β-Glucuronidase levels in patients with fibrocystic breast disease

Certain enzymes in tissues and body fluids may, through reversal of the detoxification process, influence the composition and availability of steroid hormones, toxins, and carcinogens. The ubiquitous enzyme -glucuronidase, which hydrolyzes glucuronide conjugates, thereby reversing one of the main detoxification and excretion pathways, was found to vary in concentration in different cysts over a 300-fold range. The distribution was a continuum, devoid of discrete sub-populations. Evidence obtained on selected cyst fluids of high and low -glucuronidase activities indicated that the level of the enzyme significantly influenced the ratio of unconjugated: glucuronidated estradiol. The patients with fibrocystic breast disease fell into 2 distinct subpopulations on the basis of their serum -glucuronidase activity. In one group the activity was near normal, while in the second group the average serum -glucuronidase activity was 3-fold higher than in the women who did not have benign breast disease.     

Effects of human breast fibroblasts on growth and 17β-estradiol dehydrogenase activity of MCF-7 cells in culture

Summary The effect of conditioned medium from human breast fibroblasts on growth and 17-estradiol dehydrogenase (E2DH) activity of the MCF-7 human breast cancer cell line has been investigated. Fibroblasts were derived from normal and tumorous (benign and malignant) breast tissue. Conditioned medium from normal derived fibroblasts inhibited the growth of MCF-7 cells, the effect being statistically significant by day 3 of culture. In contrast, conditioned medium from benign and malignant derived fibroblasts significantly enhanced the growth of MCF-7 cells by day 9 of culture. When added to MCF-7 cells for three days, conditioned medium from all three types of fibroblasts increased E2DH activity in the reductive direction (estrone (E1) estradiol (E2)) 2–3 fold. There was little or no effect on the oxidative direction (E2 E1). After 12 days, enzyme activity in the reductive direction was still markedly increased, the effect being greatest in conditioned medium from fibroblasts derived from malignant breast tissue, and least in conditioned medium from fibroblasts derived from benign breast tumors. These results demonstrate that human breast fibroblasts may have paracrine influences on neighbouring epithelial cellsin vivo.     

Response of multicellular tumor spheroids to liposomes containing TNF-α

Summary This publication describes a new model to investigate the influence of tumor necrosis factor- (TNF-) on a three-dimensional glial cell aggregate under defined, standardized, reproducible conditions using the glioma cell line A 172.The cells are initially grown as normal monolayer culture until they reach a cell density of up to 1×10⁶. Subsequently they are grown as spheroids by the liquid overlay technique. Spheroids grown in this way were divided into ten groups of more than 50 cell aggregates. Three groups were coincubated with free TNF- in increasing dosages (100 ng/ml, 200 ng/ml and 1000 ng/ml); three groups were incubated with empty liposomes (0.2 mg/ml, 0.4 mg/ml and 2 mg/ml); three groups received liposomes which had been loaded with TNF-, and one group, which received no treatment, served as control.The diameter of the spheroids ranged from 80 m to 350 m. There was no significant difference in growth between the 3 groups treated with free TNF-. Comparing spheroids treated with TNF- with those which had been coincubated with empty liposomes, there was a significant difference (p<0.001) in growth, which correlated with the amount of liposomes. Similarly, free TNF- had a significantly (P<0.001) stronger growth-inhibiting effect as compared to liposomes loaded with TNF-. Comparing the groups treated with liposomes only to those treated with liposomes loaded with TNF-, the latter exhibited a more marked (although not significantly) growth-inhibiting effect.The preliminary conclusion is that the major growth-inhibiting effect seems to be mediated by the liposomes. This phenomenon is in agreement with results obtained in monolayer cultures.     

