To Tamoxifen Research Articles

Number needed to treat (NNT) to compare benefits of letrozole (LET) with adjuvant chemotherapy (CT) in patients (pts) with node-positive (N+) breast cancer (BC)

581 Background: Adjuvant CT is an accepted treatment for N+ BC. The addition of taxanes to anthracycline based adjuvant CT has been demonstrated to improve overall survival (OS) in all but one study as in table below. BIG 1–98 has reported a survival gain with the aromatase inhibitor LET compared to tamoxifen (TAM). NNT (the reciprocal of absolute benefit) is the number of pts that need to be treated with a new intervention to avoid 1 additional event over the benefit provided by standard therapy. For multiple numerical outcomes from randomized trials, NNT is an effective method to express results in a clinically meaningful way. To evaluate the degree of benefit of LET over TAM in women with early stage N+ BC in BIG 1–98, we compared the NNT for survival benefit from LET to that needed to see benefit from the addition of a taxane to anthracycline based CT based on clinical trial data in BC pts with N+ disease. Methods: 5-yr survival data were taken from the pivotal randomized controlled trial (RCT) for LET (N+ pts from BIG 1–98, TAM arm censored for crossover to LET) and from 4 RCTs of adjuvant CT trials, AC-T, FEC-D, and DAC. NNT was calculated with respect to OS at 5 yrs; outcome is presented as the NNT to save a life (see Table). Results: The NNT to save a life for adjuvant LET vs TAM is comparable to the NNT for the addition of a taxane to anthracycline based CT in women with N+ BC. Conclusions: The magnitude of survival benefit of LET over TAM in terms of NNT is comparable to that needed to see a survival benefit from paradigm- changing modern adjuvant CT regimens. F= fluorouracil, E = epirubicin, C = cyclophosphamide, D = docetaxel, T = paclitaxel, A = doxorubicin [Table: see text] [Table: see text]

An economic analysis of arthralgia/myalgia (A/M) versus recurrence in women with hormone receptor-positive (HR+) breast cancer (BC) on aromatase inhibitors (AIs)

e11601 Background: The symptoms of A/M are associated with estrogen suppression and are more common with AIs relative to tamoxifen (TAM). AIs have demonstrated superior efficacy when compared with TAM. In BIG 1–98, letrozole (LET) treatment provides a significant reduction in the risk of distant metastasis (DM); the updated 76 month BIG 1–98 analysis now suggests that early reduction in DM ultimately leads to overall survival (OS) benefit, 40 fewer deaths with LET compared with TAM. [Mouridsen, 2008] It has been reported that women reporting new joint symptoms at 3 months after initiating an AI have a significantly lower risk for recurrence compared with women not reporting these symptoms [Cuzick, 2008]. Methods: Data from the Henry Ford Health System (1995–2005) were used to identify postmenopausal HR+ early BC patients with at least 1 year of AI therapy follow-up after surgery. Total health care costs of managing A/M associated with hormone therapy as well as BC recurrences were estimated from charges incurred during health care encounters for these conditions. Results: In the 834 patients eligible, the treated A/M incidence was 21% and the total health care cost ∼ $429 per symptomatic patient/yr. The average annual cost of any BC recurrence was previously reported at ∼$131,000/yr, with greatest cost seen with DM at ∼ $265,783/yr [Weiderkehr, 2008]. Conclusions: The economic costs of treating A/M are nominal, particularly in light of the superior efficacy of AIs and the economic burden of BC recurrences. DM is the most costly of the recurrences, and in BIG 1–98, reducing early DM appears to impact OS. Discontinuation of AIs, or switching to less effective therapies in an effort to manage A/M, should be weighed against the benefit of AI therapy in reducing BC recurrences, particularly DM, as well as the high costs associated the managing these recurrences. No significant financial relationships to disclose.

