RESUMO
This paper describes the development, implementation, and students' perceptions of a new trilayer approach of teaching (TLAT). The TLAT model involved blending lecture, in-class group activities, and out-of-class assignments on selected content areas and was implemented initially in a first-year integrated pharmacy course. Course contents were either delivered by traditional lectures or by the TLAT. A survey instrument was distributed by SurveyMonkey to determine students' perceptions of the TLAT model. Descriptive statistics were used for data analysis. Students' performance in a total of 225 examination and quiz questions was analyzed to evaluate whether the TLAT model improved students' learning. Students' (n = 98) performance scores for TLAT-based and lecture-based questions were 83.3 ± 10.2 and 79.5 ± 14.0, respectively (P < 0.05). Ninety-three percent of students believed that in-class group activities enhanced conceptual understanding of course materials, helped them take responsibility of their own learning, and enhanced their overall learning experiences. More than 80% of respondents felt that solving cases and developing concept maps helped them sharpen creative and critical thinking skills. In addition, 90% of the respondents indicated that the homework throughout the semester helped them stay up to date and focused with the progress of the course. The use of the TLAT model led to an improvement in student learning of complex concepts. Moreover, the results suggest that this model improves students' self-reliance and attitudes toward learning. Our findings should serve as an impetus for inclusion of diverse active learning strategies in pharmacy education.
Assuntos
Educação em Farmácia/métodos , Educação em Farmácia/normas , Farmacologia/educação , Fisiologia/educação , Ensino/normas , Currículo/normas , Currículo/tendências , Avaliação Educacional , Humanos , Aprendizagem , Ensino/tendênciasRESUMO
Resistance to aromatase inhibitors (AIs) involves increased HER2. One mechanism by which HER2 may mediate resistance is through expansion of the tumor initiating cell (TIC) population. This study investigates whether combining all-trans retinoic acid (ATRA) and histone deacetylase inhibitor entinostat (ENT) can inhibit TICs and HER2 in AI-resistant cells and tumors. Modulation of cell viability and HER2 expression were assessed in AI-resistant cells treated with ATRA + ENT. Letrozole-resistant LTLT-Ca cells treated with ATRA + ENT were assayed for changes in TIC characteristics, such as TIC markers (BCRP, ALDH, and BMI-1), side population (SP), and mammosphere formation. Xenograft tumors of MCF-7Ca cells made resistant to letrozole were treated with ATRA, ATRA + letrozole, ATRA + ENT, or ATRA + ENT + letrozole. Resulting tumors were assayed for changes in TIC characteristics. Patient samples taken pre- and post-AI treatment were analyzed for changes in ERα and HER2 protein expression. Treatment with ATRA + ENT reduced HER2 expression and viability (P < 0.001) in AI-resistant cells, as well as decreased SP (P < 0.0001), mammosphere formation (P < 0.01), and expression of TIC molecular markers (P < 0.01) in LTLT-Ca. A reduction in tumor growth rate was observed in mice treated with ENT + ATRA + letrozole when compared to mice treated with single agents (P < 0.0001) or ENT + ATRA (P = 0.02). Decreased TIC characteristics, including mammosphere formation (P < 0.05), were observed in tumors from the triple combination. An increase in HER2 and downregulation in ERα protein expression was observed in patients upon resistance to AI (P < 0.005). These studies indicate that the combination of ATRA and ENT inhibits the TIC population of AI-resistant cells and may be effective in reducing tumor recurrence.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Receptor ErbB-2/metabolismo , Animais , Inibidores da Aromatase/farmacologia , Benzamidas/administração & dosagem , Benzamidas/farmacologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Inibidores de Histona Desacetilases/administração & dosagem , Humanos , Letrozol , Camundongos Nus , Nitrilas/farmacologia , Piridinas/administração & dosagem , Piridinas/farmacologia , Tretinoína/administração & dosagem , Triazóis/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
INTRODUCTION: Although aromatase inhibitors (AIs; for example, letrozole) are highly effective in treating estrogen receptor positive (ER+) breast cancer, a significant percentage of patients either do not respond to AIs or become resistant to them. Previous studies suggest that acquired resistance to AIs involves a switch from dependence on ER signaling to dependence on growth factor-mediated pathways, such as human epidermal growth factor receptor-2 (HER2). However, the role of HER2, and the identity of other relevant factors that may be used as biomarkers or therapeutic targets remain unknown. This study investigated the potential role of transcription factor hypoxia inducible factor 1 (HIF-1) in acquired AI resistance, and its regulation by HER2. METHODS: In vitro studies using AI (letrozole or exemestane)-resistant and AI-sensitive cells were conducted to investigate the regulation and role of HIF-1 in AI resistance. Western blot and RT-PCR analyses were conducted to compare protein and mRNA expression, respectively, of ERα, HER2, and HIF-1α (inducible HIF-1 subunit) in AI-resistant versus AI-sensitive cells. Similar expression analyses were also done, along with chromatin immunoprecipitation (ChIP), to identify previously known HIF-1 target genes, such as breast cancer resistance protein (BCRP), that may also play a role in AI resistance. Letrozole-resistant cells were treated with inhibitors to HER2, kinase pathways, and ERα to elucidate the regulation of HIF-1 and BCRP. Lastly, cells were treated with inhibitors or inducers of HIF-1α to determine its importance. RESULTS: Basal HIF-1α protein and BCRP mRNA and protein are higher in AI-resistant and HER2-transfected cells than in AI-sensitive, HER2- parental cells under nonhypoxic conditions. HIF-1α expression in AI-resistant cells is likely regulated by HER2 activated-phosphatidylinositide-3-kinase/Akt-protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway, as its expression was inhibited by HER2 inhibitors and kinase pathway inhibitors. Inhibition or upregulation of HIF-1α affects breast cancer cell expression of BCRP; AI responsiveness; and expression of cancer stem cell characteristics, partially through BCRP. CONCLUSIONS: One of the mechanisms of AI resistance may be through regulation of nonhypoxic HIF-1 target genes, such as BCRP, implicated in chemoresistance. Thus, HIF-1 should be explored further for its potential as a biomarker of and therapeutic target.