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.
In this work, we have investigated the antiproliferative effect of AdoMet and Doxorubicin (Doxo), alone or in combination, on different breast cancer cell lines. For the evaluation of synergism, we ...have calculated the combination index (CI) by the Calcusyn software and we have evaluated the effects of the combination on apoptosis occurrence at FACS analysis in hormone-dependent CG5 cell line. We have found that AdoMet and Doxo given in combination were strongly synergistic in the hormone-dependent CG5 and MCF-7 human breast cancer cell line, as a CI50 < 0.5 was found after 72 h of treatment while the effect was only additive in hormone-independent MDA-MB 231 cells. On the basis of our results, we have selected a combination of AdoMet and Doxo, that was highly synergistic and we have found that the AdoMet in combination with Doxo increased apoptosis induced by Doxo alone, suggesting that the synergism on growth inhibition was largely due to apoptosis. Notably, the AdoMet/Doxo combination induced a significant activation of caspases 3, and 8, while no effect was found on caspase 9 cleavage. In contrast, no significant changes of the expression of cleaved caspase 8 and 9 were found in cells treated with AdoMet and Doxo alone. Moreover, the combination induced a significant increase of Fas and FasL expression. These results highlight the importance of the synergistic effect of AdoMet with Doxo in the regulation of hormone-dependent breast cancer cell proliferation and emphasize the anti-tumor activity of these molecules.
► We studied the effects of four AIs in sensitive and resistant breast cancer cells. ► We examined the AIs anti-aromatase activity in sensitive breast cancer cells. ► The double bond in C-4, 5 ...positions is important for the AIs biological effects. ► AIs have different anti-proliferative effects in resistant breast cancer cells. ► Autophagy inhibition sensitizes resistant cancer cells to AIs.
Several therapeutic approaches are used in estrogen receptor positive (ER+) breast cancers, being one of them the use of aromatase inhibitors (AIs). Although AIs demonstrate higher efficacy than tamoxifen, they can also exhibit de novo or acquired resistance after prolonged treatment. Recently, we have described the synthesis and biochemical evaluation of four steroidal AIs, 3β-hydroxyandrost-4-en-17-one (1), androst-4-en-17-one (12), 4α,5α-epoxyandrostan-17-one (13a) and 5α-androst-2-en-17-one (16), obtained from modifications in the A-ring of the aromatase substrate, androstenedione. In this study, it was investigated the biological effects of these AIs in different breast cancer cell lines, an ER+ aromatase-overexpressing human breast cancer cell line (MCF-7aro cells), an estrogen-receptor negative (ER−) human breast cancer cell line (SK-BR-3 cells), and a late stage of acquired resistance cell line (LTEDaro cells). The effects of an autophagic inhibitor (3-methyladenine) plus AIs 1, 12, 13a or exemestane in LTEDaro cells were also studied to understand the involvement of autophagy in AI acquired resistance. Our results showed that these steroids inhibit aromatase of MCF-7aro cells and decrease cell viability in a dose- and time-dependent manner. The new AI 1 is the most potent inhibitor, although the AI 12 demonstrates to be the most effective in decreasing cell viability. Besides, and in advantage over exemestane, AIs 12 and 13a also reduced LTEDaro cells viability. The use of the autophagic inhibitor allowed AIs to diminish viability of LTEDaro cells, presenting a similar behavior to the sensitive cells. Thus, inhibition of autophagy may sensitize hormone-resistant cancer cells to anti-estrogen therapies.