Antiovulatory effect of a single injection of pure antiestrogen ZK 191703 at early stage of rat estrus cycle

The effects of ZK 191703 (ZK), a pure antiestrogen, on ovulation, follicle development and peripheral hormone levels were investigated in rats with 4-day estrus cycle and gonadotropin-primed immature rats in comparison to tamoxifen (TAM)-treatment. In adult rats, a single s.c. injection of ZK (5 mg/kg) or TAM (5 mg/kg) at an early stage of the estrus cycle (diestrus 9:00) inhibited ovulation, and was associated with suppression of the surge of preovulatory LH, FSH and progesterone. In rats treated with ZK or TAM at a late stage of the estrus cycle (proestrus 9:00), no inhibitory effects on ovulation, the gonadotropin and progesterone surge were detected. ZK treatment at diestrus 9:00, in contrast to TAM, increased the baseline LH level. When immature rats were treated with antiestrogens in the earlier stage of follicular development, 6 and 30 h but not 48 h or later after injection of gonadotropin (PMSG), ovulation was attenuated, associated with a lowered progesterone level. Unruptured preovulatory follicles were found in most of the ovaries from anovulatory animals treated with ZK or TAM. Antiestrogens, ZK and TAM administered at an early phase of the estrus cycle delay the follicular development functionally and inhibit ovulation in rats and suppression of the preovulatory progesterone surge.

Safely preventing early distant metastases (DM) with adjuvant aromatase inhibitor (AI) therapy

Background: A recent analysis of UK patients treated with tamoxifen following surgery, along with older studies, reports of an early peak of recurrence seen at about 2 years post-surgery in patients with hormone receptor-positive (HR+) early breast cancer (EBC). During this early peak of recurrence DM comprise the majority of recurrence events. AIs, the current standard of care for HR+ EBC, are superior to tamoxifen (TAM) in reducing recurrence risk.

Increased expression of Nrf2/ARE-dependent anti-oxidant proteins in tamoxifen-resistant breast cancer cells

Acquired resistance to tamoxifen (TAM) is a serious therapeutic problem in breast cancer patients. In this study, we found that the expressions of anti-oxidant proteins (γ-glutamylcysteine ligase heavy chain (γ-GCL h), heme oxygenase-1, thioredoxin and peroxiredoxin1) in TAM-resistant MCF-7 (TAMR-MCF-7) cells were higher than control MCF-7 cells. Molecular analyses using antioxidant response element (ARE)-containing reporters and gel-shift supported the critical role of NF-E2-related factor2 (Nrf2)/ARE in the overexpression of antioxidant proteins in TAMR-MCF-7 cells. Intracellular peroxide production was significantly decreased in TAMR-MCF-7 cells and TAM resistance was partially reversed by Nrf2 siRNA. The basal phosphorylation of extracellular signal-regulated kinase (ERK) and p38 kinase were increased in the TAMR-MCF-7 cells and the inhibition of ERK significantly decreased the activity of minimal ARE reporter and γ-GCL h protein expression in TAMR-MCF-7 cells. However, exposure of TAMR-MCF-7 cells to 17-β-estradiol or ICI-182,780 did not significantly change γ-GCL h expression. These results suggest that the persistent activation of Nrf2/ARE is critical for the enhanced expression of anti-oxidant proteins in TAM-resistant breast cancer cells and the pathway of ERK, but not of estrogen receptor signaling are involved in the up-regulation of Nrf2/ARE.

Reflecting time dependency in hazard rates to analyse the cost-effectiveness of letrozole or anastrozole versus tamoxifen as adjuvant therapies for early breast cancer in hormone receptor-positive (HR+) postmenopausal women: The U.S. perspective