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Inibidores da Aromatase/farmacologia , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Proteínas de Neoplasias/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Hipóxia Celular , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Letrozol , Células MCF-7 , Nitrilas/farmacologia , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/biossíntese , Receptor ErbB-2/antagonistas & inibidores , Receptores de Estrogênio/antagonistas & inibidores , Receptores de Estrogênio/metabolismo , Esferoides Celulares , Serina-Treonina Quinases TOR/metabolismo , Triazóis/farmacologia , Células Tumorais CultivadasRESUMO
Loss of ERα in breast cancer correlates with poor prognosis, increased recurrence rates, and higher incidence of metastasis. Epigenetic silencing of E-cadherin (loss of which is associated with more invasive phenotype) is observed in metastatic cell lines and invasive breast cancers. Here, we are showing that entinostat (ENT) can reverse epithelial to mesenchymal transition (EMT), which is considered to be a first step in the process of metastases formation. Triple-negative breast cancer cells such as MDA-MB-231 and Hs578T show a basal phenotype characterized by loss of E-cadherin expression and higher expression of mesenchymal markers such as N-cadherin and vimentin along with transcriptional repressors such as twist and snail. When MDA-MB-231 and Hs578T cells or tumors were treated with ENT, E-cadherin transcription was increased along with reduction in N-cadherin mRNA expression. Chromatin immunoprecipitation assay showed that treatment of MDA-MB-231 and Hs578T cells increased the activation of E-cadherin promoter by reducing the association of twist and snail with the E-cadherin (CDH1) promoter and downregulated both the snail and twist. ENT also inhibited cell migration in vitro. In addition, phosphorylation of vimentin was increased, as well as remodeling of vimentin filaments. ENT treatment also reduced formation of tubulin-based microtentacles, which help floating cells attach to other surfaces. These findings suggest that ENT can reverse EMT and may reduce the formation of metastasis.
Assuntos
Benzamidas/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Caderinas/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Inibidores de Histona Desacetilases/farmacologia , Piridinas/farmacologia , Neoplasias da Mama/metabolismo , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismo , Proteína 1 Relacionada a Twist/metabolismoRESUMO
Most breast cancer (BC) patients have tumors that express hormone receptors (HRs). Although endocrine therapy, such as aromatase inhibitors, is very effective, most patients with metastatic HR-positive (HR(+)) BC become resistant to endocrine therapy at some point in their treatment and subsequently require chemotherapy. The PI3K/mTOR pathway is often upregulated in endocrine-resistant BC patients and, therefore, has been one of the targets for development of new agents. Recently, a Phase III trial (BOLERO-2) in aromatase inhibitor-resistant BC patients showed a significant improvement in time to progression with the combination of everolimus and exemestane compared with exemestane alone, confirming the importance of the PI3K/mTOR pathway in endocrine-resistant BC. Side effects from mTOR inhibitors are manageable, but early detection and proactive management are required to ensure patients' safety, compliance and continuity of treatment. Thus, mTOR inhibitors offer a new hope and promise for patients with HR(+) BC.
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Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Antineoplásicos Hormonais/farmacologia , Sinergismo Farmacológico , Everolimo , Feminino , Humanos , Indóis/farmacologia , Indóis/uso terapêutico , Purinas/farmacologia , Purinas/uso terapêutico , Sirolimo/análogos & derivados , Sirolimo/farmacologia , Sirolimo/uso terapêutico , Serina-Treonina Quinases TOR/antagonistas & inibidoresRESUMO
INTRODUCTION: Podcasts are a popular way to learn and engage at the convenience of the listener. Education is incorporating podcasts to supplement and reinforce students learning inside and outside the classroom. METHODS: The authors created a podcast covering the Top 200 commonly prescribed medications. This was to help students recall and reinforce medication knowledge they typically must learn on their own. RESULTS: Student performance on post-tests improved (p = 0.0011) compared to pre-tests with an effect size r of 0.39 (0.37, 0.32, and 0.42 for P1, P2 and P3 respectively). Students reported the content was easy to follow, and they enjoyed learning from other students. The total number of plays for the podcast as of 19 July 2023 were 882. Each episode had a range of one to 89 number of plays. DISCUSSION: The podcast was well received by students, and drug knowledge increased. While the podcasts were shorter in time, they still provided the foundational information for a first-year pharmacy student to know. Overall, podcasts provide another way to help students retain and reinforce material learned inside and outside the classroom.
