Ex vivo interaction between blood components and hormone-dependent breast cancer cells induces alterations associated with epithelial-mesenchymal transition and thrombosis.

The prevalence of breast cancer is steadily increasing with metastasis and thromboembolic complications identified as the most common causes of death. The acquisition of an aggressive phenotype by hormone-dependent breast cancers is mediated by Transforming Growth Factor Beta 1 (TGF-ß1) expression and is associated with epithelial-mesenchymal transition (EMT) and, potentially, increased propensity for thrombosis. We investigated this phenomenon by assessing the effect of platelet-rich plasma (PRP) and whole blood (WB) on parameters of EMT and hypercoagulation in vitro. MCF-7 breast cancer cells were cultured under standard conditions, followed by co-culture with PRP or WB. Cells were processed for real-time PCR (TGF-ß1 and vimentin), electron microscopy or immunocytochemistry (TGF-ß1). Micrographs were qualitatively assessed, and real-time PCR data analyzed with PAST Statistical Software. The addition of blood components heightened TGF-ß1 immunolocalization and significantly increased corresponding gene expression. Both PRP and WB significantly increased vimentin expression and induced a shape change from a typical epithelial phenotype to a spindle-shape morphology, indicative of EMT. Fibrin fiber, network and plaque formation indicated a hypercoagulatory environment. The results thus show that in preparation for hematogenous metastasis, hormone-dependent breast cancer cells assume an aggressive phenotype associated with EMT, simultaneously increasing the propensity for the formation of thrombo-emboli.

Novel Selective Estrogen Receptor Ligand Conjugates Incorporating Endoxifen-Combretastatin and Cyclofenil-Combretastatin Hybrid Scaffolds: Synthesis and Biochemical Evaluation.

Nuclear receptors such as the estrogen receptors (ERα and ERß) modulate the effects of the estrogen hormones and are important targets for design of innovative chemotherapeutic agents for diseases such as breast cancer and osteoporosis. Conjugate and bifunctional compounds which incorporate an ER ligand offer a useful method of delivering cytotoxic drugs to tissue sites such as breast cancers which express ERs. A series of novel conjugate molecules incorporating both the ER ligands endoxifen and cyclofenil-endoxifen hybrids covalently linked to the antimitotic and tubulin targeting agent combretastatin A-4 were synthesised and evaluated as ER ligands. A number of these compounds demonstrated pro-apoptotic effects, with potent antiproliferative activity in ER-positive MCF-7 breast cancer cell lines and low cytotoxicity. These conjugates displayed binding affinity towards ERα and ERß isoforms at nanomolar concentrations e.g., the cyclofenil-amide compound 13e is a promising lead compound of a clinically relevant ER conjugate with IC50 in MCF-7 cells of 187 nM, and binding affinity to ERα (IC50 = 19 nM) and ERß (IC50 = 229 nM) while the endoxifen conjugate 16b demonstrates antiproliferative activity in MCF-7 cells (IC50 = 5.7 nM) and binding affinity to ERα (IC50 = 15 nM) and ERß (IC50 = 115 nM). The ER binding effects are rationalised in a molecular modelling study in which the disruption of the ER helix-12 in the presence of compounds 11e, 13e and 16b is presented These conjugate compounds have potential application for further development as antineoplastic agents in the treatment of ER positive breast cancers.

Molecular docking, QSAR, pharmacophore modeling, and dynamics studies of some chromone derivatives for the discovery of anti-breast cancer agents against hormone-dependent breast cancer.

