetric analysis. Results  Fifteen cases (65.2%) demonstrated NIS-positivity with 11 tumors (47.8%) exhibiting strong expression. Plasma membrane immunoreactivity was observed in four breast cancers and was equivocal in another four tumors. Tumor-specific radioiodide uptake was demonstrated by ¹²³I scintigraphy in a patient with a large primary breast cancer unresponsive to neoadjuvant therapy. The tumor concentrated 2.05, 1.53, and 1.96 times more isotope than normal breast tissue at 1, 5, and 21 h. The relative increased uptake is consistent with positive NIS expression in the tumor on definitive surgery; however, the cumulative concentration in the tumor was not sufficient to achieve a therapeutic effect, had the isotope been ¹³¹I. Conclusions  NIS is strongly expressed in a significant proportion of triple-negative breast cancer cells, suggesting a potential role for NIS-directed ¹³¹I-radioablative strategies in this patient population. Kent Nowles—Posthumous.     

Interactions between ipriflavone and the estrogen receptor

Estrogen replacement therapy is effective in the prevention of postmenopausal osteoporosis, and a direct action of 17--estradiol (17E₂) on osteoblastic and osteoclastic cells has been demonstrated. The inhibition of bone resorption by ipriflavone (IP), an isoflavone derivative devoid of estrogenic properties but active in potentiating the effects of estroge on bone tissue, has been shown in in vitro and in vivo studies and confirmed by clinical data. To investigate the molecular mechanisms that underlie IP effect, we studied the possible interactions of IP and its four main in vivo metabolites (I, II, III, and V) with the estrogen receptor (ER) in the human preosteoclastic cell line FLG 29.1, whose growth and function are modulated by the compound. In parallel experiments, the human breast cancer cell line MCF7 was also analyzed. IP binding sites were demonstrated in the nuclear fraction of FLG 29.1 cells. 17E₂ and other steroid compounds failed to displace IP binding to intact FLG 29.1 cells. Similarly, IP and metabolites I, III, and V were not able to displace 17E₂ binding to intact MCF7 cells, whereas metabolite II showed an IC₅₀ of 61 nM. 17E₂ binding to FLG 29.1 cells was increased after preincubation with metabolites I, III, and V. IP and its metabolites did not induce FR-dependent gene expression in FLG 29.1 and MCF7 cells transfected with a reporter gene and an estrogen response element (ERE). These results suggest that IP effects on osteoclast precursors are not mediated by a direct interaction with the ER, even if a crosstalk between the mechanisms of action of IP and 17E₂ cannot be excluded.     

Preoperative Profiling of Symptomatic Breast Cancer by Diagnostic Core Biopsy

Background Precise preoperative profiling of breast tumors could facilitate fuller consideration of (neo)adjuvant therapies. Methods Diagnostic core biopsy (DCB) accuracy in profiling the primary tumor was prospectively studied in 95 patients with operable breast cancer. The histological type and grade (hematoxylin and eosin staining) and membrane receptor status (semiquantitative immunohistochemistry for estrogen [ER] and progesterone [PR] receptors, as well as Her-2 antigen expression) were assigned by the DCB before surgery. These measures were then compared with those of the definitive surgical specimen available after operation. Results DCB correctly ascribed tumor type and grade and ER, PR, and Her-2 receptor status in most cases (correlating exactly in 97.5%, 77%, 68%, 71%, and 60%, respectively) with at least moderate concordance (weighted κ, >.41). When miscategorized, DCB consistently tended to upscore the receptor stain intensity compared with the surgical specimen (22%, 19%, and 27% had higher ER, PR, and Her-2 categorical scores, respectively). ER H-scores correlated best in specimens that stained strongly (224.4 ± 3 vs. 215.5 ± 5) and were significantly higher on DCB in those that stained either moderately (195.6 ± 8.2 vs. 156.8 ± 5.1; P < .0001) or weakly (157.1 ± 24.8 vs. 81.4 ± 4; P = .02). DCB accurately identified all tumors with clinically important ER and Her-2 expression. Furthermore, it promoted three patients into the therapeutically significant range of ER (n = 1) or Her-2 (n = 2) expression. ER negativity on DCB (n = 25) indicated a high-grade tumor (88%), although 11 (44%) patients also overexpressed Her-2. Significant Her-2 expression (n = 16) on DCB predicted the tumor as being poorly differentiated (80%) and both ER and PR negative (67%). Conclusions DCB accurately profiles clinically relevant measures of primary tumor cell differentiation. It also reliably categorizes patients with regard to (neo)adjuvant therapy before radical surgery is attempted.     