Glycogen‐rich carcinomas of the breast display unique characteristics with respect to proliferation and the frequency of oligonucleosomal fragments

We determined the proliferation rate and apoptotic activity of glycogenrich carcinomas of the breast as opposed to nonclear cell tumors by means of MIB1 immunohistochemistry and in situ detection of oligonucleosomal fragments (TUNEL reaction). The retrospective biopsy series included six invasive clear cell carcinomas of the glycogenrich type as well as 15 randomly selected cases of invasive ductal carcinoma without evidence of glycogen storage. Three patients in the clear cell group and seven patients in the control cohort developed lymphnode metastasis. The MIB1 labeling index of glycogenrich carcinomas averaged 9.05%, while that of the controls was 30.03%. Apoptotic nuclei were present in a mean of 1.26% of glycogenrich carcinoma cells. The control tumors exhibited an average apoptotic frequency of 5.85%. Tumor size, hormone receptor status, and presence or absence of lymph node involvement were found not to correlate with either proliferation or apoptosis. We conclude that glycogenrich breast carcinomas are characterized by a peculiar low proliferationlow apoptosis cell kinetic profile. The aggressive clinical behavior of these neoplasms may possibly be accounted for by an ineffective apoptotic elimination of otherwise slowly proliferating tumor cells.     

IFN-β Gene Therapy Induces Systemic Antitumor Immunity Against Malignant Glioma

We previously demonstrated that intratumoral administration of liposomes containing the murine interferon beta (IFN-) gene [lip(pSV2muIFN-)] resulted in stronger growth-inhibitory effect on GL261 (H-2^b) mouse glioma inoculated in brains of syngeneic C57BL/6 mice than conventional exogenous IFN- administration, and histologic evaluation revealed the massive infiltration of T lymphocytes (CD8 > CD4) within the residual tumor. The present study was aimed at determining whether such tumor-infiltrating lymphocytes (TIL) have any tumor-specific cytotoxic effects. Intratumoral administration of lip(pSV2muIFN-) resulted in prolonged survival time and a 50% tumor-free incidence in the mice treated. The surviving animals were subsequently re-challenged with either subcutaneous or intracranial injection of GL261 cells, and no tumors were found to develop over a 50-day period. In vivo depletion of CD8, but not CD4 cells decreased the efficacy of lip(pSV2muIFN-). Specific cytotoxic T lymphocytes (CTL) against GL261 cells were generated from both TIL and spleen cells of the mice treated. The results of flow cytometric analysis and antibody blocking test revealed that the bulk CTL lines thus prepared were T cell receptor (TCR) , CD8 T lymphocytes with H-2^b restriction.These findings suggest that, in addition to direct growth-inhibitory effects by the IFN- gene on the tumor cells, activation of systemic cellular immunity may participate in antitumor effects in vivo, despite the fact that central nervous system is generally regarded as an immunologically privileged site.     

Estrone sulfate: A potential source of estradiol in human breast cancer tissues

Summary Local formation of estradiol in human breast tumors could provide a more important source of estrogen than is delivered from plasma. Prior studies have suggested that estrone is primarily synthesized from estrone sulfate. The enzyme 17-hydroxysteroid dehydrogenase (HSD) would be required to convert estrone to estradiol.This study characterized HSD in 1000 × g supernatants from human breast tumors. Estradiol synthesis was linearly related to tissue concentration or time over the range studied. Cofactor requirements varied with estrone concentration. High and low affinity sites were found in 50% of tissues studied, while the remainder contained only low affinity sites. Screen assays showed measurable activity in all 42 samples tested. This activity ranged from 0.73–>100 nmol estrone synthesized/g protein/hr, with a median activity of 5.9 nmol/g/hr.We evaluated the biological relevance of the sulfatase-HSD pathway by testing the ability of estrone sulfate to stimulate colony formation in soft agar cultures of nitrosomethylurea-induced rat mammary tumors. The maximally effective concentration ranged from 10^–7 to 10^–4 M. Significant stimulation of colony formation was observed in 7 of 8 experiments. The estrone sulfate stimulation pattern was similar to that previously observed with estradiol. Of the³H-estrone sulfate added to the dishes, 20–98% was recovered as estrone and 0.2–6% as estradiol. These studies suggest that the requisite enzymes are present in human breast tumors for conversion of estrone sulfate to estradiol, and that this pathway may be biologically significant.This work presented in part at the International Congress on Endocrinology of the Breast, September 19–22, 1984, Torino, Italy.     

