RESUMEN
In the effort to identify deubiquitinating enzymes required for the growth of colorectal cancer (CRC) cells, we found that OTUB2 knockdown markedly inhibited the viability of these cancer cells in culture and in xenografted mice. It was also found that the level of OTUB2 was elevated in primary CRCs, and its high expression was a poor prognostic indicator for the patients. Interestingly, immunoprecipitation and LC-MS/MS analyses suggested that ß-Catenin was an OTUB2-interacting protein, and there was a positive correlation between OTUB2 and ß-Catenin expression in both CRC tissues and cell lines. We then performed reciprocal co-immunoprecipitations and demonstrated that OTUB2 and ß-Catenin bound to each other. Enforced expression of OTUB2 decreased ubiquitination of ß-Catenin and increased the half-life and intracellular level of ß-Catenin, whereas the catalytic inactive OTUB2 did not. OTUB2 also enhanced ß-Catenin-mediated transactivation as measured by TCF-luciferase and expression of endogenous CCND1 and MYC in CRC cells. These results indicated that OTUB2 was a potential target for therapeutic intervention for CRC.
RESUMEN
RNF6, a RING-type ubiquitin ligase, has been identified as an oncogene in various cancers but its role in multiple myeloma (MM) remains elusive. In the present study we first showed that the expression levels of RNF6 in MM were significantly elevated compared with the bone marrow cells of healthy donors. Overexpression of RNF6 in LP1 and PRMI-8266 MM cell lines promoted cell proliferation, whereas knockdown of RNF6 led to apoptosis of MM cells. Furthermore, we revealed that RNF6, as a ubiquitin ligase, interacted with glucocorticoid receptor (GR) and induced its K63-linked polyubiquitination. Different from current knowledge, RNF6 increased GR stability at both endogenous and exogenous contexts. Such an action greatly promoted GR transcriptional activity, which was confirmed by luciferase assays and by the increased expression levels of prosurvival genes including Bcl-xL and Mcl-1, two typical downstream genes of the GR pathway. Consistent with these findings, ectopic expression of RNF6 in MM cells conferred resistance to dexamethasone, a typical anti-myeloma agent. In conclusion, we demonstrate that RNF6 promotes MM cell proliferation and survival by inducing atypical polyubiquitination to GR, and RNF6 could be a promising therapeutic target for the treatment of MM.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Mieloma Múltiple/metabolismo , Receptores de Glucocorticoides/metabolismo , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Proteínas de Unión al ADN/genética , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Mieloma Múltiple/patología , Receptores de Glucocorticoides/genética , Relación Estructura-Actividad , UbiquitinaciónRESUMEN
[This corrects the article DOI: 10.18632/oncotarget.2688.].
RESUMEN
Ubiquitin specific peptidase 5 (USP5) is a ubiquitous expressed deubiquitinating enzyme (DUB). It has been shown involved in DNA repair, apoptosis, inflammation, and tumor cell growth. However, the function and molecular mechanism of USP5 in colorectal cancer (CRC) are still unclear. In the present study, we asked how it affected the growth of colorectal cancer cells. Methods: A shRNA-based high-content screening was performed to identify DUBs affecting the growth of CRC cells. CCK-8 assay and xenografts were used to assess CRC cell growth, survival and tumorigenesis. RT-qPCR, immunoblotting and immunohistochemistry were carried out to quantitate USP5 expression in CRC tissues and cell lines. Immunoprecipitation and mass spectrometry analysis were performed to identify USP5-interacting proteins. Cycloheximide chase was performed to assess Tu translation elongation factor (TUFM) stability. Dual luciferase reporter assay was utilized for USP5 promoter analysis. Results: We found that USP5 was highly expressed in a group of primary CRC tissues, and the increased USP5 was correlated with clinical stages and shorter overall survival. While USP5 knockdown effectively inhibited CRC cell growth, overexpressed USP5 promoted the growth of CRC cells and made them more resistant to doxorubicin (DOX). TUFM was discovered as a substrate of USP5. USP5 deubiquitinated TUFM and increased its level in CRC cells. Enforced expression of TUFM was able to alleviate the growth inhibition induced by USP5 knockdown. Further analyses showed that EBF transcription factor 1 (EBF1) was a major regulator for USP5 transcription, and DOX inhibited EBF1-USP5-TUFM axis in CRC cells. Conclusions: USP5 was required for CRC cells and promoted their growth and resistance to chemotherapeutics. TUFM was a USP5 deubiquitinating substrate that mediated the cellular effects of USP5. The transcription of USP5 was regulated by EBF1. Thus, targeting EBF1-USP5-TUFM axis is a potential novel strategy for CRC treatment.