596 Background: The BIG 1–98 and ATAC randomized controlled trials demonstrated that, in postmenopausal women with hormone receptor-positive (HR+) early breast cancer, initial adjuvant therapy for 5 years with the aromatase inhibitors (AIs) letrozole (LET) or anastrozole (ANA) is superior to tamoxifen (TAM). The table shows the difference in early effects of the 2 AIs. This study shows the cost-effectiveness of 5 years of initial adjuvant therapy with LET or ANA versus 5 years TAM in a postmenopausal HR+ population. Methods: Clinical trial data from BIG-198 and ATAC populated a Markov model to estimate the incremental cost per quality-adjusted life year (QALY) gained of the AIs compared to TAM. Variable annual hazard ratios for time to recurrence over the 5 year treatment period were applied to a pooled set of variable annual hazard rates for TAM. Probabilities of breast cancer event type and adverse events (AEs) (end. cancer, fractures, stroke, VTEs, and bleeding) were based on published results of the clinical trials and U.S. studies. Costs of breast cancer and AE care, and QALY weights were taken from published studies. Incremental costs and QALYs were estimated over the lifetime of HR+ women aged 60 yrs, discounted at 5% annually. Results: LET increases QALYs by 0.22 per patient compared with TAM while ANA patients gain 0.18 QALYs. Mean incremental cost per QALY gained for the AIs compared to TAM were $37,328 for LET and $40,531 for ANA. Probabilistic sensitivity analysis demonstrated that LET and ANA have around a 70% probability of being cost-effective at a QALY value of $50,000. Conclusions: Adjuvant treatment of postmenopausal HR+ women with LET or ANA for 5 years is a cost-effective use of U.S. health care resources compared with TAM. Based on an indirect comparison of mean values, LET is more cost-effective than ANA, with a higher predicted increment in quality adjusted survival. Early Effects in the ATAC and BIG-198 Trials ATAC (No. of events at 2.5 years) BIG 1–98 (No. of events at 2 years) ANA TAM Prevented (% reduction) LET TAM Prevented (% reduction) Recurrences 196 236 40 (-17%) 117 168 51 (- 30%) Distant recurrences 133 143 10 (-7%) 87 125 38 (-30%) Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Novartis Novartis Novartis

Differential regulation of ErbB2 expression by cAMP-dependent protein kinase in tamoxifen-resistant breast cancer cells

Acquired resistance to tamoxifen (TAM) is a serious therapeutic problem in breast cancer patients, and Her-2/ErbB2 expression is associated with decreased sensitivity to TAM. We previously reported that cAMP-dependent protein kinase (PKA)-mediated activator protein-2 (AP-2) activation was responsible for the expression of Her-2/ErbB2 in p53-inactivated mammary epithelial cells (Yang et al., 2006). In the present study, we tested the hypothesis that PKA plays a role in the expression of ErbB2 in tamoxifen-resistant breast cancer cells. Treatment with H-89, a specific PKA inhibitor, suppressed 4-hydroxytamoxifen-induced ErbB2 expression in control MCF-7 cells. In contrast, PKA inhibition by H-89 or cAMP-dependent protein kinase inhibitor l gamma overexpression increased the expression levels of ErbB2 in TAM-resistant MCF-7 (TAMR-MCF-7) cells. Transcriptional regulation of the erbB2 gene depends on two transcription factors, AP-2 and polyomavirus enhancer activator3 (PEA3). H-89 decreased nuclear or total levels of PEA3 in TAMR-MCF-7 cells. Chromatin immunoprecipitation assay results revealed that H-89 treatment reduced PEA3 binding to the proximal Ets binding site of the erbB2 gene promoter. Reporter gene analyses using human erbB2 gene promoter supported the critical role of PEA3 in the overexpression of ErbB2 in TAMR-MCF-7 cells treated with H-89. This deregulated PKA signaling cascades required for the ErbB2 expression may be important for the differential response of TAM-resistant breast cancer cells to EGF/ErbB2 stimuli.

Centchroman induces G 0/G 1 arrest and Caspase-dependent Apoptosis involving Mitochondrial Membrane Depolarization in MCF-7 and MDA MB-231 Human Breast Cancer Cells

