RESUMEN
The DNA damage repair enzyme, O6-methylguanine DNA methyltransferase (MGMT) is overexpressed in breast cancer, correlating directly with estrogen receptor (ER) expression and function. In ER negative breast cancer the MGMT promoter is frequently methylated. In ER positive breast cancer MGMT is upregulated and modulates ER function. Here, we evaluate MGMT's role in control of other clinically relevant targets involved in cell cycle regulation during breast cancer oncogenesis. We show that O6-benzylguanine (BG), an MGMT inhibitor decreases CDC2, CDC20, TOP2A, AURKB, KIF20A, cyclin B2, A2, D1, ERα and survivin and induces c-PARP and p21 and sensitizes ER positive breast cancer to temozolomide (TMZ). Further, siRNA inhibition of MGMT inhibits CDC2, TOP2A, AURKB, KIF20A, Cyclin B2, A2 and survivin and induces p21. Combination of BG+TMZ decreases CDC2, CDC20, TOP2A, AURKB, KIF20A, Cyclin A2, B2, D1, ERα and survivin. Temozolomide alone inhibits MGMT expression in a dose and time dependent manner and increases p21 and cytochrome c. Temozolomide inhibits transcription of TOP2A, AURKB, KIF20A and does not have any effect on CDC2 and CDC20 and induces p21. BG+/-TMZ inhibits breast cancer growth. In our orthotopic ER positive breast cancer xenografts, BG+/-TMZ decreases ki-67, CDC2, CDC20, TOP2A, AURKB and induces p21 expression. In the same model, BG+TMZ combination inhibits breast tumor growth in vivo compared to single agent (TMZ or BG) or control. Our results show that MGMT inhibition is relevant for inhibition of multiple downstream targets involved in tumorigenesis. We also show that MGMT inhibition increases ER positive breast cancer sensitivity to alkylator based chemotherapy.
RESUMEN
Antiestrogen therapy resistance remains a huge stumbling block in the treatment of breast cancer. We have found significant elevation of O(6) methylguanine DNA methyl transferase (MGMT) expression in a small sample of consecutive patients who have failed tamoxifen treatment. Here, we show that tamoxifen resistance is accompanied by upregulation of MGMT. Further we show that administration of the MGMT inhibitor, O(6)-benzylguanine (BG), at nontoxic doses, leads to restoration of a favorable estrogen receptor alpha (ERα) phosphorylation phenotype (high p-ERα Ser167/low p-ERα Ser118), which has been reported to correlate with sensitivity to endocrine therapy and improved survival. We also show BG to be a dual inhibitor of MGMT and ERα. In tamoxifen-resistant breast cancer cells, BG alone or in combination with antiestrogen (tamoxifen [TAM]/ICI 182,780 [fulvestrant, Faslodex]) therapy enhances p53 upregulated modulator of apoptosis (PUMA) expression, cytochrome C release and poly (ADP-ribose) polymerase (PARP) cleavage, all indicative of apoptosis. In addition, BG increases the expression of p21(cip1/waf1). We also show that BG, alone or in combination therapy, curtails the growth of tamoxifen-resistant breast cancer in vitro and in vivo. In tamoxifen-resistant MCF7 breast cancer xenografts, BG alone or in combination treatment causes significant delay in tumor growth. Immunohistochemistry confirms that BG increases p21(cip1/waf1) and p-ERα Ser167 expression and inhibits MGMT, ERα, p-ERα Ser118 and ki-67 expression. Collectively, our results suggest that MGMT inhibition leads to growth inhibition of tamoxifen-resistant breast cancer in vitro and in vivo and resensitizes tamoxifen-resistant breast cancer cells to antiestrogen therapy. These findings suggest that MGMT inhibition may provide a novel therapeutic strategy for overcoming antiestrogen resistance.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/enzimología , Metilasas de Modificación del ADN/antagonistas & inhibidores , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Antagonistas de Estrógenos/uso terapéutico , Receptor alfa de Estrógeno/metabolismo , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Antagonistas de Estrógenos/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Guanina/análogos & derivados , Guanina/farmacología , Guanina/uso terapéutico , Humanos , Inmunohistoquímica , Ratones , Ratones Desnudos , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Tamoxifeno/uso terapéutico , Transcripción Genética/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