Function of the IGF-I Receptor in Breast Cancer

The insulin-like growth factor-I receptor (IGF-IR)³ is a transmembrane tyrosine kinaseregulating various biological processes such as proliferation, survival, transformation, differentiation,cell-cell and cell-substrate interactions. Different signaling pathways may underlie thesepleiotropic effects. The specific pathways engaged depend on the number of activated IGF-IRs,availability of intracellular signal transducers, the action of negative regulators, and is influencedby extracellular modulators. Experimental and clinical data implicate the IGF-IR in breastcancer etiology. There is strong evidence linking hyperactivation of the IGF-IR with theearly stages of breast cancer. In primary breast tumors, the IGF-IR is overexpressed andhyperphosphorylated, which correlates with radio-resistance and tumor recurrence. In vitro,the IGF-IR is often required for mitogenesis and transformation, and its overexpression oractivation counteract effects of various pro-apoptotic treatments. In hormone-responsive breastcancer cells, IGF-IR function is strongly linked with estrogen receptor (ER) action. TheIGF-IR and the ER are co-expressed in breast tumors. Moreover, estrogens stimulate the expressionof the IGF-IR and its major signaling substrate IRS-1, while antiestrogens downregulateIGF-IR signaling, mainly by decreasing IRS-1 expression and function. On the other hand,overexpression of IRS-1 promotes estrogen-independence for growth and transformation. InER-negative breast cancer cells, usually displaying a more aggressive phenotype, the levelsof the IGF-IR and IRS-1 are often low and IGF is not mitogenic, yet the IGF-IR is stillrequired for metastatic spread. Consequently, IGF-IR function in the late stages of breastcancer remains one of the most important questions to be addressed before rationalanti-IGF-IR therapies are developed.     

Suppression of plasma testosterone and prostate carcinoma size by a redox-based,brain-targeted estrogen delivery system in the rat

Studies were undertaken to examine the effects of an estradiol-chemical delivery system (E₂-CDS) or castration (CAST) on plasma testosterone (T) and growth of the Segaloff 11095 carcinoma. Fischer 344 rats were implanted subcutaneously with the Segaloff 11095 tumor and tumor growth was monitored thereafter. After optimal tumor growth, when the average tumor size was ～25 × 15 mm (length × width; 4–5 g wet weight), rats were randomized into (1) testis-intact controls; (2) CAST; (3) intact + E₂-CDS groups (rats received weekly injection of the E₂-CDS at 0.5 mg/kg). Animals were killed 7 or 14 days after the initiation of treatments. Blood and tissue samples were collected for subsequent analysis. Plasma T levels were suppressed by 98% and 97% through 14 days after CAST or E₂-CDS treatment. CAST increased plasma gonadotropin (LH) concentrations, while E₂-CDS reduced LH compared to intact control levels. E₂-CDS treatment increased plasma E₂ levels to 24 (one injection) or 75 pg/ml (two injections) at 7 or 14 days, respectively. E₂-CDS, given once a week for 2 consecutive weeks, resulted in a decreased growth of the prostate tumor by 61%, while CAST reduced the weights of these tumors by only 20%. In response to E₂-CDS (one or two injections), weights of the in situ ventral prostate and seminal vesicles were significantly reduced by 70% and 50%, respectively, in tumorbearing rats. Similarly, CAST reduced the weights of these tissues by 80% (prostate) or 52% (seminal vesicle) at 7 or 14 days after treatment. Pituitary weight increased, while testes weight decreased by 20% with two injections of E₂-CDS, compared with intact control rats. Collectively, these data indicate that E₂-CDS is effective in reducing the growth rate of prostatic tumors in the rat. © 1993 Wiley-Liss, Inc.     