Different hormonal therapies are used for estrogen receptor positive (ER
+
) breast cancers, being the third-generation of aromatase inhibitors (AIs), an effective alternative to the classical ...tamoxifen. AIs inhibit the enzyme aromatase, which is responsible for catalyzing the conversion of androgens to estrogens. In this study, it was evaluated the effects of several steroidal AIs, namely 3β-hydroxyandrost-4-en-17-one (
1
), androst-4-en-17-one (
12
), 4α,5α-epoxyandrostan-17-one (
13a
) and 5α-androst-2-en-17-one (
16
), on cell proliferation, cell cycle progression and cell death in an ER
+
aromatase-overexpressing human breast cancer cell line (MCF-7aro). All AIs induced a decrease in cell proliferation and these anti-proliferative effects were due to a disruption in cell cycle progression and cell death, by apoptosis. AIs
1
and
16
caused cell cycle arrest in G
0
/G
1
, while AIs
12
and
13a
induced an arrest in G
2
/M. Moreover, it was observed that these AIs induced apoptosis by different pathways, since AIs
1
,
12
and
13a
activated the apoptotic mitochondrial pathway, while AI
16
induced apoptosis through activation of caspase-8. These results are important for the elucidation of the cellular effects of steroidal AIs on breast cancer cells and will also highlight the importance of AIs as inducers of apoptosis in hormone-dependent breast cancers.
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
towards both the ERα and ERβ isoforms. In results from the NCI 60-cell line screen, the lead conjugate
displayed potent and highly selective antiproliferative activity towards the MCF-7 human cancer cell line (IC
= 5 nM). In the ER-binding assays, the lead conjugate
demonstrated potent ER competitive binding in ERα (IC
value: 0.9 nM) and ERβ (IC
value: 4.7 nM). Preliminary biochemical results also demonstrate that the lead conjugate
may exhibit pure antagonism. This series makes an important addition to the class of ER antagonists and may have potential applications in anticancer therapy.
An improved steroid sulfatase inhibitor was prepared by replacing the N-propyl group of the second-generation steroid-like inhibitor (2) with a N-3,3,3-trifluoropropyl group to give (10). This ...compound is 5-fold more potent in vitro, completely inhibits rat liver steroid sulfatase activity after a single oral dose of 0.5 mg/kg, and exhibits a significantly longer duration of inhibition over (2). These biological properties are attributed to the increased lipophilicity and metabolic stability of (10) rendered by its trifluoropropyl group and also the potential H-bonding between its fluorine atom(s) and Arg(98) in the active site of human steroid sulfatase. Like other sulfamates, (10) is expected to be sequestered, and transported by, erythrocytes in vivo because it inhibits human carbonic anhydrase II (hCAII) potently (IC(50), 3 nmol/L). A congener (4), which possesses a N-(pyridin-3-ylmethyl) substituent, is even more active (IC(50), 0.1 nmol/L). To rationalize this, the hCAII-(4) adduct, obtained by cocrystallization, reveals not only the sulfamate group and the backbone of (4) interacting with the catalytic site and the associated hydrophobic pocket, respectively, but also the potential H-bonding between the N-(pyridin-3-ylmethyl) group and Nepsilon(2) of Gln(136). Like (2), both (10) and its phenolic precursor (9) are non-estrogenic using a uterine weight gain assay. In summary, a highly potent, long-acting, and nonestrogenic steroid sulfatase inhibitor was designed with hCAII inhibitory properties that should positively influence in vivo behavior. Compound (10) and other related inhibitors of this structural class further expand the armory of steroid sulfatase inhibitors against hormone-dependent breast cancer.
Human 17β-hydroxysteroid dehydrogenase (17β-HSD) type 1 is an enzyme that acts at the pre-receptor level. It catalyzes the NADPH-dependent reduction of the weak estrogen estrone into the most potent ...estrogen 17β-estradiol, which exerts proliferative effects via estrogen receptors. Overexpression of 17β-HSD type 1 in estrogen-responsive tissues is related to the development of hormone-dependent diseases, such as breast cancer and endometriosis. 17β-HSD type 1 thus represents an attractive target for development of new drugs. Recently, we discovered that substituted coumarin derivatives potently and selectively inhibit 17β-HSD type 1. In the present study, we have performed additional biochemical and biological evaluation of the most promising coumarin derivative. First, we used an efficient method for isolation and purification of the active, soluble recombinant human 17β-HSD type 1 from
Escherichia coli. This 17β-HSD type 1 showed a specific activity of 0.64
±
0.08
μmol
min
−1
mg
−1 for estrone reduction in the presence of NADPH at pH 6.5, and a
K
m of 62
nM for estrone. Next, we evaluated the best of the coumarin-derivative inhibitors, showing its reversible and competitive inhibition of 17β-HSD type 1 reductive activity with a
K
i of 53
nM. We confirmed that this coumarin inhibitor acts not only in a cell-free assay, but also decreases endogenous 17β-HSD type 1 activity in human T-47D breast cancer cells. This inhibitor also reduced estrone dependent growth of T-47D cells after 48
h of incubation.
