Cannabidivarin and cannabigerol induce unfolded protein response and angiogenesis dysregulation in placental trophoblast HTR-8/SVneo cells.

Cannabidivarin (CBDV) and cannabigerol (CBG) are minor phytocannabinoids from Cannabis sativa, whose health benefits have been reported. However, studies about the impact of these cannabinoids on fundamental cellular processes in placentation are scarce. Placental development involves physiological endoplasmic reticulum (ER) stress, however when exacerbated it can lead to altered angiogenesis and pregnancy disorders, such as intrauterine growth restriction and preeclampsia. In this work, the effects of CBDV and CBG (1-10 µM) on placental extravillous trophoblasts were studied, using the in vitro model HTR-8/SVneo cells. Both cannabinoids induced anti-proliferative effects and reactive oxygen/nitrogen species generation, which was dependent on transient receptor potential vanilloid 1 (TRPV1) activation. Moreover, CBDV and CBG significantly upregulated, in a TRPV-1 dependent manner, the gene expression of HSPA5/Glucose-regulated protein 78 (GRP78/BiP), a critical chaperone involved in ER stress and unfolded protein response (UPR) activation. Nevertheless, the UPR pathways were differentially activated. Both cannabinoids were able to recruit the IRE branch, while only CBDV enhanced the expression of downstream effectors of the PERK pathway, namely p-eIF2α, ATF4 and CHOP. It also augmented the activity of the apoptotic initiator caspases-8 and -9, though the effector caspases-3/-7 were not activated. TRB3 expression was increased by CBDV, which may hinder apoptosis termination. Moreover, both compounds upregulated the mRNA levels of the angiogenic factors VEGFA, PGF and sFLT1, and disrupted the endothelial-like behavior of HTR-8/SVneo cells, by reducing tube formation. Thus, CBDV and CBG treatment interferes with EVTs functions and may have a negative impact in placentation and in pregnancy outcome.

Influence of tumor microenvironment on the different breast cancer subtypes and applied therapies.

Despite the significant improvements made in breast cancer therapy during the last decades, this disease still has increasing incidence and mortality rates. Different targets involved in general processes, like cell proliferation and survival, have become alternative therapeutic options for this disease, with some of them already used in clinic, like the CDK4/6 inhibitors for luminal A tumors treatment. Nevertheless, there is a demand for novel therapeutic strategies focused not only on tumor cells, but also on their microenvironment. Tumor microenvironment (TME) is a very complex and dynamic system that, more than surrounding and supporting tumor cells, actively participates in tumor development and progression. During the last decades, it has become clear that the cellular and acellular components of TME differ between the various breast cancer subtypes and shape the differences regarding their severity and prognosis. The pivotal role of the TME in controlling tumor growth and influencing responses to therapy represents a potential source for novel targets and therapeutic strategies. In this review, we present a description of the multiple therapeutic options used for different breast cancer subtypes, as well as the influence that the TME may exert on the development of the disease and on the response to the distinct therapies, which in some cases may explain their failure by the occurrence of relapses and resistance. Furthermore, the ongoing studies focused on the use of TME components for developing potential cancer treatments are described.

Effects of a combination of cannabidiol and delta-9-tetrahydrocannabinol on key biological functions of HTR-8/SVneo extravillous trophoblast cells.