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Proteínas Mitocondriales/metabolismo , Factor Tu de Elongación Peptídica/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica , Neoplasias Colorrectales/genética , Femenino , Células HCT116 , Células HT29 , Humanos , Immunoblotting , Lentivirus/genética , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Proteínas Mitocondriales/genética , Factor Tu de Elongación Peptídica/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteasas Ubiquitina-Específicas/genéticaRESUMEN
Mutations and altered expression of deubiquitinating enzymes (DUBs) have been found associated with many human diseases including cancers. In this study, Ubiquitin specific protease 1 (USP1) expression was found significantly increased in some colorectal cancers (CRC). The elevated USP1 level was associated with short overall survival of patients and with advanced stages of cancers. In cultured CRC cells, knockdown of USP1 induced growth arrest at G2/M of cell cycle and reduced the expression of anti-apoptotic proteins Bcl-2 and Mcl-1. Its knockdown also led to reduction of DNA-repair related substrates FANCD2 and ID1. Further investigations found that small molecular inhibitor of USP1 ML323 sensitized CRC cells to DNA-targeting chemotherapeutics, including doxorubicin, TOPI/II inhibitors, and PARP inhibitor, but not to 5-Fu. These results indicate that USP1 plays a critical in colorectal cancer cell survival and is a promising target for anti-colorectal cancer chemotherapy. Targeting USP1 may represent an effective strategy to regulate the DNA-repairing system.
RESUMEN
Nuclear factor kappa B (NF-κB) signaling pathway is activated in many colorectal cancer (CRC) cells and in the tumor microenvironment, which plays a critical role in cancer initiation, development, and response to therapies. In the present study, we found that the widely used antimalarial drug mefloquine was a NF-κB inhibitor that blocked the activation of IκBα kinase, leading to reduction of IκBα degradation, decrease of p65 phosphorylation, and suppressed expression of NF-κB target genes in CRC cells. We also found that mefloquine induced growth arrest and apoptosis of CRC cells harboring phosphorylated p65 in culture and in mice. Furthermore, expression of constitutive active IKKß kinase significantly attenuated the cytotoxic effect of the compound. These results showed that mefloquine could exert antitumor action through inhibiting the NF-κB signaling pathway, and indicated that the antimalarial drug might be repurposed for anti-CRC therapy in the clinic as a single agent or in combination with other anticancer drugs.
Asunto(s)
Antimaláricos/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Mefloquina/farmacología , FN-kappa B/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HCT116 , Células HEK293 , Células HT29 , Humanos , Quinasa I-kappa B/metabolismo , Ratones , Fosforilación/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Ring finger protein 6 (RNF6) is a key oncogene in both prostate cancer and leukemia, but its role is elusive in breast cancer. In the present study, we found that RNF6 was overexpressed in more than 70% of breast cancer tissues and it was associated with overall survival. RNF6 increased breast cancer cell proliferation, migration and reduced cell sensitivity to doxorubicin. Further studies showed that RNF6 was closely associated with increased expression of estrogen receptor, a critical factor in the development of breast cancers. RNF6 was found to induce ERα expression and increased its stability. In doxorubicin-resistant breast cancer cells, RNF6 was found to be elevated in association with increased ERα and anti-apoptotic Bcl-xL, but not pro-apoptotic Bim-1. In consistence with this finding, overexpression of ERα led to increased Bcl-xL but had no effects on Bim-1. Therefore, this study demonstrated that there exists an RNF6/ERα/Bcl-xL axle in breast cancer which promotes cancer cell proliferation and survival. Targeting the RNF6/ERα/Bcl-xL axle could be a promising strategy in the treatment of breast cancer.