Studies with Centchroman (CC) as a candidate anti-breast cancer agent are into phase III multicentric clinical trial in stage III/IV breast cancer. We have previously demonstrated its anti-neoplastic activity in Estrogen Receptor positive (ER+ve) MCF-7 Human Breast Cancer Cells (HBCCs). We now present the basis for anti-neoplastic activity of CC, mediated through apoptosis in both ER+ve/−ve MCF-7 and MDA MB-231 HBCCs respectively, compared to Tamoxifen (TAM) as a positive control. All the experiments were performed with 48 h estrogen-deprived cells exposed to CC/TAM for the subsequent 48 h. Cytotoxic potential of CC was assessed through SRB assay. Cell-cycle analysis, Time-dependent cytotoxicity, Reactive Oxygen Species (ROS) and Mitochondrial Membrane Permeability were investigated by Flow Cytometry. Early-stage apoptosis was detected by Annexin–PI staining. Caspases were assayed colorimetrically whereas nuclear derangements were assessed morphologically through PI staining and finally by DNA fragmentation analysis. Cell viability studies confirmed the IC 50 of CC in MCF-7 and MDA MB-231 cells to be 10 and 20 μM ( P < 0.001) respectively, suggesting enhanced susceptibility of the former cell type to CC. FACS data reveals CC mediated G 0/G 1 arrest ( P < 0.01) along with the presence of prominent sub-G 0/G 1 peak ( P < 0.001) in both the cell types suggesting ongoing apoptosis. Phosphatidylserine externalization, mitochondrial events, caspase evaluation and nuclear morphology changes reveal initiation/progression of caspase-dependent apoptosis even at a dose of 1 μM which eventually leads to DNA fragmentation in both the cell types. Results demonstrate that CC induces caspase-dependent apoptosis in MCF-7 and MDA MB-231 cells irrespective of ER status similar to TAM in terms of anti-neoplastic activity.

Aromatase inhibitor-induced joint symptom in patients with breast cancer

11070 Background: Aromatase inhibitor (AI) are superior to tamoxifen (TAM) for postmenopausal women with estrogen receptor (ER)-positive breast cancer. Toxic profiles are different between AI and TAM. The frequency of bone fracture and joint symptom are high in AI compared with TAM. However, there is little report concerning a detailed joint symptom. Methods: From January 2002 to March 2006, 120 postmenopausal women with ER-positive breast cancer were treated with anastrozole 1mg or exemestane 25mg daily at our hospital. We studied retrospectively the incidence, onset, type, sites, grade, and change of joint symptom. We also investigated the incidence of discontinuation due to joint symptoms and correlation between patient’s characteristics and joint symptoms. Results: Median follow-up time is 590 days and median age is 63 years old. Forty-seven (39.1%) of 120 patients complained of joint symptom. Median onset time of the symptom was 90 days of starting AI. All cases with the symptom complained of joint stiffness and 33 (70.2%) of 47 patients with the symptom have joint pain. In decreasing order, the most commonly affected sites were hand, knee, wrist, ankle, shoulder and elbow. The joint symptom was mostly grade 1/2, and most symptom ware resolved with exercise. Most common feature is morning digital stiffness like initial symptom of rheumatoid arthritis. Some patients experienced severe joint symptom. Subsequently, 15 (12.5%) patients had to discontinue AI because of the severe symptom. Discontinuation of AI led to improvement of symptoms, and patients received TAM in stead of AI also led to resolution of symptoms. AI-induced joint symptom was significantly correlated with young age, prior chemotherapy and switching from TAM to AI. Conclusions: Most AI-induced joint symptom is modest, and this symptom can lead to resolution with exercise. However, some patients lead to discontinue AI because of AI-induced severe joint symptom. We should inform patients treated with AI about AI- induced joint symptom and observe patient’s symptom in detail because severe joint symptom can prevent maintenance of quality of life and compliance. No significant financial relationships to disclose.

Induction of multidrug resistance associated protein 2 in tamoxifen-resistant breast cancer cells

Acquired resistance to tamoxifen (TAM) is a serious therapeutic problem in breast cancer patients. The transition from chemotherapy-responsive breast cancer cells to chemotherapy-resistant cancer cells is mainly accompanied by the increased expression of multidrug resistance-associated proteins (MRPs). In this study, it was found that TAM-resistant MCF-7 (TAMR-MCF-7) cells expressed higher levels of MRP2 than control MCF-7 cells. Molecular analyses using MRP2 gene promoters supported the involvement of the pregnane X receptor (PXR) in MRP2 overexpression in TAMR-MCF-7 cells. Although CCAAT/enhancer-binding protein beta was overexpressed continuously in TAMR-MCF-7 cells, this might not be responsible for the transcriptional activation of the MRP2 gene. In addition, the basal activities of phosphatidylinositol 3-kinase (PI3-kinase) were higher in the TAMR-MCF-7 cells than in the control cells. The inhibition of PI3-kinase significantly reduced both the PXR activity and MRP2 expression in TAMR-MCF-7 cells. Overall, MRP2 induction plays a role in the additional acquisition of chemotherapy resistance in TAM-resistant breast cancer.