In recent years, cannabis use has increased among pregnant women. In addition, the phytocannabinoids cannabidiol (CBD) and delta-9-tetrahydrocannabinol (THC) alone or in combination are being used for therapeutical applications. THC and CBD are able to cross the placenta and a lot remains unknown concerning their impact on angiogenesis and extravillous trophoblasts' (EVTs) migration and invasion, which are essential processes for placentation. Thus, in this study, the HTR-8/SVneo cell line was employed to evaluate the effects of CBD, THC and of their combination (1:1, 2 µM). Cannabinoids affected epithelial-mesenchymal transition, as showed by increased expression of the epithelial protein marker E-cadherin for CBD and CBD plus THC treatments, and decrease of mesenchymal intermediate filament vimentin for all treatments. The gene expression of the metalloproteinases MMP2 and MMP9, and of their inhibitors TIMP1 and TIMP2 was increased, except the latter for THC treatment. Moreover, CBD reduced cell migration and invasion, an effect that was enhanced by its combination with THC. CBD with or without THC also upregulated the gene expression of PGF, while the anti-angiogenic factor sFLT1 was increased for all treatments. VEGFA and FLT1 were not affected. Alone or combined CBD and THC also decreased tube segments' length. Additionally, ERK1/2 and STAT3 phosphorylation was increased in the CBD and CBD plus THC-treated cells, while THC only activated STAT3. AKT activation was only affected by CBD. This work demonstrates that the exposure to cannabinoid-based products containing CBD and/or THC, may interfere with key processes of EVTs differentiation. Therefore, crucial phases of placental development can be affected, compromising pregnancy success.

In Vitro Effects of Combining Genistein with Aromatase Inhibitors: Concerns Regarding Its Consumption during Breast Cancer Treatment.

INTRODUCTION: The third-generation of aromatase inhibitors (AIs)-Exemestane (Exe), Letrozole (Let), and Anastrozole (Ana)-is the main therapeutic approach applied for estrogen receptor-positive (ER+) breast cancer (BC), the most common neoplasm in women worldwide. Despite their success, the development of resistance limits their efficacy. Genistein (G), a phytoestrogen present in soybean, has promising anticancer properties in ER+ BC cells, even when combined with anticancer drugs. Thus, the potential beneficial effects of combining G with AIs were investigated in sensitive (MCF7-aro) and resistant (LTEDaro) BC cells. METHODS: The effects on cell proliferation and expression of aromatase, ERα/ERß, and AR receptors were evaluated. RESULTS: Unlike the combination of G with Ana or Let, which negatively affects the Ais' therapeutic efficacy, G enhanced the anticancer properties of the steroidal AI Exe, increasing the antiproliferative effect and apoptosis relative to Exe. The hormone targets studied were not affected by this combination when compared with Exe. CONCLUSIONS: This is the first in vitro study that highlights the potential benefit of G as an adjuvant therapy with Exe, emphasizing, however, that soy derivatives widely used in the diet or applied as auxiliary medicines may increase the risk of adverse interactions with nonsteroidal AIs used in therapy.

Cannabidiol as a Promising Adjuvant Therapy for Estrogen Receptor-Positive Breast Tumors: Unveiling Its Benefits with Aromatase Inhibitors.

BACKGROUND: Estrogen receptor-positive (ER+) breast cancer is the most diagnosed subtype, with aromatase inhibitors (AIs) being one of the therapeutic drug types used in the clinic. However, endocrine resistance may develop after prolonged treatment, and different approaches, such as combining endocrine and targeted therapies, have been applied. Recently, we demonstrated that cannabidiol (CBD) induces anti-tumor actions in ER+ breast cancer cells by targeting aromatase and ERs. Considering this, we studied, in vitro, whether CBD when combined with AIs could improve their effectiveness. METHODS: MCF-7aro cells were used and the effects on cell viability and on the modulation of specific targets were investigated. RESULTS: CBD when combined with anastrozole (Ana) and letrozole (Let) caused no beneficial effect in comparison to the isolated AIs. In contrast, when combined with the AI exemestane (Exe), CBD potentiated its pro-cell death effects, abolished its estrogen-like effect, impaired ERα activation, and prevented its oncogenic role on the androgen receptor (AR). Moreover, this combination inhibited ERK1/2 activation, promoting apoptosis. The study of the hormonal microenvironment suggests that this combination should not be applied in early stages of ER+ breast tumors. CONCLUSIONS: Contrary to Ana and Let, this study highlights the potential benefits of combining CBD with Exe to improve breast cancer treatment and opens up the possibility of new therapeutic approaches comprising the use of cannabinoids.