Robust Ability of IFN-γ to Upregulate Class II MHC Antigen Expression in Tumor Bearing Rat Brains

T cells are attractive for delivering therapy to brain tumor, especially disseminated micro-tumor. However, to trigger effector function, tumor antigen must be re-presented to T cells, via major histocompatibility complex (MHC) proteins, at the tumor site. In normal brain, MHC⁺ antigen-presenting cells (APC) are rare, but abundant after gamma interferon (IFN-) injection. Here we studied tumor-bearing brains. IFN- (or buffer) was injected stereotactically into brains with established tumors from a panel of immunologically varied glioma cell lines, some expressing b-galactosidase as a micro-tumor marker. Four days later, cryostat sections were stained for tumor and MHC proteins. In phosphate-buffered saline-injected controls, class II MHC⁺ potential APC (microglia, macrophages) were seen only at (some) tumor sites. In rats that received IFN-, class II⁺ potential APC were widespread, including all actual and potential micro-tumor sites and all tumor-free areas. In the same slides, neither class I nor class II MHC antigen was detected in neural cells or most tumor cells. This MHC pattern favors indirect re-presentation of tumor antigen, by tumor-adjacent APC. The robust response to IFN- might also be exploited in other ways: activated microglia and macrophages can attack tumor directly, and class II⁺ APC may help mark micro-tumor sites.     

Phase II study of cisplatin and 5‐fluorouracil in previously treated metastatic breast cancer: an Eastern Cooperative Oncology Group study (PA 185)

Purpose: The present study was conducted to investigate the efficacy and toxicity of a cisplatin and 5fluorouracil (5FU) combination in previously treated advanced breast cancer.Methods: Thirtysix women with recurrent metastatic breast cancer were entered on a phase II study of 5FU 1000mg/m²/day given intravenously as a continuous infusion on days 1–3 and cisplatin 30mg/m²/day given intravenously over 1h on days 2–4, repeated every 21 days. All subjects had received one previous chemotherapy regimen for metastatic disease and either progressed during treatment or relapsed after responding to previous chemotherapy. Fourteen patients had also received previous adjuvant chemotherapy, 17 patients had previous radiation therapy, and 29 patients had previous hormonal therapy.Results: Among 32 responseevaluable patients, there were 10 partial remissions (31%) and 1 complete remission (3%), with an overall objective response rate of 34%. Median duration of response was 4 months. Median survival was 10.5 months for responders and 9.5 months for the entire group. Toxicity was mild to moderate in most patients. Overall twelve patients experienced grade 3 toxicity (10 hematologic, 1 mucositis, and 2 nausea). There were no grade 4 or 5 toxicities.Conclusion: Infusional cisplatin and 5FU is a well tolerated and active regimen in women with previously treated advanced breast cancer.     

Aromatase and prostaglandin inter-relationships in breast adipose tissue: significance for breast cancer development

The role of prostaglandins in the development of breast cancer is a topic of growing interest. Stimulation of aromatase expression within the breast has been proposed as a mechanism whereby prostaglandins could influence breast cancer growth. In the present study, we show that PGE₂ is a powerful stimulator of aromatase expression in human breast adipose stromal cells. Moreover, TNF, which also stimulates aromatase expression in breast adipose stromal cells, acts to increase the secretion of PGE₂ by these cells, as well as the expression of COX 2 and PGE synthase, but not that of COX 1. On the other hand, class I cytokines had no effect, either by themselves or in the presence of estradiol. These factors had little influence on secretion of 15-deoxy-^12,14-PGJ₂, which is inhibitory of aromatase expression by breast adipose stromal cells. These results further substantiate an important role for PGE₂ to stimulate estrogen biosynthesis within the local environment of the breast.     

Inhibition of hormone-dependent and independent breast cancer cell growthin vivo andin vitro with the antiestrogen toremifene and recombinant human interferon-α2