Disruption of estrogen receptor DNA-binding domain and related intramolecular communication restores tamoxifen sensitivity in resistant breast cancer

A serious obstacle to successful treatment of estrogen receptor (ER)-positive human breast cancer is cell resistance to tamoxifen (TAM) therapy. Here we show that the electrophile disulfide benzamide (DIBA), an ER zinc finger inhibitor, blocks ligand-dependent and -independent cell growth of TAM-resistant breast cancer in vitro and in vivo. Such inhibition depends on targeting disruption of the ER DNA-binding domain and its communication with neighboring functional domains, facilitating ERalpha dissociation from its coactivator AIB1 and concomitant association with its corepressor NCoR bound to chromatin. DIBA does not affect phosphorylation of HER2, MAPK, AKT, and AIB1, suggesting that DIBA-modified ERalpha may induce a switch from agonistic to antagonistic effects of TAM on resistant breast cancer cells.

Cost-effectiveness of letrozole and anastrozole as adjuvant therapy for hormone receptor positive early breast cancer in postmenopausal women

10577 Background: The BIG 1–98 and ATAC studies demonstrated that, in postmenopausal women with hormone receptor positive (HR+) early breast cancer, 5 years of initial adjuvant therapy with the aromatase inhibitors (AIs) letrozole (LET) or anastrozole (ANA) is superior to tamoxifen (TAM). The cost-effectiveness TAM, LET, and ANA have not been previously evaluated using a consistent methodology. Methods: A Markov model was used to estimate the incremental cost per quality-adjusted life year (QALY) gained with initial adjuvant therapy with LET vs TAM, ANA vs TAM, and LET vs ANA in postmenopausal women with HR+ early stage breast cancer from the US healthcare system perspective. Probabilities of recurrence (including contralateral tumor) and adverse events (endometrial cancer, thromboembolism, fractures, hypercholesterolemia, MI, and stroke) for TAM were based primarily on published US population-based studies and trials of prophylactic TAM vs placebo. Corresponding probabilities for LET and ANA were calculated by multiplying probabilities for TAM by estimated relative risks of LET vs TAM and ANA vs TAM from the BIG 1–98 and ATAC trials respectively. Other probabilities, costs, and health-state utilities were obtained from published studies. Expected lifetime costs and QALYs were estimated for a cohort of HR+ postmenopausal women with early breast cancer, aged 61 years at therapy initiation and discounted at 3% annually. Probabilistic sensitivity analyses were conducted to assess precision of results. Results: Incremental cost per QALY gained for LET vs TAM is $33,536 (95% CI $20,409 to $70,566) and for ANA vs TAM is $38,967 (95% CI $23,826 to $81,904). Compared with ANA, LET is less costly ($9,647 vs $10,190) and gains more QALYs (0.29 vs 0.26), although differences in costs (95% CI -$1,669 to $671) and QALYs (95% CI -0.16 to 0.22) are not statistically significant. Conclusions: In postmenopausal women with HR+ early breast cancer, adjuvant therapy with either LET or ANA is cost-effective from a US healthcare system perspective. Although LET dominates ANA in our base-case analysis, definitive conclusions regarding the cost-effectiveness of LET vs ANA must await results of comparative clinical studies. [Table: see text]

Evaluation of leukocyte arylsulphatase a, serum interleukin-6 and urinary heparan sulphate following tamoxifen therapy in breast cancer