Assuntos
Educação em Farmácia , Avaliação Educacional , Estudantes de Farmácia , Webcasts como Assunto , Humanos , Webcasts como Assunto/estatística & dados numéricos , Educação em Farmácia/métodos , Educação em Farmácia/normas , Educação em Farmácia/estatística & dados numéricos , Estudantes de Farmácia/estatística & dados numéricos , Estudantes de Farmácia/psicologia , Avaliação Educacional/métodos , Avaliação Educacional/estatística & dados numéricos , Aprendizagem , Currículo/tendências , Currículo/normasRESUMO
Despite significant improvement in the treatment outcome of hormone responsive postmenopausal breast cancer, some patients eventually acquire resistance to aromatase inhibitors (AIs). Using our MCF-7Ca xenograft model, we observed that although AIs such as anastrozole initially inhibit tumor growth effectively, tumors eventually began to grow. Our previous data show that anastrozole-resistant tumors upregulate growth factor receptor pathways as they adapt to grow in the low estrogen environment. Therefore, in the current study, we investigated the effect of inhibiting the growth factor receptor pathways with a MEK-1/2 inhibitor selumetinib (AZD6244, ARRY-142866). We treated the mice with anastrozole-resistant tumors with selumetinib alone or in combination with anastrozole. MCF-7Ca cells were inoculated sc into ovariectomized athymic nude mice supplemented throughout the experiment with androstenedione (100 µg/day), the substrate for aromatase conversion to estrogen. Once the tumors reached a measurable size (~300 mm(3)), the mice were treated with anastrozole (200 µg/day), supplemented with androstenedione (Δ(4)A). The tumors in the anastrozole group doubled in volume after 6 weeks, at which time the animals were regrouped to receive the following treatments: (i) anastrozole, (ii) anastrozole withdrawal (Δ(4)A alone), (iii) selumetinib (25 mg/kg/d, bid, po), and (iv) selumetinib + anastrozole, (n = 10 mice/group). The treatments were given for 6 weeks (till week 12) and then the mice were euthanized, the tumors were collected and analyzed. The tumors of mice treated with selumetinib + anastrozole had significantly lower growth rates than those treated with single agents (p = 0.008). Western blot analysis of the tumors showed that treatment with anastrozole resulted in upregulation of proteins in the growth factor receptor cascade such as p-mTOR, pAkt, pMEK, and pMAPK. This was accompanied by downregulation of ERα protein, consistent with previous findings. The treatment of mice with selumetinib resulted in downregulation of activated MAPK, along with p-mTOR, which likely resulted in upregulation of ERα. Our results suggest that inhibition of the growth factor receptor pathway with selumetinib can reverse anastrozole resistance.
Assuntos
Benzimidazóis/farmacologia , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Nitrilas/farmacologia , Triazóis/farmacologia , Anastrozol , Androstenodiona/farmacologia , Animais , Antineoplásicos Hormonais/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Inibidores da Aromatase/farmacologia , Benzimidazóis/administração & dosagem , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , Camundongos , Camundongos Nus , Nitrilas/administração & dosagem , Ovariectomia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Triazóis/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Aromatase inhibitors (AIs) are an effective therapy in treating estrogen receptor-positive breast cancer. Nonetheless, a significant percentage of patients either do not respond or become resistant to AIs. Decreased dependence on ER-signaling and increased dependence on growth factor receptor signaling pathways, particularly human epidermal growth factor receptor 2 (EGFR2/HER2), have been implicated in AI resistance. However, the role of growth factor signaling remains unclear. This current study investigates the possibility that signaling either through HER2 alone or through interplay between epidermal growth factor receptor 1 (EGFR/HER1) and HER2 mediates AI resistance by increasing the tumor initiating cell (TIC) subpopulation in AI-resistant cells via regulation of stem cell markers, such as breast cancer resistance protein (BCRP). TICs and BCRP are both known to be involved in drug resistance. Results from in vitro analyses of AI-resistant versus AI-sensitive cells and HER2-versus HER2+ cells, as well as from in vivo xenograft tumors, indicate that (1) AI-resistant cells overexpress both HER2 and BCRP and exhibit increased TIC characteristics compared to AI-sensitive cells; (2) inhibition of HER2 and/or BCRP decrease TIC characteristics in letrozole-resistant cells; and (3) HER2 and its dimerization partner EGFR/HER1 are involved in the regulation of BCRP. Overall, these results suggest that reducing or eliminating the TIC subpopulation with agents that target BCRP, HER2, EGFR/HER1, and/or their downstream kinase pathways could be effective in preventing and/or treating acquired AI resistance.