More than 120 human papillomaviruses (HPVs) have now been identified and have been associated with a variety of clinical lesions. To understand the molecular differences among these viruses that result in lesions with distinct pathologies, we have begun a MS-based proteomic analysis of HPV-host cellular protein interactions and have created the plasmid and cell line libraries required for these studies. To validate our system, we have characterized the host cellular proteins that bind to the E7 proteins expressed from 17 different HPV types. These studies reveal a number of interactions, some of which are conserved across HPV types and others that are unique to a single HPV species or HPV genus. Binding of E7 to UBR4/p600 is conserved across all virus types, whereas the cellular protein ENC1 binds specifically to the E7s from HPV18 and HPV45, both members of genus alpha, species 7. We identify a specific interaction of HPV16 E7 with ZER1, a substrate specificity factor for a cullin 2 (CUL2)-RING ubiquitin ligase, and show that ZER1 is required for the binding of HPV16 E7 to CUL2. We further show that ZER1 is required for the destabilization of the retinoblastoma tumor suppressor RB1 in HPV16 E7-expressing cells and propose that a CUL2-ZER1 complex functions to target RB1 for degradation in HPV16 E7-expressing cells. These studies refine the current understanding of HPV E7 functions and establish a platform for the rapid identification of virus-host interactions.
Asunto(s)
Proteínas Oncogénicas Virales/metabolismo , Papillomaviridae/metabolismo , Secuencia de Aminoácidos , Western Blotting , Línea Celular , Humanos , Datos de Secuencia Molecular , Proteínas Oncogénicas Virales/química , Papillomaviridae/clasificación , Unión Proteica , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Espectrometría de Masas en TándemRESUMEN
Laminin α2 (LAMA2)-deficient congenital muscular dystrophy is a severe, early-onset disease caused by abnormal levels of laminin 211 in the basal lamina leading to muscle weakness, transient inflammation, muscle degeneration and impaired mobility. In a Lama2-deficient mouse model for this disease, animal survival is improved by muscle-specific expression of the apoptosis inhibitor Bcl-2, conferred by a MyoD-hBcl-2 transgene. Here we investigated early disease stages in this model to determine initial pathological events and effects of Bcl-2 on their progression. Using quantitative immunohistological and mRNA analyses we show that inflammation occurs very early in Lama2-deficient muscle, some aspects of which are reduced or delayed by the MyoD-hBcl-2 transgene. mRNAs for innate immune response regulators, including multiple Toll-like receptors (TLRs) and the inflammasome component NLRP3, are elevated in diseased muscle compared with age-matched controls expressing Lama2. MyoD-hBcl-2 inhibits induction of TLR4, TLR6, TLR7, TLR8 and TLR9 in Lama2-deficient muscle compared with non-transgenic controls, and leads to reduced infiltration of eosinophils, which are key death effector cells. This congenital disease model provides a new paradigm for investigating cell death mechanisms during early stages of pathogenesis, demonstrating that interactions exist between Bcl-2, a multifunctional regulator of cell survival, and the innate immune response.
