Mechanism Of Tamoxifen Action Research Articles

Tamoxifen: From Anti-cancer to Antifungal Drug

Tamoxifen (TAM) is an important drug for treatment of breast cancer. It is most effective against estrogen receptor-positive and negative breast cancer. Protective adjuvant is another applied of TAM for women at risk of development of breast cancer. Anti-cancer activity of TAM can take various pathways of action. Antagonistic with estrogen receptor and oxidation reaction are the most proposed mechanism of action of TAM in cancer cells. Recently, many studies focused on the potential antimicrobial action of TAM. Fungi are demonstrated to affect by TAM through various mechanism of action. Yeasts, especially Candida albicans, are the most common type of fungi used to test the antifungal action of TAM. Inhibitory action on some components of the calcium-calcineurin pathway in fungal cells is most acceptable mechanism of TAM action. TAM can also play a synergistic role to increase the antifungal activity of other standard agents. This review will discuss the most recent information about antifungal action of TAM.

Tamoxifen Directly Interacts with the Dopamine Transporter.

The selective estrogen receptor modulator tamoxifen increases extracellular dopamine in vivo and acts as a neuroprotectant in models of dopamine neurotoxicity. We investigated the effect of tamoxifen on dopamine transporter (DAT)-mediated dopamine uptake, dopamine efflux, and [3H]WIN 35,428 [(-)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane] binding in rat striatal tissue. Tamoxifen dose-dependently blocked dopamine uptake (54% reduction at 10 μM) and amphetamine-stimulated efflux (59% reduction at 10 μM) in synaptosomes. It also produced a small but significant reduction in [3H]WIN 35,428 binding in striatal membranes, indicating a weak interaction with the substrate binding site in the DAT. Biotinylation and cysteine accessibility studies indicated that tamoxifen stabilizes the outward-facing conformation of the DAT in a cocaine-like manner and does not affect surface expression of the DAT. Additional studies with mutant DAT constructs D476A and I159A suggested a direct interaction between tamoxifen and a secondary substrate binding site of the transporter. Locomotor studies revealed that tamoxifen attenuates amphetamine-stimulated hyperactivity in rats but has no depressant or stimulant activity in the absence of amphetamine. These results suggest a complex mechanism of action for tamoxifen as a regulator of the DAT. Due to its effectiveness against amphetamine actions and its central nervous system permeant activity, the tamoxifen structure represents an excellent starting point for a structure-based drug-design program to develop a pharmacological therapeutic for psychostimulant abuse.

Abstract 1797: Loss of FoxO1 expression is associated with decreased IGF-1R levels and alters IGFBP-1 induction in tamoxifen-resistant breast cancer cells

Abstract Breast cancer is one of the leading causes of cancer-related deaths in the world. The majority of breast cancers express estrogen receptor alpha (ERα) and tamoxifen, a selective estrogen receptor modulator, is commonly prescribed as adjuvant therapy for ERα-positive breast cancer patients. The development of chemoresistance is a common occurrence in treated patients, however, the molecular mechanisms associated with acquired tamoxifen resistance are not well defined. Previous work from our lab identified a novel mechanism of tamoxifen action that involved the accumulation of extracellular Insulin-like growth factor binding protein-1 (IGFBP-1) and modulates IGF-1-dependent signaling transduction. Tamoxifen-induced IGFBP-1 induction in this model was mediated by G protein-coupled estrogen receptor (GPER)-dependent CREB activation as shown by phosphorylation on serine 133. To determine IGFBP-1 induction is altered during the development of chemoresistance, this mechanism of action was studied in tamoxifen resistant MCF-7 (TamR) cells. Whereas CREB was constitutively phosphorylated in TamR cells, IGFBP-1 levels were not elevated when compared to parental cells and IGFBP-1 was not induced by tamoxifen treatment in TamR cells. These data suggest that another transcription factor(s) are also necessary for the induction of IGFBP-1 in breast cancer cells. FoxO1 is a known regulator of IGFBP-1 transcription and we tested whether FoxO1 contributes to tamoxifen-induced IGFBP-1 transcription. Our results indicate that FoxO1 expression levels were significantly reduced in TamR cells compared with parental cells. Furthermore, FoxO1 knockdown experiments demonstrated this transcription factor mediates tamoxifen-induced IGFBP-1 induction in MCF-7 cells. Consistent with previously published observations, IGF-1R expression was reduced in TamR cells compared to parental MCF-7 cells. To determine if loss of IGF-1R expression in involved in the loss of FoxO1 expression in TamR cells, IGF-1R was exogenously expressed and FoxO1 expression was measured. IGF-1R expression increases FoxO1 levels and partially restores tamoxifen-induced IGFBP-1 induction in TamR cells. These results suggest that FoxO1-mediated IGFBP-1 accumulation is altered during the development of tamoxifen resistance and is associated with decreased IGF-1R expression. Citation Format: Ali Vaziri-Gohar, Kevin D. Houston. Loss of FoxO1 expression is associated with decreased IGF-1R levels and alters IGFBP-1 induction in tamoxifen-resistant breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1797.