In search of new anti-breast cancer agents, the present study envisaged the design and synthesis of a series of benzopyran-chalcones. All the synthesized compounds were assayed for their in-vitro anticancer activity against ER + MCF-7 and triple-negative MDA-MB-231 breast cancer cell lines using SRB assay. The synthesized compounds were found active against ER + MCF-7 cell lines. Based on the in-vitro data, in-silico analysis was performed using hormone-dependent breast cancer targets such as hER-α and aromatase because the compounds showed activity against MCF-7 cells and none was active against MDA-MB-231. The in-silico results supported the in-vitro anticancer activity suggesting the affinity of compounds toward hormone-dependant breast cancer. Compounds 4A1 to 4A3 were found to be most cytotoxic to MCF-7 cells with IC50 values of 31.87, 22.95, and 20.34 µg/ml, respectively (Doxorubicin IC50: <10 µg/ml). In addition, they showed the interactions with the amino acid residues of a binding cavity of an hER-α. Furthermore, quantitative structure-activity relationship (QSAR) studies were performed to reveal the vital structural features required for anticancer activity against breast cancer. Molecular dynamic simulation studies of hER-α and 4A3 in comparison with the raloxifene complex ensure the appropriate refinement of compounds in the dynamic system. Additionally, a generated pharmacophore model explored the essential pharmacophoric features of the synthesized scaffolds with respect to clinically used drug molecules for optimal hormone-dependant anti-breast cancer activity.Communicated by Ramaswamy H. Sarma.

Novel synthetic derivatives of cinnamic and p-coumaric acids with antiproliferative effect on breast MCF-7 tumor cells.

p-Coumaric acid is derived from cinnamic acid and is one of the major compounds in the Brazilian green propolis extract. Studies have shown that both p-coumaric acid and cinnamic acid have promising antiproliferative effects. In this context, aiming to increase the complexity of these active natural products and their activities, we performed coupling reactions with propargylamine and benzylamine, as well as with threonine, phenylalanine and lysine amino acids, aiming to enhance their antiproliferative effects towards the hormone-dependent breast cancer MCF-7 cells. Overall, the p-coumaric acid coupling with L-threonine amino acid (compound 15) had the best selectivity index (SI = 5.1), with half-maximal inhibitory concentration of 39.6 ± 1 µM, showing a high selectivity against hormone-dependent breast cancer cell lines MCF-7 and low cytotoxicity against the normal breast cell lines MCF-10A. Thus, this new natural product derivative may represent a prototype for the future development of antiproliferative agents, especially against hormone-dependent breast cancer.

Dual Aromatase-Sulphatase Inhibitors (DASIs) for the Treatment of Hormone Dependent Breast Cancer.

Breast cancer is the most frequently diagnosed cancer in women and the second most common form of cancer, causing death after lung cancer, all across the globe at an alarming rate. The level of estrogens in breast cancer tissues of postmenopausal women is 10-40 folds higher than the non-carcinogenic breast tissues. As a result of this greater level of estrogen, breast tissue becomes more prone to develop breast cancer; mainly, estradiol plays a significant role in the initiation and development of hormone-dependent breast cancer. Androstenedione, Adrenal dehydroepiandrosterone sulfate, and estrone-sulfate also play an important role as precursors for estrogen biosynthesis. Estrogen deprivation exhibits an attractive phenomenon in the advancement of ideal therapeutics for the treatment of breast cancer. Inhibition of aromatase and sulphatase emerged as an attractive therapy for the treatment of hormone-dependent breast cancer via deprivation of estrogen by different pathways. The cocktail of aromatase and sulphatase inhibitors known as Dual Aromatase-sulphatase Inhibitors (DASIs) emerged as an attractive approach for effective estrogen deprivation. The present review article focused on the journey of dual aromatase-sulphatase inhibitors from the beginning to date (2020). Keeping in view the key observations, this review may be helpful for medicinal chemists to design and develop new and efficient dual aromatase-sulphatase inhibitors for the possible treatment of hormone- related breast cancer.

Nuclear PDCD4 Expression Defines a Subset of Luminal B-Like Breast Cancers with Good Prognosis.