Leukocyte arylsulphatase A (AS-A) was shown to be significantly high in newly-diagnosed breast cancer patients. Previous reports imply a connection between serum interleukin-6 (IL-6) and breast cancer, possibly through a modulation of enzymes involved in estrogen synthesis. Abnormal distribution of heparan sulphate proteoglycans (HSPGs) in malignant breast epithelial cells suggests that they play a key role in the regulation of cell growth. Estradiol is believed to be effective in modulating glycosaminoglycans (GAGs) and their depolymerizing enzymes. Therefore, in this study, attempts were made to evaluate the activity of leukocyte arylsulphatase A, serum interleukin-6, urinary GAGs and heparan sulphate (HS) in response to tamoxifen (TAM) therapy in mastectomised breast cancer patients. Thirty-four patients (aged 30–82 years) were administered TAM (20 mg twice daily). Blood and urine samples of each patient were collected three times (at the beginning, and in third and sixth month of TAM therapy), and biochemical parameters were measured. There was no difference between baseline leukocyte AS-A activity and that measured after three months. At the end of six months, enzyme activity was significantly higher than the former values ( p = 0.022), but within the reference intervals reported in the literature. Although this increase might imply a normalization, the duration of TAM therapy is not long enough to make a decision about either regression or aggravation of the disease. TAM did not have any effect on serum IL-6, urinary HS and GAG levels which may be due to insensitivity of these variables to TAM during the short period of therapy. Both urinary GAG and HS levels measured at sixth month exhibited a positive correlation with the baseline level of leukocyte AS-A ( p = 0.005 and 0.009, respectively), suggesting that positive responses to the drug might be seen in patients with low AS-A activity.

L’acide zolédronique et métastases osseuses : revue systématique de la littérature

Invasive lobular breast cancer (ILC) is an understudied malignancy with distinct clinical, pathological, and molecular features that distinguish it from the more common invasive ductal carcinoma (IDC). Mounting evidence suggests that estrogen receptor-alpha positive (ER+) ILC has a poor response to Tamoxifen (TAM), but the mechanistic drivers of this are undefined. In the current work, we comprehensively characterize the SUM44/LCCTam ILC cell model system through integrated analysis of gene expression, copy number, and mutation, with the goal of identifying actionable alterations relevant to clinical ILC that can be co-targeted along with ER to improve treatment outcomes. We show that TAM has several distinct effects on the transcriptome of LCCTam cells, that this resistant cell model has acquired copy number alterations and mutations that impinge on MAPK and metabotropic glutamate receptor (GRM/mGluR) signaling networks, and that pharmacological inhibition of either improves or restores the growth-inhibitory actions of endocrine therapy.

Economic evaluation of adjuvant hormonal options in postmenopausal women with breast cancer: Tamoxifen vs tamoxifen then exemestane vs anastrazole

6040 Background: Adjuvant anastrazole (ANA) for 5 years or tamoxifen followed by exemestane (TAM-EX) for 2.5 years each have become acceptable alternatives to tamoxifen (TAM) for 5 years in postmenopausal women with breast cancer. An economic evaluation was undertaken to compare the cost-effectiveness (CE) of ANA and TAM-EX relative to TAM alone. Methods: A Markov model was developed to calculate cumulative costs and disease free survival years (DFSY) for each alternative over 5- and 20-year time horizons. The analysis took a direct payer perspective. Drug costs were based on average wholesale prices in Canada in 2004. Costs of adverse events and relapses were not included in the primary analysis. The baseline TAM DFS rate and hazard ratios associated with ANA and TAM-EX were taken from the published ATAC and IES trials. Beyond the 5-year treatment period, hazard ratios for ANA and TAM-EX were set to 1.0, reflecting conservative estimates of benefit. Both costs and benefits were discounted at 3%. Results: Both ANA and TAM-EX were associated with gains in DFS. Per 1,000 patients treated, ANA resulted in an incremental gain of 56.4 DFSY after 5 years and 162.3 DFSY after 20 years relative to TAM alone. TAM-EX had an incremental gain of 31.6 and 164.9 DFSY per 1,000 treated relative to TAM alone after 5 and 20 years, respectively. Incremental costs were $6.35 million per 1,000 treated with ANA and $2.90 million per 1,000 treated with TAM-EX. The CE of ANA relative to TAM alone was $112,510 per DFSY after 5 years and $39,124 per DFSY after 20 years. The CE of TAM-EX relative to TAM alone was $91,604 and $17,570 per DFSY after 5 and 20 years. Comparing TAM-EX and ANA directly, ANA had an incremental cost of $139,174 per DFSY gained at 5 years and was dominated by TAM-EX at 20 years. Conclusions: Although the short-term CE of ANA and TAM-EX is unfavourable, projecting the benefits over a longer horizon results in CE ratios well below $50,000 per DFSY gained. Both alternatives appear to be cost-effective options for postmenopausal women with breast cancer. These results will be updated based on the recently reported DFS and adverse events rates of the ATAC and IES trials. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration AstraZeneca Pfizer