Abstract 5510: Tamoxifen induces estrogen receptor-independent bioenergetic stress: A synthetic lethality approach to target tumor metabolism

Abstract Tamoxifen, a selective estrogen receptor modulator (SERM) is clinically used for adjuvant treatment of estrogen receptor (ER) positive breast cancer. Recent data from our lab has shown that tamoxifen inhibits oxygen consumption at high, yet clinically relevant, doses. We hypothesize that this occurs through a non-ER dependent, direct effect on the mitochondria, and causes metabolic and signaling changes that can be exploited for therapeutic benefit. ER+, MCF7 breast cancer cells have a higher basal respiratory rate and lower glycolytic rate than ER, MDA231, cells. However, tamoxifen inhibited oxygen consumption to a similar degree in both cell lines Pharmacological inhibition of ERα expression or CRISPR-Cas9 mediated knock out of the ERα did not affect the ability of tamoxifen to decrease oxygen consumption. In response to tamoxifen inhibition of oxygen consumption, tumor cells increased dependence on glycolysis. Glucose deprivation or treatment with glycolytic inhibitors, 2-deoxy glucose (2DG) or 3-bromo-pyruvate significantly sensitized breast cancer cells to tamoxifen treatment. Tamoxifen combined with glucose deprivation or treatment with 2-DG led to robust activation of the AMPK signaling pathway. Knockout of AMPKα1 in breast cancer cells ameliorated cytotoxicity. Based on these data we’ve also demonstrated clinical usefulness of tamoxifen in a non-breast cancer model. BRAF inhibitor resistant melanoma exhibits a higher rate of oxidative phosphorylation over to the BRAF inhibitor sensitive melanoma cells. Tamoxifen-induced inhibition of oxygen consumption increased cytotoxicity in the OxPhos-addicted, BRAF inhibitor-resistant, population compared to the BRAF inhibitor sensitive cells. Our results indicate a new and unanticipated mechanism of action for tamoxifen in ER+ and ER-breast cancers. Moreover, since this effect is not ER dependent, tamoxifen may have clinical use in a wider spectrum of cancers especially when combined with inhibitors of glycolysis. Citation Format: Natalie A. Daurio, Stephen Tuttle, Constantinos Koumenis. Tamoxifen induces estrogen receptor-independent bioenergetic stress: A synthetic lethality approach to target tumor metabolism. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5510. doi:10.1158/1538-7445.AM2015-5510

Tamoxifen Directly Disrupts Platelet Angiogenic Potential and Inhibits Platelet-Mediated Metastasis