An Exemestane Derivative, Oxymestane-D1, as a New Multi-Target Steroidal Aromatase Inhibitor for Estrogen Receptor-Positive (ER+) Breast Cancer: Effects on Sensitive and Resistant Cell Lines.

Around 70-85% of all breast cancer (BC) cases are estrogen receptor-positive (ER+). The third generation of aromatase inhibitors (AIs) is the first-line treatment option for these tumors. Despite their therapeutic success, they induce several side effects and resistance, which limits their efficacy. Thus, it is crucial to search for novel, safe and more effective anti-cancer molecules. Currently, multi-target drugs are emerging, as they present higher efficacy and lower toxicity in comparison to standard options. Considering this, this work aimed to investigate the anti-cancer properties and the multi-target potential of the compound 1α,2α-epoxy-6-methylenandrost-4-ene-3,17-dione (Oxy), also designated by Oxymestane-D1, a derivative of Exemestane, which we previously synthesized and demonstrated to be a potent AI. For this purpose, it was studied for its effects on the ER+ BC cell line that overexpresses aromatase, MCF-7aro cells, as well as on the AIs-resistant BC cell line, LTEDaro cells. Oxy reduces cell viability, impairs DNA synthesis and induces apoptosis in MCF-7aro cells. Moreover, its growth-inhibitory properties are inhibited in the presence of ERα, ERß and AR antagonists, suggesting a mechanism of action dependent on these receptors. In fact, Oxy decreased ERα expression and activation and induced AR overexpression with a pro-death effect. Complementary transactivation assays demonstrated that Oxy presents ER antagonist and AR agonist activities. In addition, Oxy also decreased the viability and caused apoptosis of LTEDaro cells. Therefore, this work highlights the discovery of a new and promising multi-target drug that, besides acting as an AI, appears to also act as an ERα antagonist and AR agonist. Thus, the multi-target action of Oxy may be a therapeutic advantage over the three AIs applied in clinic. Furthermore, this new multi-target compound has the ability to sensitize the AI-resistant BC cells, which represents another advantage over the endocrine therapy used in the clinic, since resistance is a major drawback in the clinic.

Design, synthesis, biological activity evaluation and structure-activity relationships of new steroidal aromatase inhibitors. The case of C-ring and 7ß substituted steroids.

In this work, new steroidal aromatase inhibitors (AIs) were designed, synthesized, and tested. In one approach, C-ring substituted steroids namely those functionalized at C-11 position with an α or ß hydroxyl group or with a carbonyl group as well as C-9/C-11 steroidal olefins and epoxides were studied. It was found that the carbonyl group at C-11 is more beneficial for aromatase inhibition than the hydroxyl group, and that the C-ring epoxides were more potent than the C-ring olefins, leading to the discovery of a very strong AI, compound 7, with an IC50 of 0.011 µM, better than Exemestane, the steroidal AI in clinical use, which presents an IC50 of 0.050 µM. In another approach, we explored the biological activity of A-ring C-1/C-2 steroidal olefins and epoxides in relation to aromatase inhibition and compared it with the biological activity of C-ring C-9/C-11 steroidal olefins and epoxides. On the contrary to what was observed for the C-ring olefins and epoxides, the A-ring epoxides were less potent than A-ring olefins. Finally, the effect of 7ß-methyl substitution on aromatase inhibition was compared with 7α-methyl substitution, showing that 7ß-methyl is better than 7α-methyl substitution. Molecular modelling studies showed that the 7ß-methyl on C-7 seems to protrude into the opening to the access channel of aromatase in comparison to the 7α-methyl. This comparison led to find the best steroidal AI (12a) of this work with IC50 of 0.0058 µM. Compound 12a showed higher aromatase inhibition capacity than two of the three AIs currently in clinical use.

Bisphenols A, F, S and AF trigger apoptosis and/or endoplasmic reticulum stress in human endometrial stromal cells.