The hormone receptor-positive (estrogen and/or progesterone receptor (PR)-positive) and HER2-negative breast cancer (BC) subtype is a biologically heterogeneous entity that includes luminal A-like (LumA-like) and luminal B-like (LumB-like) subtypes. Decreased PR levels is a distinctive biological feature of LumB-like tumors. These tumors also show reduced sensitivity to endocrine therapies and poorer prognosis than LumA-like tumors. Identification of biomarkers to accurately predict disease relapse in these subtypes is crucial in order to select effective therapies. We identified the tumor suppressor PDCD4 (programmed cell death 4), located in the nucleus (NPDCD4), as an independent prognostic factor of good clinical outcome in LumA-like and LumB-like subtypes. NPDCD4-positive LumB-like tumors presented overall and disease-free survival rates comparable to those of NPDCD4-positive LumA-like tumors, indicating that NPDCD4 improves the outcome of LumB-like patients. In contrast, NPDCD4 loss increased the risk of disease recurrence and death in LumB-like compared with LumA-like tumors. This, along with our results showing that LumB-like tumors present lower NPDCD4 positivity than LumA-like tumors, suggests that NPDCD4 loss contributes to endocrine therapy resistance in LumB-like BCs. We also revealed that PR induces PDCD4 transcription in LumB-like BC, providing a mechanistic explanation to the low PDCD4 levels in LumB-like BCs lacking PR. Finally, PDCD4 silencing enhanced BC cell survival in a patient-derived explant model of LumB-like disease. Our discoveries highlight NPDCD4 as a novel biomarker in LumA- and LumB-like subtypes, which could be included in the panel of immunohistochemical markers used in the clinic to accurately predict the prognosis of LumB-like tumors.

Incidence of comorbidities in women with breast cancer treated with tamoxifen or an aromatase inhibitor: an Australian population-based cohort study.

BACKGROUND: The development of comorbidities has become increasingly relevant with longer-term cancer survival. OBJECTIVE: To assess the pattern of comorbidities among Australian women with breast cancer treated with tamoxifen or an aromatase inhibitor. DESIGN: Retrospective cohort study using Pharmaceutical Benefits Scheme (PBS) data (10% sample) from January 2003 to December 2014. Dispensing claims data were used to identify comorbidities and classified with the Rx-Risk-V model. The breast cancer cohort had tamoxifen or an aromatase inhibitor dispensed between 2004 and 2011 with no switching between types of endocrine therapy. Comparisons were made between the breast cancer cohort and specific control groups (age- and sex-matched at 1:10 ratio without any dispensing of anti-neoplastic agents during the study period) for the development of five individual comorbidities over time using Cox regression models. RESULTS: Women treated with tamoxifen had a higher incidence of cardiovascular conditions, diabetes, and pain or pain-inflammation, but a lower incidence of hyperlipidaemia compared with non-cancer control groups, as indicated by PBS data. Women treated with aromatase inhibitors were more likely to develop cardiovascular conditions, osteoporosis, and pain or pain-inflammation compared with non-cancer control groups. The risks of hyperlipidaemia and osteoporosis were significantly lower among tamoxifen users compared with aromatase inhibitor users. CONCLUSIONS: Women with hormone-dependent breast cancer treated with an endocrine therapy had a higher risk of developing specified comorbid conditions than women without cancer, with different comorbidity profiles for those on tamoxifen versus aromatase inhibitors. Further research into the causes and mechanism of development and management of comorbidities after cancer is needed.

Design, Synthesis and Biochemical Evaluation of Novel Selective Estrogen Receptor Ligand Conjugates Incorporating an Endoxifen-Combretastatin Hybrid Scaffold.

Nuclear-receptors are often overexpressed in tumours and can thereby be used as targets when designing novel selective chemotherapeutic agents. To date, many conjugates incorporating an estrogen receptor (ER) ligand have been synthesised in order to direct chemical agents to tissue sites containing ERs. A series of ER ligand conjugates were synthesised incorporating an antagonistic ER ligand scaffold based on endoxifen, covalently-bound via an amide linkage to a variety of combretastatin-based analogues, which may act as antimitotic agents. These novel endoxifen-combretastatin hybrid scaffold analogues were biochemically evaluated in order to determine their antiproliferative and cytotoxicity effects in both the ER-positive MCF-7 and the ER-negative MDA-MB-231 human breast cancer cell lines. ER competitive binding assays were carried out to assess the binding affinity of the lead conjugate 28 towards both the ERα and ERß isoforms. In results from the NCI 60-cell line screen, the lead conjugate 28 displayed potent and highly selective antiproliferative activity towards the MCF-7 human cancer cell line (IC50 = 5 nM). In the ER-binding assays, the lead conjugate 28 demonstrated potent ER competitive binding in ERα (IC50 value: 0.9 nM) and ERß (IC50 value: 4.7 nM). Preliminary biochemical results also demonstrate that the lead conjugate 28 may exhibit pure antagonism. This series makes an important addition to the class of ER antagonists and may have potential applications in anticancer therapy.