The antiproliferative action of the antiestrogen toremifene and recombinant human interferon-2a (IFN-2a) were examined on human breast cancer cell lines grown in culture and in the athymic mouse.Solid tumors grew from an inoculation of a 99:1 ratio of hormone dependent (MCF-7) and hormone independent (MDA-MB-231) breast cancer cells without estrogen administration. However, estradiol supplementation significantly increased the rate of tumor growth. The daily administration of 1.35 × 10⁶ U of recombinant human IFN-2a resulted in a marked reduction of tumor growth in both estradiol-treated and non-treated mice. Toremifene administration (130 µg/day from a sustained release preparation) markedly inhibited estradiol stimulation of mouse uterine weight and partially reduced estradiol-stimulated tumor growth. The combination of IFN-2a (1.35 × 10⁶ u/day) with toremifene (130 µg/day) reduced estradiol-stimulated growth much below that of toremifene alone but not below that seen with interferon alone.Toremifene (10^–10-10^–6M) did not inhibit the growth of hormone-independent MDA-MB-231 breast cancer cellsin vitro whereas it did inhibit the growth of hormone-dependent MCF-7 cells in phenol red containing media. IFN-2a (1–10,000 u) inhibited the growth of both MCF-7 and MDA-MB-231 cells in culture; however, MCF-7 cells were approximately 10-fold more sensitive to interferon inhibition. This was consistent with the MCF-7 cells showing a greater sensitivity to interferon than MDA-MB-231 cells in the induction of 25-oligoadenylate synthetase.The heterogeneous tumor model in the athymic mouse suggests that differential sensitivities of breast cancer cells to the antiproliferative actions of interferon may influence the effectiveness of combination therapies.     

Relationship between the morphological and biological characteristics of intraductal components accompanying invasive ductal breast carcinoma and patient age

We divided 324 cases with invasive ductal breast carcinoma into three age groups, and investigated the differences in proliferative activity and extension of the intraductal components among the age cohorts. Proliferative activity was expressed as the number of MIB1-positive nuclei per 1000 cancer cells in the intraductal components (MLI), and the intraductal component extension farthest from the invasive focus was defined as the maximum distance of ductal spread (MXDS). Moreover, analyses were conducted for three grade types, classified according to the classification system of ductal carcinoma in situ. The under-40 age group had significantly higher MXDS values than the other two age groups (p=0.0280), and this trend was more marked in those with the non-high grade without necrosis type (p=0.0045). The under-40 age group had higher MLIs, but the differences did not reach statistical significance (p=0.0793). In regard to those with the high grade type, the under-40 age group had significantly higher MLIs than the other two age groups (p=0.0269), and this trend was not significant in the cases with any other grade types. Associations between the age group and the margin status of the lumpectomy specimens were investigated in the 143 cases in which breast conserving surgery was tried. The under-40s had a significantly higher margin-positive rate in their lumpectomy specimens than the other two age groups (p=0.0362), and this trend was also seen in the groups with the non-high grade without necrosis type (p=0.0256). These results confirm the importance of considering patient age when designing surgical procedures for breast conserving therapy.     

Transformation of estrone and estradiol in hormone-dependent and hormone-independent human breast cancer cells

Summary Using different hormone-dependent (MCF-7, T-47D) and hormone-independent (MDA-MB-231, Hs-578S, MDA-MB-436) human breast cancer cells, the interconversion estrone (E₁) estradiol (E₂) was explored. The data show very clearly that in the hormone-dependent cells the tendency is to form E₂ after incubation with E₁, whereas after incubation with E₂ most of this estrogen remains unchanged. In the hormone-independent cells, in contrast most of E₁ remains E₁, while E₂ is converted into E₁. The tendency of the reductive oxidative direction is supported by the analysis of estrogens in the culture medium. To explore the possible action of different drugs on the 17-hydroxysteroid dehydrogenase (17-HSD) activity, it was observed that the potent antiestrogen ICI 164,384 inhibits the conversion of E₁ to E₂, while a lesser effect is observed with Danazol and only weak inhibition is obtained with the progestagen Promegestone (R-5020). It is concluded that the orientation of 17-HSD activity for the interconversion E₁ E₂ in hormone-dependent and -independent cells is related to the hormonal status of the cells.     

Genetic polymorphisms of platelet adhesive molecules: association with breast cancer risk and clinical presentation