Disruption of non-differentiated endometrial stromal cells could have noxious consequences in female reproduction, impairing endometrial remodelling and implantation. Following the classification of bisphenol A (BPA) as an endocrine disrupting chemical, it started to be gradually withdrawn from the market, being substituted by structural analogues, whose effects in human health are not fully understood. This work used a telomerase-immortalized human endometrial stromal cell line (St-T1b) to study the effects of BPA and its three most commercialized structural analogues (ranked: bisphenols S, F and AF) on endometrial stromal cells to understand their effects on female reproductive function. Bisphenols showed dissimilar effects. All four compounds generated endoplasmic reticulum (ER) stress. In addition, bisphenols A, F and AF induced apoptosis through different mechanisms, with bisphenol AF causing cell cycle arrest at G2/M phase. Bisphenol AF decreased mitochondrial transmembrane potential and bisphenols A, F and AF produced oxidative stress.

Effects of PI3K inhibition in AI-resistant breast cancer cell lines: autophagy, apoptosis, and cell cycle progression.

INTRODUCTION: Breast cancer is the leading cause of cancer death in women. The aromatase inhibitors (AIs), Anastrozole (Ana), Letrozole (Let), and Exemestane (Exe) are a first-line treatment option for estrogen receptor-positive (ER+) breast tumors, in postmenopausal women. Nevertheless, the development of acquired resistance to this therapy is a major drawback. The involvement of PI3K in resistance, through activation of the PI3K/AKT/mTOR survival pathway or through a cytoprotective autophagic process, is widely described. MATERIALS AND METHODS: The involvement of autophagy in response to Ana and Let treatments and the effects of the combination of BYL-719, a PI3K inhibitor, with AIs were explored in AI-resistant breast cancer cell lines (LTEDaro, AnaR, LetR, and ExeR). RESULTS: We demonstrate that Ana and Let treatments do not promote autophagy in resistant breast cancer cells, contrary to Exe. Moreover, the combinations of BYL-719 with AIs decrease cell viability by different mechanisms by nonsteroidal vs. steroidal AIs. The combination of BYL-719 with Ana or Let induced cell cycle arrest while the combination with Exe promoted cell cycle arrest and apoptosis. In addition, BYL-719 decreased AnaR, LetR, and ExeR cell viability in a dose- and time-dependent manner, being more effective in the ExeR cell line. This decrease was further exacerbated by ICI 182,780. CONCLUSION: These results corroborate the lack of cross-resistance between AIs verified in the clinic, excluding autophagy as a mechanism of resistance to Ana or Let and supporting the ongoing clinical trials combining BYL-719 with AIs.

Differential biological effects of aromatase inhibitors: Apoptosis, autophagy, senescence and modulation of the hormonal status in breast cancer cells.

Estrogen receptor-positive (ER+) breast carcinomas are the most common subtype, corresponding to 60% of the cases in premenopausal and 75% in postmenopausal women. The third-generation of aromatase inhibitors (AIs), the non-steroidal Anastrozole (Ana) and Letrozole (Let) and the steroidal Exemestane (Exe), are considered a first-line endocrine therapy for postmenopausal women. Despite their clinical success, the development of resistance is the major setback in clinical practice. Nevertheless, the lack of cross-resistance between AIs hints that these drugs may act through distinct mechanisms. Therefore, this work studied the different effects induced by AIs on biological processes, such as cell proliferation, death, autophagy and senescence. Moreover, their effects on the regulation of the hormonal environment were also explored. The non-steroidal AIs induce senescence, through increased YPEL3 expression, on aromatase-overexpressing breast cancer cells (MCF-7aro), whereas Exe promotes a cytoprotective autophagy, thus blocking senescence induction. In addition, in a hormone-enriched environment, the non-steroidal AIs prevent estrogen signaling, despite up-regulating the estrogen receptor alpha (ERα), while Exe down-regulates ERα and maintains its activation. In these conditions, all AIs up-regulate the androgen receptor (AR) which blocks EGR3 transcription in Exe-treated cells. On the other hand, in hormone-depleted conditions, a crosstalk between AR and ERα occurs, enhancing the estrogenic effects of Exe. This indicates that Exe modulates both ERα and AR, while Ana and Let act as pure AIs. Thus, this study highlights the potential clinical benefit of combining AR antagonists with Exe and discourages the sequential use of Exe as second-line therapy in postmenopausal breast cancer.