Assuntos
Inibidores da Aromatase/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Células-Tronco Neoplásicas/metabolismo , Nitrilas/farmacologia , Receptor ErbB-2/metabolismo , Triazóis/farmacologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Inibidores da Aromatase/uso terapêutico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/metabolismo , Feminino , Humanos , Letrozol , Células MCF-7 , Camundongos , Camundongos Nus , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Aromatase inhibitors (AI) that inhibit breast cancer cell growth by blocking estrogen synthesis have become the treatment of choice for post-menopausal women with estrogen receptor positive (ER+) breast cancer. However, some patients display de novo or acquired resistance to AI. Interactions between estrogen and growth factor signaling pathways have been identified in estrogen-responsive cells as one possible reason for acquisition of resistance. Our laboratory has characterized an autocrine growth factor overexpressed in invasive ductal carcinoma named PC-Cell Derived Growth Factor (GP88), also known as progranulin. In the present study, we investigated the role GP88 on the acquisition of resistance to letrozole in ER+ breast cancer cells METHODS: We used two aromatase overexpressing human breast cancer cell lines MCF-7-CA cells and AC1 cells and their letrozole resistant counterparts as study models. Effect of stimulating or inhibiting GP88 expression on proliferation, anchorage-independent growth, survival and letrozole responsiveness was examined. RESULTS: GP88 induced cell proliferation and conferred letrozole resistance in a time- and dose-dependent fashion. Conversely, naturally letrozole resistant breast cancer cells displayed a 10-fold increase in GP88 expression when compared to letrozole sensitive cells. GP88 overexpression, or exogenous addition blocked the inhibitory effect of letrozole on proliferation, and stimulated survival and soft agar colony formation. In letrozole resistant cells, silencing GP88 by siRNA inhibited cell proliferation and restored their sensitivity to letrozole. CONCLUSION: Our findings provide information on the role of an alternate growth and survival factor on the acquisition of aromatase inhibitor resistance in ER+ breast cancer.
Assuntos
Inibidores da Aromatase/farmacologia , Aromatase/metabolismo , Neoplasias da Mama/enzimologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Nitrilas/farmacologia , Triazóis/farmacologia , Aromatase/genética , Neoplasias da Mama/genética , Neoplasias da Mama/fisiopatologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Feminino , Inativação Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Letrozol , Progranulinas , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , RNA Interferente Pequeno/metabolismoRESUMO
INTRODUCTION: In fall 2017, West Coast University School of Pharmacy implemented ExamSoft for testing. Three courses in each didactic year employed ExamSoft. Prior to this, courses had Scantron-based exams. We surveyed the students to assess their perception of ExamSoft. We hypothesized that students' inherent bias towards technology affected their perception of ExamSoft. METHODS: To assess this hypothesis, we conducted a survey of all students. The survey contained questions about comfort with technology and nine questions on students' perceptions of ExamSoft and its usefulness. RESULTS: The survey responses were stratified according to the preference of respondents towards technology and its use in exams. Respondents were stratified into three groups: tech-embracers, tech-skeptics, and neutral. Our results showed that respondents classified as tech-skeptics tended to have a more negative view of ExamSoft and its perceived impact on their grades than students stratified as tech-embracers or neutral. CONCLUSIONS: Our study suggests that students' inherent bias towards technology plays an important role in their perception of computer-based testing. Assessing incoming students' comfort with technology and student orientation activities to help acquaint students with new technology could help improve their acceptance of educational technology used for testing.
Assuntos
Computadores , Estudantes , Tecnologia Educacional , Humanos , Percepção , Inquéritos e QuestionáriosRESUMO
Treatment of hormone sensitive breast cancer tumors with endocrine therapy such as antiestrogens or aromatase inhibitors has improved the outcome significantly. Studies including our own have shown that downregulation of ERα with pure antiestrogen fulvestrant in combination with aromatase inhibitors may prolong responsiveness of the tumors to endocrine therapy. Fulvestrant has been studied as second line or first line treatment for post-menopausal hormone receptor positive breast cancers as a single agent or in combination with AIs. Studies have also suggested that further escalation of dose may improve benefit. However, dose escalation of fulvestrant, which is administered via intramuscular injection, is difficult due to its poor solubility. To overcome this shortcoming of an injectable drug, a novel orally active antiestrogen, AZD9496 was developed. In addition to being orally active, AZD9496 is designed as a selective ERα downregulator (SERD). In the current study, we compared the effect of AZD9496 and fulvestrant on the growth of MCF-7Ca (human estrogen receptor positive MCF-7 cells stably transfected with human placental aromatase gene) xenografts grown in ovariectomized athymic nude mice. AZD9496 was also compared to fulvestrant in vitro as a single agent or in combination with anastrozole. Our current study shows that AZD9496 is equally effective as fulvestrant at controlling the growth of hormone sensitive human breast cancer tumors. Similar to fulvestrant, AZD9496 inhibits cellular aromatase activity through ERα mediated signaling. However, unlike fulvestrant, combination of AZD9496 with anastrozole did not produce increased tumor inhibition. Our results show that AZD9496 was significantly better at inhibiting cellular aromatase which contributed to its anticancer activity. Next, we measured the effect of AZD9496 on the mouse uterus. Uterine weight of mice treated with AZD9496 was significantly lower than that for mice treated with androstenedione. This reduction in uterine weight was due to AZD9496 mediated inhibition of aromatase activity and not a direct effect on uterine ERα expression. We also observed that anti-cancer efficacy of AZD9496 depended on its ability to inhibit cellular aromatase. These results suggest that AZD9496 may be a better alternative to fulvestrant due to its selectivity for mammary ER and ability to inhibit aromatase in addition of downregulating ERα that can be obtained upon oral administration. As such, AZD9496 may prove to be a better option than fulvestrant for the treatment of hormone sensitive human breast cancer.