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Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/secundario , Proliferación Celular , Proteínas de Unión al ADN/metabolismo , Receptor alfa de Estrógeno/metabolismo , Proteína bcl-X/metabolismo , Apoptosis , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Estudios de Casos y Controles , Proteínas de Unión al ADN/genética , Receptor alfa de Estrógeno/genética , Femenino , Estudios de Seguimiento , Humanos , Metástasis Linfática , Persona de Mediana Edad , Clasificación del Tumor , Invasividad Neoplásica , Estadificación de Neoplasias , Pronóstico , Tasa de Supervivencia , Células Tumorales Cultivadas , Proteína bcl-X/genéticaRESUMEN
The activated JAK2-STAT3 signaling pathway is a high risk factor for multiple myeloma (MM), a fatal malignancy of plasma cells. In the present study, SC09, a potential inhibitor of cholesterol absorption, was identified in a STAT3-targeted drug screen. SC09 suppressed the activation of STAT3 in a time-course and concentration-dependent manner but did not affect its family members STAT1 and STAT5. SC09 inhibited STAT3 transcriptional activity and downregulated the expression of STAT3-regulated genes. Further studies showed that SC09 selectively inhibited JAK2 activation but not other kinases including c-Src, ERK, p38 and mTOR that are all associated with STAT3 activation. Moreover, SC09 obviously induced MM cell death in vitro and delayed MM tumor growth in vivo. SC09-induced MM cell death was dependent on the endogenous STAT3 status, and this effect could be attenuated by enforced expression of STAT3. All the results collectively indicated that SC09 blocks the JAK2-STAT3 signaling thus displaying anti-MM activity. Given its well tolerance and anti-MM potency, SC09 is credited for further investigation as a promising drug for MM treatment.
Asunto(s)
Antineoplásicos/farmacología , Colesterol/metabolismo , Janus Quinasa 2/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Mieloma Múltiple/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Modelos Animales de Enfermedad , Doxorrubicina/farmacología , Humanos , Janus Quinasa 2/química , Ratones , Ratones Desnudos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Fosforilación , Factor de Transcripción STAT3/genética , Activación Transcripcional/efectos de los fármacosRESUMEN
RNF6 is a little-studied ring finger protein. In the present study, we found that RNF6 was overexpressed in various leukemia cells and that it accelerated leukemia cell proliferation, whereas knockdown of RNF6 delayed tumor growth in xenografts. To find out the mechanism of RNF6 overexpression in leukemia, we designed a series of truncated constructs of RNF6 regulatory regions in the luciferase reporter system. The results revealed that the region between -144 and -99 upstream of the RNF6 transcription start site was critical and that this region contained a PBX1 recognition element (PRE). PBX1 modulated RNF6 expression by binding to the specific PRE. When PRE was mutated, RNF6 transcription was completely abolished. Further studies showed that PBX1 collaborated with PREP1 but not MEIS1 to modulate RNF6 expression. Moreover, RNF6 expression could be suppressed by doxorubicin, a major anti-leukemia agent, via down-regulating PBX1. This study thus suggests that RNF6 overexpression in leukemia is under the direction of PBX1 and that the PBX1/RNF6 axis can be developed as a novel therapeutic target of leukemia.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/metabolismo , Regulación Leucémica de la Expresión Génica , Leucemia/metabolismo , Proteínas Proto-Oncogénicas/biosíntesis , Elementos de Respuesta , Animales , Proteínas de Unión al ADN/genética , Doxorrubicina/farmacología , Células HL-60 , Xenoinjertos , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Células Jurkat , Células K562 , Leucemia/tratamiento farmacológico , Leucemia/genética , Leucemia/patología , Ratones , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Trasplante de Neoplasias , Factor de Transcripción 1 de la Leucemia de Células Pre-B , Proteínas Proto-Oncogénicas/genéticaRESUMEN
The oncogenic STAT3 signaling pathway is emerging as a promising target for the treatment of multiple myeloma (MM). In the present study, we identified a novel STAT3 inhibitor SC99 in a target-based high throughput screen. SC99 inhibited JAK2-STAT3 activation but had no effects on other transcription factors such as NF-κB, and kinases such as AKT, ERK, and c-Src that are in association with STAT3 signaling pathway. Furthermore, SC99 downregulated the expression of STAT3-modulated genes, including Bcl-2, Bcl-xL, VEGF, cyclin D2, and E2F-1. By inhibiting the STAT3 signaling, SC99 induced MM cell apoptosis which could be partly abolished by the ectopic expression of STAT3. Furthermore, SC99 displayed potent anti-MM activity in two independent MM xenograft models in nude mice. Oral administration of SC99 led to marked decrease of tumor growth within 10 days at a daily dosage of 30 mg/kg, but did not raise toxic effects. Taken together, this study identified a novel oral JAK2/STAT3 inhibitor that could be developed as an anti-myeloma agent.