The anti-cancer potential of crotoxin in estrogen receptor-positive breast cancer: Its effects and mechanism of action.

Estrogen receptor-positive (ER+) breast cancer is the most diagnosed subtype of breast cancer. Currently, aromatase inhibitors (AIs) are used as first-line treatment option in this type of tumors, however they cause several side effects, which is why new therapeutic approaches are demanding. The South American rattlesnake Crotalus durissus terrificus produces a venom enriched in several bioactive substances, like phospholipases A2 (PLA2). One of those is crotoxin, a ß-neurotoxin, that has already been reported for its anti-cancer properties in different cancers. Recently, its clinical interest has emerged and, in fact, a clinical trial in patients with advanced cancer is underway. Considering this, in this work, we studied the biological mechanisms behind the anti-cancer effects of crotoxin B (CTX) in an ER+ aromatase-overexpressing breast cancer cell line (MCF-7aro cells). Results revealed that CTX impairs MCF-7aro cells growth, through a cell cycle arrest at G2/M phase, inhibition of ERK1/2 pathway and by apoptosis through activation of caspase-8. In addition, it can be considered a safe natural compound as did not affect non-cancerous cells and only showed anti-growth effects in breast cancer cells. Therefore, this study represents an important landmark to better understand the effects and mechanisms of action of crotoxin in ER+ breast cancer.

Cannabidiol disrupts apoptosis, autophagy and invasion processes of placental trophoblasts.

Cannabidiol (CBD) is a constituent of Cannabis sativa without psychotropic activity, whose medical benefits have been recognised. However, little is known about the potential toxic effects of CBD on reproductive health. Placental development involves tightly controlled processes of cell proliferation, differentiation, apoptosis, autophagy and migration/invasion of trophoblast cells. Cannabis use by pregnant women has been increasing, mainly for the relief of nausea associated with the first trimester, which raises great concern. Regarding the crucial role of cytotrophoblast cells (CTs) and extravillous trophoblasts (EVTs) in placentation, the effects of CBD (1-10 µM) were studied, using in vitro model systems BeWo and HTR-8/SVneo cell lines, respectively. CBD causes cell viability loss in a dose-dependent manner, disrupts cell cycle progression and induces apoptosis through the mitochondrial pathway, on both cell models. Moreover, CBD induces autophagy only in HTR-8/SVneo cells, being this process a promoter of apoptosis. Hypoxia-responsive genes HIF1A and SPP1 were also increased in CBD-treated HTR-8/SVneo cells suggesting a role for HIF-1α in the apoptotic and autophagic processes. In addition, CBD was able to decrease HTR-8/SVneo cell migration. Therefore, CBD interferes with trophoblast turnover and placental remodelling, which can have a considerable impact on pregnancy outcome. Thus, from an in vitro perspective our study adds new evidence for the potential negative impact of cannabis use by pregnant women.

Unveiling the mechanism of action behind the anti-cancer properties of cannabinoids in ER+ breast cancer cells: Impact on aromatase and steroid receptors.