Assuntos
Inibidores da Aromatase/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Cinamatos/uso terapêutico , Indóis/uso terapêutico , Neoplasias Mamárias Experimentais/tratamento farmacológico , Moduladores Seletivos de Receptor Estrogênico/uso terapêutico , Anastrozol/farmacologia , Anastrozol/uso terapêutico , Animais , Antineoplásicos Hormonais/farmacologia , Antineoplásicos Hormonais/uso terapêutico , Aromatase/metabolismo , Inibidores da Aromatase/farmacologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Cinamatos/farmacologia , Antagonistas do Receptor de Estrogênio/farmacologia , Antagonistas do Receptor de Estrogênio/uso terapêutico , Receptor alfa de Estrogênio , Feminino , Fulvestranto/farmacologia , Fulvestranto/uso terapêutico , Humanos , Indóis/farmacologia , Neoplasias Mamárias Experimentais/metabolismo , Camundongos Nus , Pós-Menopausa , Moduladores Seletivos de Receptor Estrogênico/farmacologiaRESUMO
OBJECTIVES: To improve treatment for patients with breast and prostate cancer. METHODS: A number of novel inhibitors of steroidogenic enzymes have been developed. Their biological effects have been evaluated in a variety of preclinical models. Aromatase (estrogen synthetase) inhibitors have now been extensively tested in clinical trials in breast cancer patients. Inhibitors of 17alpha-hydroxylase/lyase have also been studied in preclinical models and are beginning trials in prostate cancer patients. RESULTS: The enzyme aromatase (CYP19) has proven to be an important therapeutic target. Inhibitors of aromatase (AIs) are showing greater benefit than antiestrogens in the treatment of breast cancer. Although effective in other conditions in both women and men, AIs have not been useful in benign prostatic hypertrophy or prostate cancer. However inhibitors of 17alphahydroxylase/lyase (CYP17) to block synthesis of androgens may be effective for prostate cancer. Recent clinical trials with abiraterone and preclinical studies with other novel CYP17 inhibitors, which also interact with the androgen receptor and cause its down-regulation, could provide a new approach for treating this disease. In further studies, we optimized treatment with aromatase inhibitors and antiestrogens utilizing an intratumoral aromatase xenograft model. AIs were more effective and sustained growth inhibition was longer than antiestrogens. However, inevitably tumors eventually began to grow despite continued treatment. Analysis of breast tumors from mice treated with letrozole revealed up-regulation of HER-2 and MAP Kinase signaling proteins and down-regulation of the estrogen receptor. Our studies showed that tumors adapt to AI treatment by activating alternate signaling pathways, thus enabling them to proliferate in the absence of estrogen. When mice bearing resistant tumors were treated with trastuzumab, the anti-HER-2 antibody (herceptin), HER-2 was decreased in the tumor but the estrogen receptor and aromatase were restored. Tumor growth was significantly inhibited by treatment with trastuzumab in addition to letrozole. CONCLUSIONS: Aromatase inhibitors are proving to be an effective new class of agents for the treatment of breast cancer. Compounds inhibiting 17alphahydroxylase/lyase have potential for the treatment of prostate cancer. Our results suggest that strategies to overcome resistance to these types of agents can restore sensitivity of the tumors to hormone therapy.