17β-Estradiol Rapidly Activates Calcium Release from Intracellular Stores via the GPR30 Pathway and MAPK Phosphorylation in Osteocyte-Like MLO-Y4 Cells

Estrogen regulates critical cellular functions, and its deficiency initiates bone turnover and the development of bone mass loss in menopausal females. Recent studies have demonstrated that 17β-estradiol (E₂) induces rapid non-genomic responses that activate downstream signaling molecules, thus providing a new perspective to understand the relationship between estrogen and bone metabolism. In this study, we investigated rapid estrogen responses, including calcium release and MAPK phosphorylation, in osteocyte-like MLO-Y4 cells. E₂ elevated [Ca²⁺] _i and increased Ca²⁺ oscillation frequency in a dose-dependent manner. Immunolabeling confirmed the expression of three estrogen receptors (ERα, ERβ, and G protein-coupled receptor 30 [GPR30]) in MLO-Y4 cells and localized GPR30 predominantly to the plasma membrane. E₂ mobilized calcium from intracellular stores, and the use of selective agonist(s) for each ER showed that this was mediated mainly through the GPR30 pathway. MAPK phosphorylation increased in a biphasic manner, with peaks occurring after 7 and 60 min. GPR30 and classical ERs showed different temporal effects on MAPK phosphorylation and contributed to MAPK phosphorylation sequentially. ICI182,780 inhibited E₂ activation of MAPK at 7 min, while the GPR30 agonist G-1 and antagonist G-15 failed to affect MAPK phosphorylation levels. G-1-mediated MAPK phosphorylation at 60 min was prevented by prior depletion of calcium stores. Our data suggest that E₂ induces the non-genomic responses Ca²⁺ release and MAPK phosphorylation to regulate osteocyte function and indicate that multiple receptors mediate rapid E₂ responses.     

The Effect of 17β-Estradiol at Doses of 0.5, 1 and 2 mg Compared with Placebo on Early Postmenopausal Bone Loss in Hysterectomized Women

In a randomized double-masked placebo-controlled parallel-group trial 166 hysterectomized (± oophorectomy) perimenopausal and postmenopausal women aged 45–55 years with a follicle stimulating hormone level above 20 IU/l were treated with one daily dose of either 0.5 mg 17β-estradiol (E₂), 1 mg E₂, 2 mg E₂ or placebo for 2 years. Bone mineral density (BMD) and biochemical bone markers were determined. All three doses of E₂ were significantly better than placebo with respect to change in BMD at the lumbar spine (L1–L4) (p<0.0001 for all pairwise comparisons) and hip (femoral neck, trochanter, Ward’s triangle). The mean percentage change from baseline at the lumbar spine was −0.2%, 0.8% and 1.8% in the 0.5, 1 and 2 mg E₂ groups respectively compared with −3.5% in the placebo group. Both 1 and 2 mg E₂ were significantly better than placebo in increasing the BMD at the femoral neck (p<0.001), trochanter (p<0.01) and Ward’s triangle (p<0.0001), while 0.5 mg E₂ was significantly better than placebo at the femoral neck (p<0.001) and Ward’s triangle (p<0.0001). The overall difference in mean percentage change in BMD at the femoral neck versus placebo (−0.2%) was 3.8% for 0.5 mg, 4.0% for 1 mg and 3.9% for 2 mg E₂; the corresponding numbers for trochanter were −0.3%, 1.3%, 3.3% and 3.2%, respectively, and −2.2%, 2.9%, 2.9% and 4.0%, respectively, for Ward’s triangle. More than half the women who received placebo presented with a decrease in BMD at the hip. The percentage of women in the 0.5 mg E₂ group who maintained or increased BMD at the femoral neck, trochanter and Ward’s triangle was 69%, 56% and 44%, respectively. For 1 mg E₂ the numbers were 69%, 78% and 61% respectively, and for 2 mg E₂ were 59%, 68% and 59% respectively. Osteocalcin, serum pyridinium crosslinks, urinary pyridinium crosslinks and urinary hydroxyproline/creatinine decreased significantly (p<0.0001, p<0.05) in the 0.5, 1 and 2 mg E₂ groups compared with the placebo group after 6 and 24 months of treatment. Received: 28 May 2001 / Accepted: 11 October 2001     