Asunto(s)
Antineoplásicos/uso terapéutico , Hidrazonas/uso terapéutico , Janus Quinasa 2/antagonistas & inhibidores , Mieloma Múltiple/tratamiento farmacológico , Factor de Transcripción STAT3/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Células 3T3 , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Ciclina D2/biosíntesis , Factor de Transcripción E2F1/biosíntesis , Activación Enzimática/efectos de los fármacos , Femenino , Células HeLa , Humanos , Ratones , Ratones Desnudos , Mieloma Múltiple/patología , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína bcl-X/biosíntesisRESUMEN
The mammalian target of rapamycin (mTOR) is extensively involved in multiple myeloma (MM) pathophysiology. In the present study, we reported a novel small molecule SC06 that induced MM cell apoptosis and delayed MM xenograft growth in vivo. Oral administration of SC06 to mice bearing human MM xenografts resulted in significant inhibition of tumor growth at doses that were well tolerated. Mechanistic studies revealed that SC06 selectively inhibited the mTOR signaling pathway but had no effects on other associated kinases, such as AKT, ERK, p38, c-Src and JNK. Further studies showed that SC06-decreased mTOR activation was associated with the downregulation of Raptor, a key component of the mTORC1 complex. SC06 also suppressed the phosphorylation of 4E-BP1 and P70S6K, two typical substrates in the mTORC1 signaling pathway. Notably, expression of Raptor, phosphorylation of mTOR and phosphorylated 4E-BP1 was also decreased in the tumor tissues from SC06-treated mice, which was consistent with the cellular studies. Therefore, given the potency and low toxicity, SC06 could be developed as a potential anti-MM drug candidate by disrupting the mTOR signaling.
Asunto(s)
Aminopiridinas/uso terapéutico , Hidrazonas/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Aminopiridinas/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Femenino , Células HEK293 , Humanos , Hidrazonas/farmacología , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones Desnudos , Mieloma Múltiple/patología , Complejos Multiproteicos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteína Reguladora Asociada a mTOR , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Recent clinical trials have demonstrated targeting PI3K pathway is a promising strategy for the treatment of blood cancers. To identify novel PI3K inhibitors, we performed a high throughput virtual screen and identified several novel small molecule compounds, including PIK-C98 (C98). The cell-free enzymatic studies showed that C98 inhibited all class I PI3Ks at nano- or low micromolar concentrations but had no effects on AKT or mTOR activity. Molecular docking analysis revealed that C98 interfered with the ATP-binding pockets of PI3Ks by forming H-bonds and arene-H interactions with specific amino acid residues. The cellular assays demonstrated that C98 specifically inhibited PI3K/AKT/mTOR signaling pathway, but had no effects on other kinases and proteins including IGF-1R, ERK, p38, c-Src, PTEN, and STAT3. Inhibition of PI3K by C98 led to myeloma cell apoptosis. Furthermore, oral administration of C98 delayed tumor growth in two independent human myeloma xenograft models in nude mice but did not show overt toxicity. Pharmacokinetic analyses showed that C98 was well penetrated into myeloma tumors. Therefore, through a high throughput virtual screen we identified a novel PI3K inhibitor that is orally active against multiple myeloma with great potential for further development.