Assuntos
Inibidores Enzimáticos/farmacologia , Esteroides/metabolismo , Anastrozol , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Aromatase/metabolismo , Inibidores da Aromatase/farmacologia , Neoplasias da Mama/terapia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Nitrilas/farmacologia , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , Trastuzumab , Triazóis/farmacologiaRESUMO
This study was carried out to determine the mechanisms associated with loss of androgen dependency and disease progression in prostate cancer. We investigated the role of the androgen receptor and its relationship to other signal transduction proteins. A hormone-refractory prostate cancer cell line [high-passage LNCaP (HP-LNCaP)] was established in vitro. Cells were treated with inhibitors of mammalian target of rapamycin and tyrosine kinase receptors. Expression of these proteins and the androgen receptor were measured by Western immunoblotting. Analysis of the model and various treatments was also assessed through proliferation assays, luciferase activation assays, binding assays, and ELISA. Our novel antiandrogen, VN/124-1, effectively inhibited proliferation of hormone-resistant prostate cancer cell lines (HP-LNCaP), which were no longer sensitive to bicalutamide and had increased expression of the androgen receptor. Treatment with everolimus or gefitinib resulted in an increase in protein expression and activation of the androgen receptor. Conversely, inhibition of the androgen receptor resulted in increased expression of IGFR1beta, pHER2, pmTOR, and pAkt. The addition of bicalutamide to everolimus or gefitinib inhibited cell proliferation in HP-LNCaP cells. However, the addition of VN/124-1 has proven to be superior to bicalutamide, and the combination was synergistic (P<0.05) compared with either agent alone. This study suggests that compensatory cross-talk between the androgen receptor and various signaling pathways may account for decreased sensitivity to androgen receptor antagonists and the progression to hormone-resistant prostate cancer. Furthermore, these findings suggest that inhibition of both pathways may provide effective control in hormone-resistant prostate cancer and restore sensitivity to androgen antagonists in hormone-refractory patients.
Assuntos
Antagonistas de Androgênios/farmacologia , Androstadienos/farmacologia , Benzimidazóis/farmacologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Transdução de Sinais/efeitos dos fármacos , Androgênios/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Masculino , Modelos Biológicos , Antígeno Prostático Específico/metabolismo , Ligação ProteicaRESUMO
Since most breast cancers occur in postmenopausal women and are hormone dependent, we developed a model system that mimics this situation. In this model, tumors of human estrogen receptor (ER) positive breast cancer cells stably transfected with aromatase (Ac-1) are grown in immune-compromised mice. Using this model we have explored a number of therapeutic strategies to maximize the antitumor efficacy of antiestrogens (AEs) and aromatase inhibitors (AIs). This intratumoral aromatase xenograft model has proved accurate in predicting the outcome of several clinical trials. In this current study we compared the effect of an AE toremifene and steroidal AI atamestane, alone or in combination, on growth of hormone-dependent human breast cancer. We have also compared toremifene plus atamestane combination with tamoxifen in this study. The growth of Ac-1 cells was inhibited by tamoxifen, toremifene and atamestane in vitro with IC(50) values of 1.8+/-1.3 microM, 1+/-0.3 microM and 60.4+/-17.2 microM, respectively. The combination of toremifene plus atamestane was found to be better than toremifene or atamestane alone in vitro. The effect of this combination was then studied in vivo using Ac-1 xenografts grown in ovariectomized female SCID mice. The mice were injected with toremifene (1000 microg/day), atamestane (1000 microg/day), tamoxifen (100 microg/day), or the combination of toremifene plus atamestane. In this study, our results indicate that the combination of toremifene plus atamestane was as effective as toremifene or tamoxifen alone but may not provide any additional benefit over toremifene alone or tamoxifen alone.
Assuntos
Androstenodiona/análogos & derivados , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Aromatase/genética , Neoplasias da Mama/tratamento farmacológico , Modelos Animais de Doenças , Toremifeno/administração & dosagem , Androstenodiona/administração & dosagem , Animais , Antineoplásicos Hormonais/administração & dosagem , Aromatase/metabolismo , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Humanos , Camundongos , Camundongos SCID , Prognóstico , Transfecção , Resultado do Tratamento , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Aromatase inhibitors that block the synthesis of estrogen are proving to be superior to antiestrogens and may replace tamoxifen as first-line treatment for postmenopausal estrogen receptor (ER)-positive breast cancer patients. However, acquisition of resistance to all forms of treatments is inevitable and a major clinical concern. In this study, we have investigated the effects of long-term estrogen deprivation in the breast cancer xenograft model and whether sensitivity to antiestrogens can be restored in vivo. We also compared whether combining wortmannin with tamoxifen or