Breast cancer is the leading cause of cancer-related death in women worldwide. In the last years, cannabinoids have gained attention in the clinical setting and clinical trials with cannabinoid-based preparations are underway. However, contradictory anti-tumour properties have also been reported. Thus, the elucidation of the molecular mechanisms behind their anti-tumour efficacy is crucial to better understand its therapeutic potential. Considering this, our work aims to clarify the molecular mechanisms underlying the anti-cancer properties of the endocannabinoid anandamide (AEA) and of the phytocannabinoids, cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC), in estrogen receptor-positive (ER+) breast cancer cells that overexpress aromatase (MCF-7aro). Their in vitro effects on cell proliferation, cell death and activity/expression of aromatase, ERα, ERß and AR were investigated. Our results demonstrated that cannabinoids disrupted MCF-7aro cell cycle progression. Unlike AEA and THC that induced apoptosis, CBD triggered autophagy to promote apoptotic cell death. Interestingly, all cannabinoids reduced aromatase and ERα expression levels in cells. On the other hand, AEA and CBD not only exhibited high anti-aromatase activity but also induced up-regulation of ERß. Therefore, all cannabinoids, albeit by different actions, target aromatase and ERs, impairing, in that way, the growth of ER+ breast cancer cells, which is dependent on estrogen signalling. As aromatase and ERs are key targets for ER+ breast cancer treatment, cannabinoids can be considered as potential and attractive therapeutic compounds for this type of cancer, being CBD the most promising one. Thus, from an in vitro perspective, this work may contribute to the growing mass of evidence of cannabinoids and cannabinoids-based medicines as potential anti-cancer drugs.

Cannabinoids in Breast Cancer: Differential Susceptibility According to Subtype.

Although cannabinoids have been used for centuries for diverse pathological conditions, recently, their clinical interest and application have emerged due to their diverse pharmacological properties. Indeed, it is well established that cannabinoids exert important actions on multiple sclerosis, epilepsy and pain relief. Regarding cancer, cannabinoids were first introduced to manage chemotherapy-related side effects, though several studies demonstrated that they could modulate the proliferation and death of different cancer cells, as well as angiogenesis, making them attractive agents for cancer treatment. In relation to breast cancer, it has been suggested that estrogen receptor-negative (ER-) cells are more sensitive to cannabinoids than estrogen receptor-positive (ER+) cells. In fact, most of the studies regarding their effects on breast tumors have been conducted on triple-negative breast cancer (TNBC). Nonetheless, the number of studies on human epidermal growth factor receptor 2-positive (HER2+) and ER+ breast tumors has been rising in recent years. However, besides the optimistic results obtained thus far, there is still a long way to go to fully understand the role of these molecules. This review intends to help clarify the clinical potential of cannabinoids for each breast cancer subtype.

Discovery of a multi-target compound for estrogen receptor-positive (ER+) breast cancer: Involvement of aromatase and ERs.

Despite intense research, breast cancer remains the leading cause of cancer-related death in women worldwide, being estrogen receptor-positive (ER+) the most common subtype. Nowadays, aromatase inhibitors (AIs), the selective estrogen receptor modulator (SERM) tamoxifen and the selective estrogen receptor down-regulator (SERD) fulvestrant are used as therapeutic options for ER+ breast cancer, since they interfere directly with the production of estrogens and with the activation of estrogen-dependent signaling pathways. Despite the success of these treatments, the occurrence of resistance limits their clinical efficacy, demanding the development of novel therapies. Recently, multi-target compounds emerged as promising therapeutic strategies for ER+ breast cancer, as they can potentially modulate several important targets simultaneously. In line with this, in this work, the anti-cancer properties and multi-target action of 1,1-Bis(4-hydroxyphenyl)-2-phenylbut-1-ene, tamoxifen bisphenol (1,1-BHPE), were evaluated in an ER+ breast cancer cell model (MCF-7aro cells). Molecular docking analysis predicted that 1,1-BHPE was able to bind to aromatase, ERα and ERß. In vitro studies showed that, although it did not present anti-aromatase activity, 1,1-BHPE reduced aromatase protein levels and interfered with ERα and ERß signaling pathways, acting as an ERα antagonist and inducing ERß up-regulation. Through these mechanisms, 1,1-BHPE was able to impair breast cancer growth and induce apoptosis. This represents an important therapeutic advantage because the main players responsible for estrogen production and signaling are modulated by a single compound. To the best of our knowledge, this is the first study describing the anti-cancer properties of 1,1-BHPE as a multi-target compound specific for ER+ breast cancer.