Unravelling exemestane: From biology to clinical prospects.

Aromatase inhibitors (AIs) are anti-tumor agents used in clinic to treat hormone-dependent breast cancer. AIs block estrogens biosynthesis by inhibiting the enzyme aromatase, preventing tumor progression. Exemestane, a third-generation steroidal AI, belongs to this class of drugs and is currently used in clinic to treat postmenopausal women, due to its high efficacy and good tolerability. Here, its pharmacological and biological aspects as well as its clinical applications and comparison to other endocrine therapeutic agents, are reviewed. It is also focused the benefits and risks of exemestane, drawbacks to be overcome and aspects to be explored.

Exemestane metabolites: Synthesis, stereochemical elucidation, biochemical activity and anti-proliferative effects in a hormone-dependent breast cancer cell line.

Exemestane is a third-generation steroidal aromatase inhibitor that has been used in clinic for hormone-dependent breast cancer treatment in post-menopausal women. It is known that exemestane undergoes a complex metabolization, giving rise to some already identified metabolites, the 17ß-hydroxy-6-methylenandrosta-1,4-dien-3-one (17-ßHE) and the 6-(hydroxymethyl)androsta-1,4,6-triene-3,17-dione (6-HME). In this study, four metabolites of exemestane have been analyzed, three of them were synthesized (6ß-spirooxiranandrosta-1,4-diene-3,17-dione (2), 1α,2α-epoxy-6-methylenandrost-4-ene-3,17-dione (3) and 17-ßHE (4)) while one was acquired, the 6-HME (6). The stereochemistry of the epoxide group of 2 and 3 has been unequivocally elucidated for the first time on the basis of NOESY experiments. New structure-activity relationships (SAR) have been established through the observation that the substitution of the double bonds by epoxide groups led to less potent derivatives in microsomes. However, the reduction of the C-17 carbonyl group to a hydroxyl group originating 17-ßHE (4) resulted in a significant increase in activity in MCF-7aro cells when compared to exemestane (IC50 0.25 µM vs 0.90 µM, respectively). All the studied metabolites reduced MCF-7aro cells viability in a dose and time-dependent manner, and metabolite 3 was the most potent one. Altogether our results showed that not only exemestane but also its main metabolites are potent aromatase inhibitors and reduce breast cancer cells viability. This suggests that exemestane efficacy may also be due to the active metabolites that result from its metabolic transformation. Our results emphasize the importance of performing further studies to expand our understanding of exemestane actions in breast cancer cells.

Local adipocytes enable estrogen-dependent breast cancer growth: Role of leptin and aromatase.

The importance of the microenvironment in breast cancer growth and progression is becoming increasingly clear. Adipocytes are abundant in the mammary microenvironment, and recent studies show that adipocytes produce endocrine, inflammatory, and angiogenic factors that have tremendous potential to affect adjacent breast cancer cells. Yet, the extent to which local adipocyte function contributes to the pathogenesis of breast cancer is largely unexplored. Here we describe a unique animal model to study interactions between adipocytes and breast cancer cells in the tumor microenvironment. Our results suggest that local interactions between adipocytes and tumor cells are sufficient to promote the growth of hormone-dependent breast cancer. We also demonstrate that leptin signaling in adipocytes induces aromatase expression, expected to result in higher estrogen in the microenvironment thus enabling mammary tumorigenesis.