Modulation of tamoxifen sensitivity by antisense Bcl‐2 and trastuzumab in breast carcinoma cells

Because the overexpression of HER-2 and Bcl-2 is associated with resistance to tamoxifen (TAM), the authors examined the effect of antisense (AS) Bcl-2 on sensitivity to TAM compared with the effect of trastuzumab on sensitivity to TAM in breast carcinoma cell lines. Drug sensitivity was assessed in vitro using a [3-4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay with the breast carcinoma cell lines ZR-75-1, MDA-MB-453, and BT-474. AS Bcl-2 18-mer phosphorothioate oligonucleotide was applied. Apoptotic cell death was assessed with the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling method, and gene expression was evaluated with Western blot analysis. The expression of Bcl-2 was identified in ZR-75-1 and BT-474 cells and, to a lesser extent, in MDA-MB-453 cells. Overexpression of HER-2 was identified in BT-474 cells, and moderate expression was identified in MDA-MB-453 and ZR-75-1 cells. Combination treatment with trastuzumab or AS Bcl-2 enhanced TAM sensitivity in ZR-75-1 cells, which showed 50% inhibitory concentration (IC50) values of 0.9 microM (7.2-fold increase) and 0.5 microM (13.0-fold), respectively. Combination treatment with trastuzumab or AS Bcl-2 slightly enhanced TAM sensitivity of BT-474 cells, with IC50 values of 3.0 microM (1.3-fold) and 1.5 microM (2.6-fold), respectively. The sensitivity of MDA-MB-453 cells to TAM was not enhanced by combination with trastuzumab or AS Bcl-2. Modulation of TAM sensitivity by AS Bcl-2 was superior to modulation by trastuzumab in HER-2-expressing and Bcl-2-expressing breast carcinoma cells. Enhanced sensitivity in combination with AS Bcl-2 was associated with down-regulation of Bcl-2 and pAkt, which was correlated with the induction of Bax and caspase-3, leading to apoptosis. AS Bcl-2 appeared to be superior to trastuzumab with respect to regulating the signal-transduction pathways involved in breast carcinoma cells.

PHTHALATES INHIBIT TAMOXIFEN-INDUCED APOPTOSIS IN MCF-7 HUMAN BREAST CANCER CELLS

Environmental estrogens represent a class of compounds that can mimic the function or activity of the endogenous estrogen 17β-estradiol (E2). Phthalates including butyl benzyl phthalate (BBP), di(n-butyl) phthalate (DBP), and di(2-ethylhexyl) phthalate (DEHP) are used as plasticizers, and also widely used in food wraps and cosmetic formulations. Phthalates have been shown to mimic estrogen and are capable of binding to the estrogen receptor (ER). It has been demonstrated that estrogen promotes drug resistance to tamoxifen (TAM) in breast cancer. In order to further evaluate the potential role of the phthalates as environmental estrogens, the effect of phthalates was investigated on TAM-induced apoptosis in MCF-7 human breast cancer cells. Our results show that phthalates, BBP (100 μM), DBP (10 μM), and DEHP (10 μM), significantly increased cell proliferation in MCF-7, but not in MDA-MB-231 cells. In addition, BBP, DBP, and DEHP mimicked estrogen in the inhibition of TAM-induced apoptosis in MCF-7 cells. Our data suggest that the inhibitory effect of phthalates on TAM-induced apoptosis involves an increase in intracellular Bcl-2 to Bax ratio. Given that the phthalates are widely used in cosmetics mainly for women, our findings that revealed the promoting effect of BBP, DBP, and DEHP on chemotherapeutic drug resistance to TAM in breast cancer may be of biological relevance.