fulvestrant inhibited tumor growth better than either drug alone. EXPERIMENTAL DESIGN: Long-term estrogen-deprived aromatase-transfected human ER-positive breast cancer cells (UMB-1Ca) were grown as tumors in ovariectomized athymic nude mice. Twelve weeks after inoculation, when tumors reached 300 mm(3), animals were grouped and injected with vehicle, Delta(4)A, letrozole, tamoxifen, fulvestrant, wortmannin, tamoxifen plus wortmannin, and wortmannin plus fulvestrant. Tumor volumes were measured weekly. RESULTS: Tumors of UMB-1Ca cells grew equally well with and without androstenedione, indicating the ability of the cells to proliferate in the absence of estrogen. The combination of wortmannin with tamoxifen or fulvestrant inhibited tumor growth better than either drug alone. The combination of wortmannin plus fulvestrant was the most effective treatment that maintained tumor regression for a prolonged time. CONCLUSION: These results suggest that blocking both ER and growth factor receptor pathways could provide effective control over tumor growth of long-term estrogen-deprived human breast cancers.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Moduladores de Receptor Estrogênico/uso terapêutico , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Androstadienos/uso terapêutico , Androstenodiona/uso terapêutico , Animais , Inibidores da Aromatase/uso terapêutico , Neoplasias da Mama/enzimologia , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/metabolismo , Humanos , Letrozol , Camundongos , Camundongos Nus , Nitrilas/uso terapêutico , Tamoxifeno/uso terapêutico , Triazóis/uso terapêutico , WortmaninaRESUMO
Previous work has shown that androgens inhibit breast cancer cells and tumor growth. On the other hand, androgens can be converted to mitogenic estrogens by aromatase in breast cancer cells. Here, we report that androgens, such as the aromatizable androstenedione and the non-aromatizable 5alpha-dihydrotestosterone, inhibit MCF-7 cell proliferation. This effect is observed only in the absence or at a low concentration of estrogens and is evident in cells with low aromatase activity. Growth of a new aromatase stably transfected MCF-7 cell line (Ac1) was stimulated by conversion of androstenedione into estrogens and was sensitive to aromatase inhibitors. We show that blockade of the androgen receptor (AR) in these cells by the antiandrogen casodex or by the anti-AR small interfering RNA inhibited the antiproliferative effect of dihydrotestosterone and letrozole (aromatase inhibitor). We also show that suppression of the estrogen-induced antiapoptotic protein Bcl-2 may be involved in the antiproliferative effects of androgens and letrozole. These effects can be reversed by casodex. In conclusion, the results suggest that aromatase inhibitors may exert their antiproliferative effect not only by reducing the intracellular production of estrogens but also by unmasking the inhibitory effect of androgens acting via the AR.
Assuntos
Androgênios/fisiologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Neoplasias Hormônio-Dependentes/patologia , Nitrilas/farmacologia , Triazóis/farmacologia , Antagonistas de Receptores de Andrógenos , Androgênios/farmacologia , Androstenodiona/farmacologia , Aromatase/genética , Aromatase/metabolismo , Neoplasias da Mama/genética , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Di-Hidrotestosterona/antagonistas & inibidores , Di-Hidrotestosterona/farmacologia , Estradiol/farmacologia , Receptor alfa de Estrogênio/biossíntese , Humanos , Letrozol , Neoplasias Hormônio-Dependentes/genética , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , RNA Interferente Pequeno/genética , Receptores Androgênicos/biossíntese , TransfecçãoRESUMO
To study the long-term effects of estrogen deprivation on breast cancer, MCF-7Ca human estrogen receptor-positive breast cancer cells stably transfected with human aromatase gene were cultured in the steroid-depleted medium for 6 to 8 months until they had acquired the ability to grow. Proliferation of these cells (UMB-1Ca) was accompanied by increased expression of human epidermal growth factor receptor 2, increased activation of AKT through phosphorylation at Ser473 and Thr308, and increased invasion compared with parental MCF-7Ca cells. Estrogen receptor expression was also increased 5-fold. Although growth was inhibited by the antiestrogen fulvestrant, the IC50 was 100-fold higher than for parental MCF-7Ca cells. Aromatase inhibitor letrozole also inhibited growth at 10,000-fold higher concentration than required for MCF-7Ca cells, whereas anastrozole, exemestane, formestane, and tamoxifen were ineffective at 100 nmol/L. Growth of UMB-1Ca cells was inhibited by phosphatidylinositol 3-kinase inhibitor wortmannin (IC50 approximately 25 nmol/L) and epidermal growth factor receptor kinase inhibitor gefitinib (ZD 1839; IC50 approximately 10 micromol/L) whereas parental MCF-7Ca cells were insensitive to these agents. Concomitant treatment of UMB-1Ca cells with the signal transduction inhibitors and anastrozole and tamoxifen restored their growth inhibitory effects. These studies show that estrogen deprivation results in up-regulation of growth factor signaling pathways, which leads to a more aggressive and hormone refractory phenotype. Cross-talk between ER and growth factor signaling was evident as inhibition of these pathways could restore estrogen responsiveness to these cells.