The main platelet adhesive receptors integrin 21, integrin IIb3 and glycoprotein (GP) Ib are also expressed in breast carcinoma cells. They play a key role in tumor cell-induced platelet aggregation and in adhesive interactions necessary for tumoral invasion and metastasis. Several polymorphisms affecting these molecules, two in integrin 2 (C807T and G1648A), one in integrin 3 (T1565C) and one in GP Ib (VNTR), influencing their levels, structure, and possibly their function, have been previously described and associated with cardiovascular diseases. In this study, we investigated the association of these polymorphisms with breast cancer risk or clinical presentation. We studied 101 patients with invasive breast cancer. The main prognostic variables were recorded, and genomic PCR analysis of these polymorphisms was performed. A group of 101 control subjects matched on age and sex was studied and compared with patients. No association was found between VNTR (GP Ib) polymorphism and breast cancer risk or presentation. Genotype and allele frequencies of C807T and G1648A polymorphisms of integrin 2 were not statistically different in breast cancer patients and controls, although we found an association between the 1648G/G genotype and higher disease stages (III and IV) (p = 0.02). Breast cancer risk was higher in carriers of 3 integrin T/T genotype (OR = 2.08, 95% CI = 1.04–4.16, p = 0.04). Furthermore, genotype 1565T/T was also associated with axillary nodal metastasis (p = 0.017) and with tumoral diameter greater than 2 cm (p = 0.02). Although confirmatory studies are needed, our results suggest that polymorphic genetic variation of integrins expressed in platelets and epithelial breast cells could modify the risk and the biological aggressiveness of breast carcinomas.     

A Japanese phase I study of continuous oral capecitabine in patients with malignant solid tumors

Background This study aimed to evaluate the tolerability and pharmacokinetics of capecitabine, given twice daily for 6 weeks without interruption, and to identify the maximum tolerated dose (MTD) and the suggested phase II schedule.Methods The initial dose of capecitabine was 251mg/m² twice daily, without interruption, and a dose escalation schedule was planned according to a modified Fibonacci scheme. Patients received oral capecitabine twice daily for at least 6 weeks unless grade 3 or 4 toxicity was observed. Blood and urine samples were collected for pharmocokinetic analysis on days 1 and 15.Results Sixteen patients with malignant solid tumors (seven breast, seven colorectal, and two gastric) were enrolled, all of whom were evaluable. Among 4 patients treated with capecitabine 1255mg/m² twice daily, one experienced grade 4 hemorrhagic gastric ulcer and one experienced grade 3 skin toxicity. Consequently, this dose was defined as the MTD, and gastrointestinal and cutaneous effects were identified as dose-limiting toxicities. There was no grade 3/4 diarrhea at any dose level. There was also no grade 4 hematologic toxicity at doses below the MTD, and there were no treatment-related deaths. Two patients with breast cancer had partial responses, at capecitabine doses of 502mg/m² and 1255mg/m², twice daily. Pharmacokinetic data show that high concentrations of doxifluridine (5-DFUR) occur in tumor cells within 2h after administration of capecitabine.Conclusion The MTD of continuous oral capecitabine over 6 weeks was 1255mg/m² twice daily. Because of the higher occurrence of skin disorders reported in this study with a continuous treatment regimen, an intermittent treatment regimen of 828mg/m², administered twice daily for 3 weeks, followed by a 1-week rest period, was recommended for phase II studies. This regimen is being evaluated in phase II studies in Japanese patients with gastric, colorectal, and breast cancers.     

Androstenedione and androst-5-ene-3β,17β-diol stimulate DMBA-induced rat mammary tumors — role of aromatase

Summary The effect of the adrenal steroids androst-5-ene-3,17-diol (⁵-diol) and androstenedione (⁴-dione) was studied on the growth of mammary carcinoma induced in the rat by dimethylbenz[a]anthracene (DMBA). The plasma levels of the two steroids were maintained at values within the range of those found in the circulation of post-menopausal women by constant release from osmotic pumps in ovariectomized animals. ⁵-diol and ⁴-dione, at the daily release rate of 500µg, led to plasma levels of 1.26±0.19 and 1.72 ± 0.75 ng/ml, respectively. At these physiologically relevant plasma concentrations, both ⁵-diol and ⁴-dione caused a marked stimulation of tumor growth while having minimal or no effect on uterine weight or on plasma prolactin and LH levels.Concomitant treatment with the aromatase inhibitor aminoglutethimide completely blocked the stimulatory effect of ⁴-dione released from silastic implants on tumor growth, while simultaneous administration of the antiandrogen flutamide had no significant effect. On the other hand, when aminoglutethimide was administered with ⁵-diol, the stimulatory effect of the adrenal steroid on tumor growth was not affected. Such data indicate that, under the present experimental conditions, transformation of ⁴-dione into androgens plays a minor role, the predominant effect of the adrenal steroid being stimulation of tumor growth through conversion into estrogens, while ⁵-diol exerts a direct estrogenic effect independent from aromatase activity. The minimal or absent effect of the same treatment on uterine weight and on plasma prolactin and LH levels indicates the tissue specificity of the effects observed, the mammary tissue being most sensitive to the action of adrenal steroids.F.L. is a MRC Career Investigator.     