Asunto(s)
Antineoplásicos/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Furanos/farmacología , Regulación Neoplásica de la Expresión Génica , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Tiocarbamatos/farmacología , Administración Oral , Animales , Apoptosis , Línea Celular Tumoral , Supervivencia Celular , Femenino , Humanos , Enlace de Hidrógeno , Ratones , Ratones Desnudos , Ratones SCID , Simulación de Dinámica Molecular , Mieloma Múltiple/tratamiento farmacológico , Trasplante de Neoplasias , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de SeñalRESUMEN
The transcription factor c-MAF could be polyubiquitinated and subsequently degraded in the proteasomes. Theoretically, any lysine residues in c-MAF could be ubiquitinated. In the present study, we tried to find out the specific lysine residue(s) mediating c-MAF ubiquitination. Through a series of mutational screens from lysine (K) to arginine (R), we found that any single lysine mutation (K to R) failed to prevent c-MAF ubiquitination, and any single lysine residue alone could not mediate c-MAF ubiquitination, which indicated that multiple lysine residues were required for c-MAF ubiquitination. Bioinformatics and computing analyses revealed that K85 and K350 could mediate c-MAF ubiquitination, which was confirmed by the cell-based assays. However, this duo was not the only pair because the K85R/K350R mutant could also be ubiquitinated. Functionally, both M12 (K85/K350) and W12 (K85R/K350R) mutants increased cyclin D2 promoter-driven luciferase activity, but they were less potent than the lysine-free counterpart (M14). In addition, M14 induced a higher level of expression of cyclin D2 at both mRNA and protein levels. Therefore, we demonstrated that c-MAF ubiquitination is mediated by multiple lysine residues, of which K85 and K350 were sufficient but not the only residues in mediating c-MAF ubiquitination. Moreover, c-MAF was found to be degraded by lysosomes. This study added a novel insight for c-MAF ubiquitination and degradation, suggesting that c-MAF stability is strictly regulated.
Asunto(s)
Lisina/metabolismo , Mieloma Múltiple/metabolismo , Proteínas Proto-Oncogénicas c-maf/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Lisina/genética , Lisosomas/metabolismo , Datos de Secuencia Molecular , Mieloma Múltiple/genética , Mutación , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas c-maf/genética , Transfección , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
Clioquinol is an anti-microbial drug, and it was recently found to induce cancer cell death. In the present study, clioquinol was found to trigger autophagy by inducing LC3 lipidation and autophagosome formation which was abolished by an autophagy inhibitor 3-methyladenine. Further study showed clioquinol displayed no effects on PI3KC3 or Beclin 1 expression but downregulated the expression and the enzymatic activity of mammalian target of Rapamycin (mTOR), a critical modulator of autophagy. Moreover, clioquinol inhibited the catalytic activity of the mTOR complex 1, thus suppressing phosphorylation of P70S6K and 4E-BP1, two major proteins associated with autophagy in the mTORC1 signaling pathway. Clioquinol induced leukemia and myeloma cell apoptosis, however, addition of autophagy inhibitor 3-methyladenine attenuated this kind of cell death. Therefore, this study demonstrated that clioquinol induces autophagy in associated with apoptosis in leukemia and myeloma cells by disrupting mTOR signaling pathway.