Platelets, which are mainly known for their role in hemostasis, are now known to play a crucial role in metastasis. Metastatic disease is the cause of roughly 90% of all cancer-related deaths and understanding the mechanisms leading to dissemination of tumor cells to distant sites remains one of the main challenges of cancer research. Tamoxifen is a selective estrogen receptor modulator that is widely used for the treatment and prevention of breast cancer. Interestingly, tamoxifen has demonstrated anti-cancer efficacy in estrogen negative breast cancers suggesting that this drug has additional mechanisms of action. Previously tamoxifen and its metabolites have been shown directly impact platelet function; however, the reported effects have been varied (Vitseva et al,. 2005, Nayak et al., 2011). Because platelets are critical for metastatic spread, we posited that tamoxifen may exert anti-tumor effects by directly altering platelet function. To explore this, we first examined the effect of tamoxifen on platelet activation. P-selectin expression was determined by flow cytometry for platelets pretreated with or without tamoxifen and then activated with ADP or MCF-7 cells. Both ADP and exposure to MCF-7 cells resulted in platelet activation and treatment with tamoxifen lead to a partial inhibition of activation. Platelets are a reservoir for angiogenic proteins that are secreted in a differentially regulated process. We have previously shown that we can manipulate the angiogenic potential of the platelet releasate through physiological (platelet agonists) and pathological activation (MCF-7 tumor cells) (Battinelli et al., 2011). We hypothesized that tamoxifen may impact malignancy by altering the release of angiogenesis regulatory proteins from platelets. To explore the impact of tamoxifen on the angiogenic potential of platelets, we analyzed the releasates from platelets exposed to tamoxifen alone or activated with ADP or MCF-7 cells in conjunction with tamoxifen. Our data reveals that platelets exposed to tamoxifen release significantly decreased amounts of VEGF in response to activation by either the platelet agonist ADP or interaction with tumor cells (MCF-7 cells). Next, in vitro angiogenesis assays were performed to further examine the effect of tamoxifen on the angiogenic potential of platelets. We observed dramatically diminished capillary tube branch point formation and decreased migration in endothelial cell cultures exposed to releasates generated from tamoxifen treated platelets compared to control releasates, demonstrating that tamoxifen inhibits the ability of activated platelets to promote angiogenesis. Platelets play a critical role in aiding the intravasation and extravasation of tumor cells in the circulation and therefore we postulated that tamoxifen could alter the ability of platelets to aid tumor cells in crossing the vascular endothelium. We performed transendothelial migration assays in which platelets were pretreated with tamoxifen or vehicle control, washed and mixed with MCF-7 tumor cells in endothelialized transwells. Platelets significantly increased the number of tumor cells that crossed the endothelial barrier; however this increase was lost when platelets were pretreated with tamoxifen. Overall our results demonstrate that tamoxifen directly alters platelet function, leading to decreased angiogenic and metastatic potential. These studies highlight a previously unknown mechanism of action for tamoxifen and may shed light on the efficacy of tamoxifen in estrogen-receptor negative cancers. Furthermore our work stresses the importance of crosstalk between platelets and cells within the tumor microenvironment for successful angiogenesis and metastatic spread and, ultimately, support the idea of utilizing targeted platelet therapies to inhibit the platelet’s role in malignancy. DisclosuresNo relevant conflicts of interest to declare.

Abstract 4460: Aromatase inhibition by active tamoxifen metabolites

Abstract Introduction: The mechanism of tamoxifen action in the treatment of breast cancer is believed to be via active metabolites that act as potent antagonists at the estrogen receptors. Despite this, few data have been published that relate the concentrations of these metabolites to their expected clinical effects. Since anti-estrogenic clinical effects may be brought about not only by estrogen antagonism but also by reduced estrogen concentrations, we tested the direct effects of tamoxifen and its active metabolites on aromatase activity. Methods: The activity of recombinant human aromatase was measured by following the generation of fluorescent metabolite from 7-methoxy-4-trifluoromethylcoumarin (MFC) in microsomal incubations in the presence and absence of multiple concentrations of tamoxifen, endoxifen, N-desmethyl-tamoxifen and 4-hydroxy-tamoxifen. Inhibition of aromatase was determined at enzyme concentrations set at 7.5 nM by measuring the percentage of enzyme activity remaining. Results: With MFC concentrations at the Km (25 µM), the IC50s for endoxifen and N-desmethyl-tamoxifen were 6.1 µM and 20.7 µM respectively. At concentrations up to 50 µM, no appreciable inhibition by tamoxifen or 4-hydroxy-tamoxifen was observed. Detailed characterization of inhibition by endoxifen and N-desmethyl-tamoxifen across a range of substrate concentrations demonstrated non-competitive kinetics for both inhibitors. The Ki values for endoxifen and N-desmethyl-tamoxifen were 4.0 µM and 15.9 µM respectively. Preincubation of these tamoxifen metabolites with aromatase did not demonstrate any irreversible effect on enzyme activity. Discussion: Among tamoxifen and its three major metabolites, only endoxifen and N-desmethyl-tamoxifen were found to be potent inhibitors of aromatase. This enzyme is the major source of estrogen in humans, and changes in its activity may thus alter estrogen-regulated physiology. Inhibition by these tamoxifen metabolites may contribute to changes in serum and tissue estrogen concentrations, and may explain in part the variability in bone density, serum lipid concentration, severity of musculoskeletal pain and breast cancer recurrence observed in patients taking tamoxifen. Relationships between tamoxifen metabolite concentrations and clinical outcomes may be complex, and mechanisms that underlie the multiple clinical effects of tamoxifen include the possibility of aromatase inhibition by endoxifen and N-desmethyl-tamoxifen. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4460. doi:10.1158/1538-7445.AM2011-4460