Expression of 3β-hydroxysteroid dehydrogenase-5,4-en isomerase activity by infiltrating ductal human breast carcinoma in vitro

In order to determine whether human mammary tumors could contribute to progesterone synthesis from pregnenolone in breast cancer patients, homogenates of infiltrating ductal primary breast tumors at different stages of malignancy (Stages II and III) obtained from pre- and post-menopausal patients (n=7, age 37–66 years) were incubated with [7n-³H]pregnenolone as substrate. Controls were heated homogenates instead of fresh homogenates. With the use of reverse-isotope dilution analysis, [³H]progesterone was isolated and characterized. No such metabolite was evident in the control incubations of heat-denatured enzymes. The extent of enzymic conversion varied from 0.02 to 4.0%. The results reveal that activity of 3-hydroxysteroid dehydrogenase-5,4-en isomerase that metabolizes pregnenolone to progesterone can be identified with the viable homogenates. It is suggested that there exists a potential for substantial progesterone synthesis in vivo. This conversion may be of considerable clinical, therapeutic, and pathophysiological significance in the patient with breast cancer. The biological impact of this conversion should be a high priority research objective.     

Coexpression of c-kit and stem cell factor in breast cancer results in enhanced sensitivity to members of the EGF family of growth factors

Insulinlike growth factor (IGF)I protects many cell types from apoptosis. As a result, it is possible that IGFIresponsive cancer cells may be resistant to apoptosisinducing chemotherapies. Therefore, we examined the effects of IGFI on paclitaxel and doxorubicininduced apoptosis in the IGFIresponsive breast cancer cell line MCF7. Both drugs caused DNA laddering in a dosedependent fashion, and IGFI reduced the formation of ladders. We next examined the effects of IGFI and estradiol on cell survival following drug treatment in monolayer culture. IGFI, but not estradiol, increased survival of MCF7 cells in the presence of either drug. Cell cycle progression and counting of trypanblue stained cells showed that IGFI was inducing proliferation in paclitaxeltreated but not doxorubicintreated cells. However, IGFI decreased the fraction of apoptotic cells in doxorubicin but not paclitaxeltreated cells. Recent work has shown that mitogenactivated protein kinase (MAPK) and phosphotidylinositol3 (PI3) kinase are activated by IGFI in these cells. PI3 kinase activation has been linked to antiapoptotic functions while MAPK activation is associated with proliferation. We found that IGFI rescue of doxorubicininduced apoptosis required PI3 kinase but not MAPK function, suggesting that IGFI inhibited apoptosis. In contrast, IGFI rescue of paclitaxelinduced apoptosis required both PI3 kinase and MAPK, suggesting that IGFImediated protection was due to enhancement of proliferation. Therefore, IGFI attenuated the response of breast cancer cells to doxorubicin and paclitaxel by at least two mechanisms: induction of proliferation and inhibition of apoptosis. Thus, inhibition of IGFI action could be a useful adjuvant to cytotoxic chemotherapy in breast cancer.     

Ultrastructural Changes of the Vascular Endothelium after Intra-arterial Administration of Tumor Necrosis Factor-alpha (TNFα) in Rat Gliomas

The ensuing ultrastructural changes in tumor vascular endothelial cells following intra-arterial administration of tumor necrosis factor- (TNF) were studied in an experimental rat glioma model. C6 glioma cells were implanted in Wistar rats and then after 14 days, 5×10³U of human natural-type TNF (1.7×10⁵U/m²) was administered through the carotid artery. The animals were sacrificed at 3 or 24h after TNF treatment. A detailed examination with transmission electron microscope revealed swelling of the tumor vascular endothelial cell nuclei and mitochondria with matrix densities at 3h. At 24h, these cells demonstrated the presence of high amplitude mitochondrial swelling or the violent blebbing characteristic of damaged mitochondria; the cytoplasm was swollen enormously and there were dissolution of cytoplasmic organelles and rupture of the plasma membrane. The observed findings were typical of cell necrosis and confirms yet another mechanism by which TNF exerts its anti-tumor effects, that is, necrotizing effects on tumor vascular endothelium. The information appears to be important in the context of clinical application of intra-arterial TNF in the treatment of malignant gliomas.     