Disease-Free Survival Advantage of Weekly Epirubicin Plus Tamoxifen Versus Tamoxifen Alone As Adjuvant Treatment of Operable, Node-Positive, Elderly Breast Cancer Patients: 6-Year Follow-Up Results of the French Adjuvant Study Group 08 Trial

To assess whether an epirubicin (EPI) -based chemotherapy plus hormonal regimen improves disease-free (DFS) in women older than 65 years, with node-positive, operable breast cancer (BC), relative to tamoxifen (TAM) alone. A total of 338 patients were randomly assigned after surgery to receive TAM 30 mg/d for 3 years (TAM, n = 164), or EPI 30 mg on days 1, 8, and 15 every 28 days for six cycles plus TAM 30 mg/d for 3 years (EPI-TAM, n = 174). In both arms, patients received radiotherapy, delivered after chemotherapy (CT) in the EPI-TAM group. The 6-year DFS rates were 69.3% with TAM and 72.6% with EPI-TAM (P = .14). The multivariate analysis shows a relative risk of relapse of 1.93 (95% CI, 1.70 to 2.17) with TAM compared with EPI-TAM (P = .005). The 6-year OS, related to disease progression, was 79.1% and 79.8%, respectively (P = .41). Compliance with CT was good: 96.9% of patients received six cycles. The acute toxicity per patient was mild: grade 2 neutropenia in 5.9%, grade 2 anemia in 2.0%, grade 3 nausea or vomiting in 4.6%, and grade 3 alopecia in 7.2%. Five cases (in five patients) of decreased left ventricular ejection fraction occurred after CT: three after adjuvant CT, and two after anthracycline-based CT for relapse. One patient died as a result of dysrhythmia related to carcinomatous lymphangitis. No secondary leukemia occurred. This study conducted in node-positive elderly patients demonstrates a significant contribution of a weekly EPI regimen in terms of DFS. Moreover, this regimen is safe for hematologic, nonhematologic, and cardiac toxicities.

Lactoferrin: A Tamoxifen-Responsive Protein in Normal and Malignant Human Endometrial Cells in Culture

We investigated the possible role of the estrogen-regulated protein lactoferrin (Lf) in the response of isolated normal human endometrial epithelial cells (NHEC) and established human endometrial carcinoma (EC) cell lines to tamoxifen (TAM). Using confocal laser scanning microscopy and a monospecific antibody, Lf was localized to the cytoplasm of normal and EC cells. Antibody neutralization of secreted Lf inhibited, whereas exogenous Lf (0–100 μg/ml) enhanced, cell proliferation in both classes of cells. Treatment of NHEC with TAM inhibited cell growth via a protein kinase-C-mediated pathway, concomitant with a reduction in the staining intensity for Lf. Importantly, in EC cells, TAM greatly enhanced the staining intensity for Lf, but did not affect cell growth. We propose that stable expression of Lf protein by EC cells may impart a survival advantage to these cells, which may, in part, account for the resistance of these cells to tamoxifen.

Characterization of the major DNA adducts in the liver of rats chronically exposed to tamoxifen for 18 months

Our previous study has shown that chronic exposure to tamoxifen (TAM) induced formation of high levels of DNA adducts in the liver, the target tissue of TAM-induced carcinogenesis in rats. One of the major DNA adducts (spot 1), as detected by 32P-postlabeling, accounted for 53% of the total adducts. To characterize this major adduct, the current study has compared spot 1 with two previously identified TAM-DNA adducts, i.e. α-TAM- N 2-deoxyguanine (α-TAM- N 2-dG) and α- N-desmethyl TAM- N 2-deoxyguanine (α- N-dmTAM- N 2-dG) by various rechromatography methods. It was found that spot 1 was further resolved into two fractions during rechromatography analysis, one fraction co-migrated with the α-TAM- N 2-dG and the other fraction co-migrated with the α- N-dmTAM- N 2–dG. These findings have demonstrated that chronic exposure to tamoxifen induced the same major DNA adducts, i.e. α-TAM- N 2-dG and α- N-dmTAM- N 2-dG as those detected in acutely exposed rats.