Tamoxifen metabolites as active inhibitors of aromatase in the treatment of breast cancer

The mechanism of tamoxifen action in the treatment of breast cancer is believed to be via active metabolites that act as potent estrogen receptor antagonists. Attempts to identify relationships between active metabolite concentrations and clinical outcomes have produced mixed results. Since anti-estrogenic effects may be brought about not only by estrogen antagonism, but also by reduced estrogen synthesis, we tested the ability of tamoxifen and its principal metabolites to inhibit aromatase in vitro. The activity of human aromatase in both recombinant and placental microsomal preparations was measured using the rate of generation of a fluorescent metabolite in the presence and absence of multiple concentrations of tamoxifen, endoxifen, N-desmethyl-tamoxifen, and Z-4-hydroxy-tamoxifen. Aromatase inhibition was further characterized by measuring the inhibition of testosterone metabolism to estradiol. The biochemical mechanisms of inhibition were documented and their inhibitory potency was compared. Using recombinant human aromatase, endoxifen, and N-desmethyl-tamoxifen were able to inhibit aromatase activity with K (i) values of 4.0 and 15.9μM, respectively. Detailed characterization of inhibition by endoxifen and N-desmethyl-tamoxifen indicated non-competitive kinetics for both inhibitors. Similarly, endoxifen-inhibited testosterone metabolism via a non-competitive mechanism. No appreciable inhibition by tamoxifen or Z-4-hydroxy-tamoxifen was observed at similar concentrations. The relative inhibitory potency was: endoxifen>N-desmethyl-tamoxifen>>>Z-4-hydroxy-tamoxifen>tamoxifen. Similar data were obtained in human placental microsomes. Endoxifen and N-desmethyl-tamoxifen were found to be potent inhibitors of aromatase. Inhibition by these tamoxifen metabolites may contribute to the variability in clinical effects of tamoxifen in patients with breast cancer. Relationships between tamoxifen metabolite concentrations and clinical outcomes may be complex, and the biologic mechanisms that underlie these relationships may include aromatase inhibition.

A new mechanism of action for tamoxifen

If taken for a 5-year period, the oestrogen-blocking drug tamoxifen significantly reduces breast-cancer recurrence;1 however, the incidence of debilitating hot flushes is substantially increased. More than half of patients who receive tamoxifen might be non-compliant 180 days after initiation of treatment.1 The oncostatic mechanism of the drug is believed to be through the reduction of oestrogen, but hydroxytamoxifen actually increases oestrogen levels; after 2 years of tamoxifen use, concentrations of serum dehydroepiandrosterone increased by 133% and mean oestradiol increased by 239%.

Regulating the tumor suppressor gene maspin in breast cancer cells: a potential mechanism for the anticancer properties of tamoxifen.