Chemopreventive and adjuvant therapeutic potential of pomegranate (Punica granatum) for human breast cancer

Fresh organically grown pomegranates (Punica granatum L.) of the Wonderful cultivar were processed into three components: fermented juice, aqueous pericarp extract and cold-pressed or supercritical CO₂-extracted seed oil. Exposure to additional solvents yielded polyphenol-rich fractions (polyphenols) from each of the three components. Their actions, and of the crude whole oil and crude fermented and unfermented juice concentrate, were assessed in vitro for possible chemopreventive or adjuvant therapeutic potential in human breast cancer. The ability to effect a blockade of endogenous active estrogen biosynthesis was shown by polyphenols from fermented juice, pericarp, and oil, which inhibited aromatase activity by 60–80%. Fermented juice and pericarp polyphenols, and whole seed oil, inhibited 17--hydroxysteroid dehydrogenase Type 1 from 34 to 79%, at concentrations ranging from 100 to 1,000g/ml according to seed oilfermented juice polyphenols>pericarp polyphenols. In a yeast estrogen screen (YES) lyophilized fresh pomegranate juice effected a 55% inhibition of the estrogenic activity of 17--estradiol; whereas the lyophilized juice by itself displayed only minimal estrogenic action. Inhibition of cell lines by fermented juice and pericarp polyphenols was according to estrogen-dependent (MCF-7)estrogen- independent (MB-MDA-231)>normal human breast epithelial cells (MCF-10A). In both MCF-7 and MB-MDA-231 cells, fermented pomegranate juice polyphenols consistently showed about twice the anti-proliferative effect as fresh pomegranate juice polyphenols. Pomegranate seed oil effected 90% inhibition of proliferation of MCF-7 at 100g/ml medium, 75% inhibition of invasion of MCF-7 across a Matrigel membrane at 10g/ml, and 54% apoptosis in MDA-MB-435 estrogen receptor negative metastatic human breast cancer cells at 50g/ml. In a %% murine mammary gland organ culture, fermented juice polyphenols effected 47% inhibition of cancerous lesion formation induced by the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). The findings suggest that clinical trials to further assess chemopreventive and adjuvant therapeutic applications of pomegranate in human breast cancer may be warranted.     

Tamoxifen and fenretinide in women with metastatic breast cancer

Background: Tamoxifen and fenretinide combination therapy has been shown to be an active treatment regimen in metastatic breast cancer patients. This pilot study sought to determine whether the addition of fenretinide to tamoxifen would be associated with antitumor activity in metastatic breast cancer patients who had been previously treated with tamoxifen or who had hormone receptor negative disease. The effect of this therapy on circulating plasma transforming growth factorbeta (TGF) levels and serum lipids was also examined.Patientsand Methods: Thirtyone patients were treated with tamoxifen (20mg po daily), and fenretinide (400mg po daily with a 3day drug holiday each month). Plasma TGF testing was performed using isoform specific sandwich ELISA.Results: Twenty four of the 31 patients were evaluable for an antitumor response including 14 estrogen receptor (ER) positive patients who had failed prior tamoxifen therapy, seven ERnegative patients, and three hormone therapy naive ERpositive patients. There were no objective antitumor responses; three patients had stable disease for 8, 8, and 24 months. Five patients (16%) discontinued therapy for toxicity (one for grade 3 skin rash and four for abnormal dark adaptation). There was a statistically significant decrease in total cholesterol (median change per patient of –13.5mg/dl; p=0.049, a 6.5% decrease), and an increase in HDL levels (median change per patient of +18mg/dl, p=0.0001, a 35% increase) with tamoxifen and fenretinide therapy.TGF-1 plasma levels were normal in 26 of 28 patients, and no changes in these levels post-treatment were demonstrated.Conclusions: Tamoxifen and fenretinide therapy is not an active combination in ER negative metastatic breast cancer or in patients whose disease has progressed on tamoxifen. This combination had a beneficial effect on total serum cholesterol and HDL levels with no associated rise in serum triglyceride levels. The 400mg dose of fenretinide was associated with symptomatic nyctalopia in one-third of patients making it an unsuitable dose for use in breast cancer prevention studies.