The Cell of Origin of <Emphasis Type="Italic">BRCA1</Emphasis> Mutation-associated Breast Cancer: A Cautionary Tale of Gene Expression Profiling

Breast tumours are highly heterogeneous with several distinct sub-types recognised according to their histological and molecular features. The biological basis for this heterogeneity is largely unknown, although there are some distinct phenotype–genotype correlations. These include BRCA1 mutation-associated breast cancers, which are typically high grade invasive ductal carcinomas of no special type (IDC-NSTs) with pushing margins that do not express estrogen receptor (ER), progesterone receptor (PR) or the HER2 receptor tyrosine kinase (‘triple negative’). Gene expression analysis of these tumours has grouped them with so called ‘basal-like’ breast cancers and this, together with evidence that knock-down of BRCA1 in vitro blocked luminal differentiation, led to speculation that these tumours arose from the normal basal stem cells within the mammary gland. Recently, however, human breast tissue from BRCA1 mutation carriers was shown to contain an expanded population of luminal progenitor cells which have increased in vitro clonogenic ability. In the mouse, targeted deletion of Brca1 in luminal ER negative progenitors resulted in the formation of mammary tumours which phenocopied human BRCA1 breast tumour pathology, while the deletion of Brca1 in basal stem cells resulted in the formation of tumours which neither resembled human BRCA1 tumours or sporadic basal-like breast tumours. Importantly, however, both sets of mouse tumours were classified as ‘basal-like’ by methods used for human tumour classification based on gene expression profiles. This demonstrates that, as it stands, expression profiling is poor at distinguishing tumour histological subtypes and is also a poor guide to the cell of tumour origin. These human and rodent studies support an origin of BRCA1-mutation associated breast cancer (and indeed of the majority of sporadic basal-like breast cancers) in a luminal ER negative mammary epithelial progenitor. This is a key finding, as identification of the cells of origin in breast cancer subtypes makes possible the identification of key processes associated with initiation, progression and maintenance of each tumour subtype, the development of novel targeted therapies and, potentially, of new preventative approaches in high risk groups.     

Factors Predicting the Axillary Lymph Node Metastasis in Breast Cancer: Is Axillary Node Clearance Indicated in Every Breast Cancer Patient?: Factors Predicting the Axillary Lymphnode Metastases in Breast Cancer

The study was carried out to find out predictors of axillary lymph node metastasis in breast cancer and to evaluate its significance in selecting the group of patients in whom axillary dissection could be avoided. Ninety-five breast cancer patients who underwent mastectomy and axillary dissection were included in the study. Factors like patient’s age, tumor size, histopathological type, histological grade and estrogen and progesterone receptor status were correlated with the axillary metastases. Out of 95 cases axillary metastasis was found in 47 (49.47%) cases. There was no correlation between patient’s age and tumor size with axillary metastasis (p > 0.05). Based on histopathological typing tumors like ductal carcinoma in situ, tubular carcinoma and mucinous carcinoma showed less tendency for axillary metastasis (p < 0.046). Association was found between histological grade and estrogen receptor and progesterone receptor positivity with presence of axillary metastasis (p < 0.001 and 0.002 respectively). The findings in this study indicate that breast cancer patients having favorable histological type, grade I tumors and estrogen and progesterone receptor negative tumor are good candidates to avoid axillary dissection.     

Docetaxel inhibits bone resorption through suppression of osteoclast formation and function in different manners