Asunto(s)
Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Clioquinol/farmacología , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Beclina-1 , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Leucemia/tratamiento farmacológico , Leucemia/patología , Diana Mecanicista del Complejo 1 de la Rapamicina , Proteínas de la Membrana/metabolismo , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/patología , Complejos Multiproteicos/metabolismo , Fosforilación , Procesamiento Proteico-PostraduccionalRESUMEN
Recent studies demonstrated that targeting the phosphatidylinositide 3-kinase (PI3K)/AKT signaling pathway is a major strategy for the treatment of androgen-independent prostate cancer. In the present study, we developed an analog BENC-511 from a recently reported PI3K inhibitor S14161 by structural optimization. Using PC3 and DU145 as the model cell lines, we found PTEN-deficient PC3 cells were more sensitive than PTEN-expressing DU145 ones in terms of cell proliferation, apoptosis, and caspase-3 activation. These findings were consistent with the inhibition on PI3K/AKT signals. BENC-511 preferably suppressed AKT activation in PC3 over DU145 cells. Notably, PTEN restoration attenuated BENC-511 induced apoptosis. Moreover, BENC-511 displayed great therapeutic efficacy in a PC3-derived prostate cancer model in nude mice. With an oral dosage of 50mg/kg, BENC-511 decreased tumor growth more than 50% in 27 days, which was accompanied with PARP cleavage, but did not show overt toxicity. This study lays a solid rationale for the development of BENC-511 as a drug for the treatment of PTEN-deficient and androgen-independent prostate cancers.
Asunto(s)
Antineoplásicos/farmacología , Benzopiranos/farmacología , Fosfohidrolasa PTEN/deficiencia , Inhibidores de las Quinasa Fosfoinosítidos-3 , Neoplasias de la Próstata/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Benzopiranos/administración & dosificación , Caspasa 3/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Activación Enzimática , Humanos , Masculino , Ratones , Ratones Desnudos , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasa/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Inhibidores de Proteínas Quinasas/administración & dosificación , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Transfección , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Dihydrorotenone (DHR) is a natural pesticide used for farming including organic produces. We recently found that DHR induces human plasma cell apoptosis by provoking endoplasmic reticulum stress. In the present study, we found that DHR arrested human plasma cancer cells at the G0/G1 phase of the cell cycle. Mechanistical studies demonstrated that cell cycle arrest was associated with downregulated cell cycle promotors including cyclin D2, cyclin D3, cyclin-dependent kinases (CDK4, CKD6), and phosphorylated-Rb. DHR inhibited cyclin D2 transactivation, thus inhibiting its mRNA expression. In addition, DHR upregulated the cell cycle repressors p21 and p53. DHR also increased the phosphorylation level of p53, suggesting the upregulated transactivation function of p53, which was confirmed by the induction of p21, a substrate of activated p53. Moreover, DHR downregulated AKT and ERK phosphorylation, an incentive of cell cycle progression. Therefore, these results collectively demonstrated that DHR disrupts the cell cycle progress, which suggests that DHR is toxic to human plasma cells. Caution is thus suggested when handling with this agent.
Asunto(s)
Puntos de Control del Ciclo Celular/efectos de los fármacos , Fase G1/efectos de los fármacos , Insecticidas/toxicidad , Células Plasmáticas/efectos de los fármacos , Fase de Descanso del Ciclo Celular/efectos de los fármacos , Rotenona/análogos & derivados , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ciclina D2/genética , Quinasas Ciclina-Dependientes/metabolismo , Humanos , Insecticidas/química , Estructura Molecular , Células Plasmáticas/enzimología , Células Plasmáticas/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rotenona/química , Rotenona/toxicidad , Transducción de SeñalRESUMEN
BACKGROUND: We previously reported a PI3K inhibitor S14161 which displays a promising preclinical activity against multiple myeloma (MM) and leukemia, but the chiral structure and poor solubility prevent its further application. METHODS: Six S14161 analogs were designed based on the structure-activity relationship; activity of the compounds in terms of cell death and inhibition of PI3K were analyzed by flow cytometry and Western blotting, respectively; anti-myeloma activity in vivo was performed on two independent xenograft models. RESULTS: Among the six analogs, BENC-511 was one of the most potent compounds which significantly inhibited PI3K activity and induced MM cell apoptosis. BENC-511 was able to inactivate PI3K and its downstream signals AKT, mTOR, p70S6K, and 4E-BP1 at 1 µM but had no effects on their total protein expression. Consistent with its effects on PI3K activity, BENC-511 induced MM cell apoptosis which was evidenced by the cleavage of Caspase-3 and PARP. Notably, addition of insulin-like growth factor 1 and interleukin-6, two important triggers for PI3K activation in MM cells, partly blocked BENC-511-induced MM cell death, which further demonstrated that PI3K signaling pathway was critical for the anti-myeloma activity of BENC-511. Moreover, BENC-511 also showed potent oral activity against myeloma in vivo. Oral administration of BENC-511 decreased tumor growth up to 80% within 3 weeks in two independent MM xenograft models at a dose of 50 mg/kg body weight, but presented minimal toxicity. Suppression of BENC-511 on MM tumor growth was associated with decreased PI3K/AKT activity and increased cell apoptosis. CONCLUSIONS: Because of its potent anti-MM activity, low toxicity (LD50 oral >1.5 g/kg), and easy synthesis, BENC-511 could be developed as a promising agent for the treatment of MM via suppressing the PI3K/AKT signaling pathway.