Cannabis sativa: Much more beyond Δ9-tetrahydrocannabinol.

Cannabis is the most used illicit drug worldwide and its medicinal use is under discussion, being regulated in several countries. However, the psychotropic effects of Δ9-tetrahydrocannabinol (THC), the main psychoactive compound of Cannabis sativa, are of concern. Thus, the interest in the isolated constituents without psychotropic activity, such as cannabidiol (CBD) and cannabidivarin (CBDV) is growing. CBD and CBDV are lipophilic molecules with poor oral bioavailability and are mainly metabolized by cytochrome P450 (CYP450) enzymes. The pharmacodynamics of CBD is the best explored, being able to interact with diverse molecular targets, like cannabinoid receptors, G protein-coupled receptor-55, transient receptor potential vanilloid 1 channel and peroxisome proliferator-activated receptor-γ. Considering the therapeutic potential, several clinical trials are underway to study the efficacy of CBD and CBDV in different pathologies, such as neurodegenerative diseases, epilepsy, autism spectrum disorders and pain conditions. The anti-cancer properties of CBD have also been demonstrated by several pre-clinical studies in different types of tumour cells. Although less studied, CBDV, a structural analogue of CBD, is receiving attention in the last years. CBDV exhibits anticonvulsant properties and, currently, clinical trials are underway for the treatment of autism spectrum disorders. Despite the benefits of these phytocannabinoids, it is important to highlight their potential interference with relevant physiologic mechanisms. In fact, CBD interactions with CYP450 enzymes and with drug efflux transporters may have serious consequences when co-administered with other drugs. This review summarizes the therapeutic advances of CBD and CBDV and explores some aspects of their pharmacokinetics, pharmacodynamics and possible interactions. Moreover, it also highlights the therapeutic potential of CBD and CBDV in several medical conditions and clinical applications.

Estrogen receptor-positive (ER+) breast cancer treatment: Are multi-target compounds the next promising approach?

Endocrine therapy is currently the main therapeutic approach for estrogen receptor-positive (ER+) breast cancer, the most frequent subtype of breast cancer in women worldwide. For this subtype of tumors, the current clinical treatment includes aromatase inhibitors (AIs) and anti-estrogenic compounds, such as Tamoxifen and Fulvestrant, being AIs the first-line treatment option for post-menopausal women. Moreover, the recent guidelines also suggest the use of these compounds by pre-menopausal women after suppressing ovaries function. However, besides its therapeutic efficacy, the prolonged use of this type of therapies may lead to the development of several adverse effects, as well as, endocrine resistance, limiting the effectiveness of such treatments. In order to surpass this issues and clinical concerns, during the last years, several studies have been suggesting alternative therapeutic approaches, considering the function of aromatase, ERα and ERß. Here, we review the structural and functional features of these three targets and their importance in ER+ breast cancer treatment, as well as, the current treatment strategies used in clinic, emphasizing the importance of the development of multi-target compounds able to simultaneously modulate these key targets, as a novel and promising therapeutic strategy for this type of cancer.

Cannabidiol (CBD) but not tetrahydrocannabinol (THC) dysregulate in vitro decidualization of human endometrial stromal cells by disruption of estrogen signaling.

Decidualization, which comprises proliferation and differentiation of endometrial stromal cells (ESCs), is essential for the establishment of a receptive endometrium and pregnancy to occur. A deregulation of decidualization has been associated with miscarriage, infertility and other pregnancy-related disorders. The role of estradiol (E2) on decidualization has already been shown, since it regulates proliferation of ESCs and expression of progesterone receptor. In this study, we investigated the effects of phytocannabinoids, tetrahydrocannabinol (THC) and cannabidiol (CBD), in proliferation and differentiation of ESCs, as well as, in E2 metabolism/signaling. We found that CBD, but not THC, inhibits ESCs differentiation. We also show that CBD prevents the increase on transcript levels of CYP19A1 gene and the elevation of E2 levels that are observed in differentiating ESCs. Moreover, we found that CBD presents anti-aromatase activity. In overall, we highlight a novel effect of CBD on human endometrial differentiation, which may lead to infertility problems.