Mammary epithelial cells and the majority of breast cancer tumors require estrogen for continued growth. Antiestrogen therapy alone, or in combination with other drugs, has long been a common procedure for breast cancer treatment and prophylaxis. Thus, there is a critical need to elucidate the mechanism(s) of action of antiestrogen treatment, especially for patients who are at risk of breast cancer development or who are currently receiving hormone therapy. In this study, we examined the ability of hormones to regulate the expression of a tumor suppressor gene, maspin, which is a serine protease inhibitor (serpin) that plays an important role in mammary gland development and is silenced during breast cancer progression. Specifically, our hypothesis tested the clinical efficacy of tamoxifen to regulate maspin expression. We used maspin promoter luciferase reporter plasmids that were transfected into normal human mammary epithelial (HMEC1331) and MCF-7 breast cancer cells, followed by determination of the effect of hormones and their antagonists on maspin promoter activity. At the protein level, cytosolic fractions from both cell types before and after hormone treatment were subjected to Western blot analysis to determine maspin level. Our studies revealed that the antiestrogen tamoxifen induces maspin promoter activity. Interestingly, antiandrogen flutamide could also induce maspin in both cell lines tested. These observations were further confirmed in patient tissues. These novel findings provide a new mechanism of action for tamoxifen under normal and pathological conditions. More significantly, these findings could have a potential impact on future therapeutic intervention strategies for breast cancer.

Tamoxifen enhances myoepithelial cell suppression of human breast carcinoma progression in vitro by two different effector mechanisms

Our previous studies have indicated that myoepithelial cells surrounding ductal and acinar epithelium of glandular organs, such as the breast, exert multiple paracrine suppressive effects on incipient and developing cancers that arise from this epithelium. Myoepithelial cells and derived cell lines (HMS 1–6) exert these effects through the secretion of a number of different effector molecules that exert anti-invasive, anti-proliferative, and anti-angiogenic activities. Since previous basic and clinical studies have examined the role of estrogen agonists and antagonists on human breast cancer cells and because issues of hormone replacement therapy (HRT) and tamoxifen chemoprevention are such timely issues in breast cancer, we wondered whether or not hormonal manipulations might affect myoepithelial cells in vitro as far as their paracrine suppressive activities on breast cancer were concerned. The present in vitro study demonstrates that treatment of myoepithelial cells with tamoxifen but not 17β-estradiol increases both maspin secretion and invasion-blocking ability. Furthermore tamoxifen but not 17β-estradiol increases inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production by myoepithelial cells when they are co-cultured with conditioned media from or breast carcinoma cells directly. This increased myoepithelial NO exerts both autocrine and paracrine antiproliferative effects which can be blocked by inhibition of iNOS. 17β-Estradiol, however, competes with all of these suppressive effects of tamoxifen suggesting that the mechanism of tamoxifen action is estrogen receptor mediated. Myoepithelial cells lack ER-α but express ER-β. Tamoxifen, but not 17β-estradiol, increases AP-1 CAT but not ERE-CAT activity. Again, 17β-estradiol competes with the transcription-activating effects of tamoxifen. These experiments collectively suggest that the actions of tamoxifen on the increased secretion of maspin and increased production of NO by myoepithelial cells are mediated through ER-β and the transcription-activation of an ER-dependent AP-1 response element.

Tamoxifen as a possible chemotherapeutic agent in endometrial adenocarcinoma

Hormone-dependent endometrial adenocarcinomas are responsive to Tamoxifen at a daily oral dosage ranging from 40 to 60 mg. This responsiveness is manifested by marked histochemical and histological transformations of the adenocarcinoma, leading finally to atrophy and disappearance of the tumor. Second-line Tamoxifen treatment achieves greater than 50% objective remission rate in patients with persisting, recurrent, or metastatic endometrial adenocarcinoma previously treated with medroxyprogesterone. A close relationship is observed between the tumor responsiveness to medroxyprogesterone and to Tamoxifen. Minor possible side-effects are rarely observed under the Tamoxifen regimen. The direct mechanism of action for Tamoxifen on the endometrial adenocarcinoma is evident, while an indirect action seems rather unlikely. Hormone-dependent endometrial adenocarcinomas are responsive to Tamoxifen at a daily oral dosage ranging from 40 to 60 mg. This responsiveness is manifested by marked histochemical and histological transformations of the adenocarcinoma, leading finally to atrophy and disappearance of the tumor. Second-line Tamoxifen treatment achieves greater than 50% objective remission rate in patients with persisting, recurrent, or metastatic endometrial adenocarcinoma previously treated with medroxyprogesterone. A close relationship is observed between the tumor responsiveness to medroxyprogesterone and to Tamoxifen. Minor possible side-effects are rarely observed under the Tamoxifen regimen. The direct mechanism of action for Tamoxifen on the endometrial adenocarcinoma is evident, while an indirect action seems rather unlikely.