Asunto(s)
Inmunidad Innata/inmunología , Distrofia Muscular Animal/inmunología , Distrofia Muscular Animal/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Western Blotting , Inmunidad Innata/genética , Etiquetado Corte-Fin in Situ , Infiltración Leucémica/genética , Infiltración Leucémica/inmunología , Infiltración Leucémica/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Distrofia Muscular Animal/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 6/genética , Receptor Toll-Like 6/metabolismo , Receptor Toll-Like 7/genética , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 8/genética , Receptor Toll-Like 8/metabolismo , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismoAsunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Cistitis Intersticial/tratamiento farmacológico , Liberación de Histamina/efectos de los fármacos , Mastocitos/metabolismo , Poliéster Pentosan Sulfúrico/uso terapéutico , Animales , Antiinflamatorios no Esteroideos/farmacología , Calcio/metabolismo , Modelos Animales de Enfermedad , Antagonistas de los Receptores Histamínicos/farmacología , Cinética , Masculino , Mastocitos/efectos de los fármacos , Poliéster Pentosan Sulfúrico/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Mast cells are involved in early- and late-phase reactions by releasing vasoactive molecules, proteases, and cytokines. Certain histamine-1 receptor antagonists and other antiallergic drugs seem to inhibit the release of mediators from rat and human mast cells. OBJECTIVE: Azelastine and olopatadine are antiallergic agents present in the ophthalmic solutions azelastine hydrochloride (Optivar, Asta Medica/Muro Pharmaceuticals, Tewksbury, MA), and olopatadine hydrochloride (Patanol, Alcon Laboratories, Fort Worth, TX), respectively. We investigated the effect of these drugs on interleukin-6 (IL-6), tryptase, and histamine release from cultured human mast cells (CHMCs). METHODS: CHMCs were grown from human umbilical cord blood-derived CD34+ cells in the presence of stem cell factor and IL-6 for 14 to 16 weeks. Sensitized CHMCs were pretreated with various concentrations of azelastine or olopatadine for 5 minutes. CHMCs were then challenged with anti-immunoglobulin E, and the released mediators were quantitated. RESULTS: The greatest inhibition of mediator release was seen with 24 microM azelastine; this level of inhibition was matched with the use of 133 microM olopatadine. At this concentration, these drugs inhibited IL-6 release by 83% and 74%, tryptase release by 55% and 79%, and histamine release by 41% and 45%, respectively. Activated CHMCs were characterized by numerous filopodia that were inhibited by both drugs as shown by electron microscopy. CONCLUSIONS: These results indicate that azelastine and olopatadine can inhibit CHMCs activation and release of IL-6, tryptase, and histamine. On an equimolar basis, azelastine was a more potent inhibitor than olopatadine.
Asunto(s)
Antialérgicos/farmacología , Dibenzoxepinas/farmacología , Interleucina-6/metabolismo , Mastocitos/efectos de los fármacos , Ftalazinas/farmacología , Serina Endopeptidasas/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Sangre Fetal , Liberación de Histamina/efectos de los fármacos , Humanos , Activación de Linfocitos , Mastocitos/metabolismo , Clorhidrato de Olopatadina , TriptasasRESUMEN
Mast cells are involved in early and late-phase reactions by releasing vasoactive molecules, proteases, and cytokines. Azelastine and olopatadine are histamine 1 receptor (H-1R) antagonists with antiallergic effects present in the ophthalmic solutions Optivar and Patanol, respectively. Because it is difficult to obtain animal or human conjunctival tissue, we first investigated the effect of these compounds on histamine and tryptase release from cultured human mast cells (CHMCs) grown out of human umbilical cord blood-derived CD34+ cells. Sensitized CHMCs were pretreated with various concentrations of azelastine or olopatadine for 5 minutes. Then, CHMCs were challenged with anti-immunoglobulin E (IgE) and the released mediators were quantitated. The greatest inhibition of mediator release was seen when CHMCs were pretreated with 24 microM of azelastine or 133 microM of olopatadine (2% dilution of azelastine or 5% olopatadine original ophthalmic solutions, respectively). We then studied the drug concentrations that gave optimal results on skin vasodilation induced by the mast cell secretagogue compound 48/80. An intradermal injection of 48/80 in rats, to which Evan's blue had been administered via the tail vein, induced substantial dye extravasation. Pretreatment of the injection site for 5 minutes with either 24 microM of azelastine or 133 microM of olopatadine completely prevented extravasation; this effect was quantitated also by fluorometric assessment of Evan's blue extracted in formamide. Evaluation of skin mast cells from injected sites showed that mast cell degranulation was inhibited greatly. These results indicate that on an equimolar basis, azelastine was a more potent inhibitor than olopatadine of both CHMC and rat skin mast cells activation.