The potential clinical benefit of targeting androgen receptor (AR) in estrogen-receptor positive breast cancer cells treated with Exemestane.

The development of acquired resistance to the aromatase inhibitors (AIs) used in clinic is being considered the major concern in estrogen-receptor positive (ER+) breast cancer therapy. Recently, androgen receptor (AR) has gained attention in the clinical setting, since it has been implicated in AIs-resistance, although, different roles for AR in cell fate have been described. In this work, our group elucidates, for the first time, the oncogenic role of AR in sensitive and resistant ER+ breast cancer cells treated with the potent third-generation steroidal AI Exemestane (Exe). We demonstrate that Exe promotes an overexpression/activation of AR, which has an oncogenic and pro-survival role in Exe-sensitive and Exe-resistant cells. Moreover, we also disclose that targeting AR with bicalutamide (CDX) in Exe-treated cells, enhances the efficacy of this AI in sensitive cells and re-sensitizes resistant cells to Exe treatment. Furthermore, by targeting AR in Exe-resistant cells, it is also possible to block the activation of the ERK1/2 and PI3K cell survival pathways, hamper ERα activation and increase ERß expression. Thus, this study, highlights a new mechanism involved in Exe-acquired resistance, implicating AR as a key molecule in this setting and suggesting that Exe-resistant cells may have an AR-dependent but ER-independent mechanism. Hence we propose AR antagonism as a potential and attractive therapeutic strategy to overcome Exe-acquired resistance or to enhance the growth inhibitory properties of Exe on ER+ breast cancer cells, improving breast cancer treatment.

Synthetic cannabinoids JWH-018, JWH-122, UR-144 and the phytocannabinoid THC activate apoptosis in placental cells.

The increasing use of synthetic cannabinoids (SCBs) in recreational settings is becoming a new paradigm of drug abuse. Although SCBs effects mimic those of the Cannabis sativa plant, these drugs are frequently more potent and hazardous. It is known that endocannabinoid signalling plays a crucial role in diverse reproductive events such as placental development. Moreover, the negative impact of the phytocannabinoid Δ9-tetrahydrocannabinol (THC) in pregnancy outcome, leading to prematurity, intrauterine growth restriction and low birth weight is well recognized, which makes women of childbearing age a sensitive group to developmental adverse effects of cannabinoids. Placental trophoblast turnover relies on regulated processes of proliferation and apoptosis for normal placental development. Here, we explored the impact of the SCBs JWH-018, JWH-122 and UR-144 and of the phytocannabinoid THC in BeWo cell line, a human placental cytotrophoblast cell model. All the cannabinoids caused a significant decrease in cell viability without LDH release, though this effect was only detected for the highest concentrations of THC. Moreover, a cell cycle arrest at the G2/M phase was also observed. JWH-018 and JWH-122 increased reactive oxygen species (ROS) production and THC, UR-144 and JWH-122 caused loss of mitochondrial membrane potential. All the compounds were able to induce caspase-9 activation. The involvement of apoptotic pathways was further confirmed through the significant increase in caspase -3/-7 activities. For UR-144, this effect was reversed by the CB1 antagonist AM281, for JWH-018 and THC this effect was mediated by both cannabinoid receptors CB1 and CB2 while for JWH-122 it was cannabinoid receptor-independent. This work demonstrates that THC and SCBs are able to induce apoptotic cell death. Although they may act through different mechanisms and potencies, the studied cannabinoids have the potential to disrupt gestational fundamental events.