Asunto(s)
Antineoplásicos/farmacología , Mieloma Múltiple/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Benzopiranos/química , Benzopiranos/farmacología , Caspasa 3/metabolismo , Línea Celular Tumoral , Femenino , Citometría de Flujo , Humanos , Immunoblotting , Factor I del Crecimiento Similar a la Insulina/farmacología , Interleucina-6/farmacología , Ratones , Ratones Desnudos , Estructura Molecular , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Carga Tumoral/efectos de los fármacosRESUMEN
The antiparasitic clioquinol (CQ) represents a class of novel anticancer drugs by interfering with proteasome activity. In the present study, we found that CQ induced blood cancer cell apoptosis by inhibiting histone deacetylases (HDACs). CQ accumulated the acetylation levels of several key proteins including histone H3 (H3), p53, HSP90, and α-tubulin. In the mechanistic study, CQ was found to down-regulate HDAC1, -3, -4, and -5 in both myeloma and leukemia cells. Computer modeling analysis revealed that CQ was well docked into the active pocket of the enzyme, where the oxygen and nitrogen atoms in CQ formed stable coordinate bonds with the zinc ion, and the hydroxyl group from CQ formed an effective hydrogen bond with Asp-267. Moreover, co-treatment with CQ and zinc/copper chloride led to decreased Ac-H3. Furthermore, CQ inhibited the activity of Class I and IIa HDACs in the cell-free assays, demonstrating that CQ interfered with HDAC activity. By inhibiting HDAC activity, CQ induced expression of p21, p27, and p53, cell cycle arrest at G1 phase, and cell apoptosis. This study suggested that the HDAC enzymes are targets of CQ, which provided a novel insight into the molecular mechanism of CQ in the treatment of hematological malignancies.
Asunto(s)
Antipruriginosos/farmacología , Apoptosis/efectos de los fármacos , Clioquinol/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Leucemia/tratamiento farmacológico , Mieloma Múltiple/tratamiento farmacológico , Cloruros/farmacología , Regulación hacia Abajo/efectos de los fármacos , Femenino , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Células K562 , Leucemia/enzimología , Leucemia/patología , Masculino , Antisépticos Bucales/farmacología , Mieloma Múltiple/enzimología , Mieloma Múltiple/patología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Células U937 , Compuestos de Zinc/farmacologíaRESUMEN
The small chemical compound 8-ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene (S14161) was recently identified as an inhibitor of the phosphoinositide 3-kinase (PI3K). In the present study, we designed a novel synthesis of S14161 and prepared a series of its analogues via the oxa-Michael-Henry reaction in the presence of catalytic amounts of l-proline and triethylamine. Further structural simplification led to the identification of 6-bromo-8-ethoxy-3-nitro-2H-chromene (BENC-511) that exhibited potent antiproliferative activities against a panel of 12 tumor cell lines. Compared with S14161, BENC-511 was more potent in blocking the AKT phosphorylation and inducing cancer cell apoptosis. BENC-511 also displayed more potent effects on human umbilical vein epithelial cells (HUVEC) migration, suggesting its anti-angiogenesis activity.