Estrogens are biosynthesised from androgens by the CYP450 enzyme complex called aromatase. Aromatase is expressed in the ovary, placenta, brain, bone, adipose tissue and breast tissue. In breast ...cancer, intratumoural aromatase is the source for local estrogen production in the tissue. Inhibition of aromatase is an important approach for reducing growth stimulatory effects of estrogens in estrogen-dependent breast cancer. The potent and selective third-generation aromatase inhibitors anastrozole, letrozole and exemestane were introduced to the market as endocrine therapy in postmenopausal patients failing anti-estrogen therapy alone, or multiple hormonal therapies. Anastrozole and letrozole are both non-steroidal aromatase inhibitors that compete with the substrate for binding to the enzyme active site. Exemestane is a mechanism-based steroidal inhibitor that mimics the substrate, is converted by the enzyme to a reactive intermediate, and results in inactivation of aromatase. These third-generation aromatase inhibitors are currently approved as first-line therapy for the treatment of postmenopausal women with metastatic estrogen-dependent breast cancer. The use of an aromatase inhibitor as initial therapy, or after treatment with tamoxifen, is now recommended as adjuvant hormonal therapy for postmenopausal women with hormone-dependent breast cancer. Several clinical studies of aromatase inhibitors focus on the use of these agents in the adjuvant setting, for the treatment of early breast cancer. Recently published results show improved responses with these agents compared with tamoxifen.
The rheumatic adverse effects accompanying treatment with aromatase inhibitors (AIs) in hormone-dependent breast cancer represent an area of clinical relevance and emerging concern. This report ...describes these rheumatic complaints detailing their clinical pattern.
During 1-year period, 18 consecutive postmenopausal women (mean age, 58.33 years; range, 52-66 years) in treatment with AIs for hormone-dependent breast cancer (mean duration of therapy, 12.0 months; range, 9.1-17.7 months) were referred for evaluation in the outpatient clinic of the rheumatology unit in relation to rheumatic complaints. According to a routine protocol planned with oncologists, patient evaluations consisted of a complete clinical examination with careful assessment of rheumatic complaints and related physical symptoms, followed by laboratory testing and a bone scintiscan. In no cases were rheumatic complaints present before AI therapy.
On the basis of clinical data and investigations and by applying accepted diagnostic criteria, a diagnosis of an undifferentiated spondyloarthropathy was reached in 10 (55.5%) of the 18 patients studied, and an oligoarthritis was shown in 2 more patients (11.1%), whereas a simple arthralgia was found in the remaining 6 patients (33.3%). In the patients meeting criteria as belonging to a spondyloarthritic subset, a family history positive for psoriasis and celiac disease was shown in 2 and 1 instance, respectively, whereas HLA-CW6 and HLA-B27 were detected in 3 and 1 case. A high serum level of anti-cyclic citrullinated peptide antibodies was shown in 1 patient with oligoarthritis. Most of the patients (16/18) were treated with nonsteroidal anti-inflammatory drugs or with corticosteroids. Methotrexate (10 mg weekly) was added in 3 of these patients, nonresponders. Aromatase inhibitor discontinuation was needed in the remaining 2 cases with spontaneous resolution of symptoms over time.
Data from the present study emphasize a previously unsuspected high prevalence of defined arthritides underlying these rheumatic complaints. Therefore, investigative efforts should be addressed to better clarify the clinical and pathogenetic significance of these important consequences of AI therapy. An accurate monitoring of rheumatic complaints has to be suggested to patients taking AI therapy, with a rapid referral to a rheumatologist in the case of consistent suspicion of an inflammatory arthritis.
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