Osteoclasts are formed from the monocyte-macrophage lineage in response to receptor activator of nuclear factor κB ligand (RANKL) expressed by osteoblasts. Bone is the most common site of breast cancer metastasis, and osteoclasts play roles in the metastasis. The taxane-derived compounds paclitaxel and docetaxel are used for the treatment of malignant diseases, including breast cancer. Here we explored the effects of docetaxel on osteoclastic bone resorption in mouse culture systems. Osteoclasts were formed within 6 days in cocultures of osteoblasts and bone marrow cells treated with 1,25-dihydroxyvitamin D₃ plus prostaglandin E₂. Docetaxel at 10⁻⁸ M inhibited osteoclast formation in the coculture when added for the entire culture period or for the first 3 days. Docetaxel, even at 10⁻⁶ M added for the final 3 days, failed to inhibit osteoclast formation. Osteoprotegerin, a decoy receptor of RANKL, completely inhibited osteoclast formation when added for the final 3 days. Docetaxel at 10⁻⁸ M inhibited the proliferation of osteoblasts and bone marrow cells. RANKL mRNA expression induced by 1,25-dihydroxyvitamin D₃ plus prostaglandin E₂ in osteoblasts was not affected by docetaxel even at 10⁻⁶ M. Docetaxel at 10⁻⁶ M, but not at 10⁻⁸ M, inhibited pit-forming activity of osteoclasts cultured on dentine. Actin ring formation and l-glutamate secretion by osteoclasts were also inhibited by docetaxel at 10⁻⁶ M. Thus, docetaxel inhibits bone resorption in two different manners: inhibition of osteoclast formation at 10⁻⁸ M and of osteoclast function at 10⁻⁶ M. These results suggest that taxanes have beneficial effects in the treatment of bone metastatic cancers. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Breast cancers: estrogen and progesterone receptor status as a predictor of in vitro chemotherapeutic response

Although clinical observations have shown that estrogen receptor-positive (ER+) breast tumors are more responsive to hormonal therapy than ER-negative (ER-) tumors, it remains controversial whether ER status can predict chemotherapeutic response. To determine if there was any correlation between estrogen and progesterone values and in vitro chemosensitivity to various anticancer drugs, clonogenic (CA), estrogen (ERA), and progesterone (PRA) assays on breast cancers were performed on 100 patients. Clonogenic assays were performed using the double-layer soft agar technique with continuous drug exposures. ERAs and PRAs were performed using the charcoal-coated dextran method. Chemosensitivity was defined as 50% inhibition of colony formation. ERA was considered positive if greater than or equal to 5 fmole/mg cytosol and PRA positive if greater than or equal to 10 fmole/mg cytosol. Significant tumor growth (greater than 30 colonies/plate) was achieved in 81/100 assays. ERA and PRA values were not predictive of colony formation in vitro. Of all agents or combinations of agents tested (L-PAM, 5-FU, MTX, adriamycin, vinblastine, cis-plat, FAC, CMF), only the response to 5-FU correlated significantly with ERA. Eight of 11 (73%) of the ER- tumors were sensitive to 5-FU, whereas only 6/20 (30%) of ER+ tumors were sensitive (P less than 0.05). ER- tumors were also more likely to be sensitive to CMF (P = 0.09) and adriamycin (P = 0.07) than ER+ tumors. PRA values were not predictive of chemosensitivity, nor did combining PRA and ERA enhance the predictive value of ERA alone.     

Estrogen Metabolism Modulates Bone Density in Men

Estrogen is a critical hormone for bone homeostasis in men, but no information is available on the role of estrogen metabolism among men. The aim of this study was to evaluate the effect of estrogen hydroxylation on male bone mineral density (BMD). Participants consisted of 61 healthy Caucasian males (mean age 66.6 ± 1.0 years). Urinary estrogen metabolites were measured by enzyme-linked immunosorbent assay, serum estradiol by ultrasensitive radioimmunoassay, sex hormone binding globulin by radioimmunoassay, and BMD of the lumbar spine and the proximal femur by dual-energy X-ray absorptiometry. Active estrogen metabolites, 16α-hydroxyestrone (16αOHE₁) and estriol (E₃), positively correlated with adjusted BMD in all regions of the proximal femur (all P < 0.05) but not at the lumbar spine, and those in the highest tertile of urinary 16αOHE₁ had the highest BMD. Free estradiol index (FEI) also positively correlated with BMD of the total hip, femoral neck, and intertrochanter (all P < 0.05), while there was no correlation between BMD with inactive metabolites (2−hydroxyestrone and 2-methoxyestrone) and serum testosterone. Multiple regression analysis showed 16αOHE₁, FEI, and body mass index are important independent predictors of BMD in all regions of the proximal femur. Estrogen metabolism may modulate BMD in men. Increased urinary 16αOHE₁ and E₃ levels are associated with high BMD at the proximal femur, and 16αOHE₁ appears to be a major determinant of BMD among the metabolites evaluated.