Assuntos
Neoplasias da Mama/patologia , Estrogênios/deficiência , Neoplasias Hormônio-Dependentes/patologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores de Estrogênio/fisiologia , Receptores de Fatores de Crescimento/fisiologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Ativação Enzimática , Estrogênios/fisiologia , Humanos , Invasividade Neoplásica , Neoplasias Hormônio-Dependentes/enzimologia , Neoplasias Hormônio-Dependentes/metabolismo , Proteínas Proto-Oncogênicas c-akt , Receptor Cross-Talk/fisiologia , Transdução de Sinais/fisiologia , Regulação para CimaRESUMO
Ovariectomized mice bearing tumor xenografts grown from aromatase-transfected estrogen receptor (ER)-positive human breast cancer cells (MCF-7Ca) were injected s.c. with 10 microg/d letrozole for up to 56 weeks. Western blot analysis of the tumors revealed that ERs (ERalpha) were increased at 4 weeks but decreased at weeks 28 and 56. Expression of erbB-2 and p-Shc increased throughout treatment, whereas growth factor receptor binding protein 2 (Grb2) increased only in tumors proliferating on letrozole (weeks 28 and 56). In cells isolated from tumors after 56 weeks and maintained as a cell line (LTLT-Ca) in 1 micromol/L letrozole, ERalpha was also decreased whereas erbB-2, adapter proteins (p-Shc and Grb2), and the signaling proteins in the mitogen-activated protein kinase (MAPK) cascade were increased compared with MCF-7Ca cells. Growth was inhibited in LTLT-Ca cells but not in MCF-7Ca cells treated with MAPK kinase 1/2 inhibitors U0126, and PD98059 (IC(50) approximately 25 micromol/L). PD98059 (5 micromol/L) also reduced MAPK activity and increased ERalpha to the levels in MCF-7Ca cells. Epidermal growth factor receptor kinase inhibitor, gefitinib (ZD1839) inhibited growth of LTLT-Ca cells (IC(50) approximately 10 micromol/L) and restored their sensitivity to tamoxifen and anastrozole. In xenografts, combined treatment with ER down-regulator fulvestrant and letrozole, prevented increases in erbB-2 and activation of MAPK and was highly effective in inhibiting tumor growth throughout 29 weeks of treatment. These results indicate that blocking both ER- and growth factor-mediated transcription resulted in the most effective inhibition of growth of ER-positive breast cancer cells.
Assuntos
Inibidores da Aromatase/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Nitrilas/farmacologia , Triazóis/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Inibidores da Aromatase/administração & dosagem , Linhagem Celular Tumoral , Esquema de Medicação , Ativação Enzimática/efeitos dos fármacos , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Letrozol , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Nitrilas/administração & dosagem , Tamoxifeno/administração & dosagem , Triazóis/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In previous studies using a xenograft model with tumors of human estrogen receptor (ER)-positive breast cancer cells transfected with aromatase (MCF-7Ca), we explored the antitumor efficacy of treatment combining the nonsteroidal aromatase inhibitor letrozole with tamoxifen. However, treatment with this combination resulted in tumor suppression similar to tamoxifen alone but was less effective than letrozole alone. Clinical findings with the nonsteroidal inhibitor anastrozole in combination with tamoxifen (ATAC trial) were consistent with our results. Although letrozole was the most effective single agent in the model, tumors ultimately began to grow during continued treatment. To investigate the mechanisms by which tumors adapted to growth during letrozole treatment, we determined the expression of proteins in tumors during letrozole treatment compared with the tumors of control mice. We found that tumors initially up-regulated the ER, but subsequently receptor levels decreased in tumors unresponsive to letrozole. Adapter proteins (p-Shc and Grb-2) as well as all of the signaling proteins in the mitogen-activated protein kinase cascade (p-Raf, p-MEK1/2, and p-MAPK) but not Akt were increased in tumors no longer responsive to letrozole. The results suggest that tumor cells adapt to estrogen deprivation during letrozole treatment by activation of alternate signaling pathways. When letrozole was combined with the pure antiestrogen fulvestrant, which down-regulates ER, the combination was extremely effective. Tumors regressed by 45% and were maintained without growth for the duration of the experiment (29 weeks). Thus, achieving more complete estrogen blockade may delay development of hormone-independent signaling pathways regulating proliferation.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Inibidores da Aromatase/uso terapêutico , Estradiol/análogos & derivados , Estradiol/uso terapêutico , Antagonistas de Estrogênios/uso terapêutico , Neoplasias Mamárias Experimentais/tratamento farmacológico , Nitrilas/uso terapêutico , Triazóis/uso terapêutico , Animais , Aromatase/genética , Modelos Animais de Doenças , Feminino , Fulvestranto , Humanos , Letrozol , Neoplasias Mamárias Experimentais/enzimologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Receptores de Estrogênio/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Estrogen deprivation has a profound effect on the female brain. One of the most obvious examples of this condition is hot flushes. Although estrogens relieve these typical climacteric symptoms, many women do not want to take them owing to unwanted side-effects impacting, for example, the uterus, breast and blood. Therefore, there is a need for developing safer estrogen therapies. We show here that treatment with 10ß,17ß-dihydroxyestra-1,4-dien-3-one (DHED), a novel brain-targeting bioprecursor prodrug of the main human estrogen, 17ß-estradiol, alleviates hot flushes in rat models of thermoregulatory dysfunction of the brain. Oral administration of DHED elicits a significant reduction of tail skin temperature (TST) rise representing hot flushes in the morphine-dependent ovariectomized rat model and results in the restoration of estrogen deprivation-induced loss of diurnal rhythm in TST. These beneficial effects occur without detrimental peripheral hormonal exposure; thus, the treatment avoids potentially harmful stimulation of estrogen-sensitive peripheral organs, including the uterus and the anterior pituitary, or the proliferation of MCF-7a breast cancer cell xenografts. Our promising preclinical assessments warrant further considerations of DHED for the development of a brain-selective 17ß-estradiol therapy to relieve hot flushes without undesirable peripheral side-effects.