RESUMO
IgE-mediated mast cell activation is a driving force in allergic disease in need of novel interventions. Statins, long used to lower serum cholesterol, have been shown in multiple large-cohort studies to reduce asthma severity. We previously found that statins inhibit IgE-induced mast cell function, but these effects varied widely among mouse strains and human donors, likely due to the upregulation of the statin target, 3-hydroxy-3-methylgutaryl-CoA reductase. Statin inhibition of mast cell function appeared to be mediated not by cholesterol reduction but by suppressing protein isoprenylation events that use cholesterol pathway intermediates. Therefore, we sought to circumvent statin resistance by targeting isoprenylation. Using genetic depletion of the isoprenylation enzymes farnesyltransferase and geranylgeranyl transferase 1 or their substrate K-Ras, we show a significant reduction in FcεRI-mediated degranulation and cytokine production. Furthermore, similar effects were observed with pharmacological inhibition with the dual farnesyltransferase and geranylgeranyl transferase 1 inhibitor FGTI-2734. Our data indicate that both transferases must be inhibited to reduce mast cell function and that K-Ras is a critical isoprenylation target. Importantly, FGTI-2734 was effective in vivo, suppressing mast cell-dependent anaphylaxis, allergic pulmonary inflammation, and airway hyperresponsiveness. Collectively, these findings suggest that K-Ras is among the isoprenylation substrates critical for FcεRI-induced mast cell function and reveal isoprenylation as a new means of targeting allergic disease.
Assuntos
Anafilaxia , Inibidores de Hidroximetilglutaril-CoA Redutases , Camundongos , Humanos , Animais , Receptores de IgE/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Farnesiltranstransferase/metabolismo , Mastócitos/metabolismo , Anafilaxia/metabolismo , Transdução de Sinais , Degranulação Celular , Imunoglobulina E/metabolismo , Inflamação/metabolismo , Colesterol/metabolismo , PrenilaçãoRESUMO
In human cancer cells that harbor mutant KRAS and WT p53 (p53), KRAS contributes to the maintenance of low p53 levels. Moreover, KRAS depletion stabilizes and reactivates p53 and thereby inhibits malignant transformation. However, the mechanism by which KRAS regulates p53 is largely unknown. Recently, we showed that KRAS depletion leads to p53 Ser-15 phosphorylation (P-p53) and increases the levels of p53 and its target p21/WT p53-activated fragment 1 (WAF1)/CIP1. Here, using several human lung cancer cell lines, siRNA-mediated gene silencing, immunoblotting, quantitative RT-PCR, promoter-reporter assays, and reactive oxygen species (ROS) assays, we demonstrate that KRAS maintains low p53 levels by activating the NRF2 (NFE2-related factor 2)-regulated antioxidant defense system. We found that KRAS depletion led to down-regulation of NRF2 and its targets NQO1 (NAD(P)H quinone dehydrogenase 1) and SLC7A11 (solute carrier family 7 member 11), decreased the GSH/GSSG ratio, and increased ROS levels. We noted that the increase in ROS is required for increased P-p53, p53, and p21Waf1/cip1 levels following KRAS depletion. Downstream of KRAS, depletion of RalB (RAS-like proto-oncogene B) and IκB kinase-related TANK-binding kinase 1 (TBK1) activated p53 in a ROS- and NRF2-dependent manner. Consistent with this, the IκB kinase inhibitor BAY11-7085 and dominant-negative mutant IκBαM inhibited NF-κB activity and increased P-p53, p53, and p21Waf1/cip1 levels in a ROS-dependent manner. In conclusion, our findings uncover an important role for the NRF2-regulated antioxidant system in KRAS-mediated p53 suppression.
Assuntos
Antioxidantes/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sistema y+ de Transporte de Aminoácidos/metabolismo , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação para Baixo , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Humanos , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteínas ral de Ligação ao GTP/antagonistas & inibidores , Proteínas ral de Ligação ao GTP/genética , Proteínas ral de Ligação ao GTP/metabolismoRESUMO
The proteasome inhibitor bortezomib is effective in hematologic malignancies such as multiple myeloma but has little activity against solid tumors, acts covalently, and is associated with undesired side effects. Therefore, noncovalent inhibitors that are less toxic and more effective against solid tumors are desirable. Structure activity relationship studies led to the discovery of PI-1840, a potent and selective inhibitor for chymotrypsin-like (CT-L) (IC50 value = 27 ± 0.14 nm) over trypsin-like and peptidylglutamyl peptide hydrolyzing (IC50 values >100 µm) activities of the proteasome. Furthermore, PI-1840 is over 100-fold more selective for the constitutive proteasome over the immunoproteasome. Mass spectrometry and dialysis studies demonstrate that PI-1840 is a noncovalent and rapidly reversible CT-L inhibitor. In intact cancer cells, PI-1840 inhibits CT-L activity, induces the accumulation of proteasome substrates p27, Bax, and IκB-α, inhibits survival pathways and viability, and induces apoptosis. Furthermore, PI-1840 sensitizes human cancer cells to the mdm2/p53 disruptor, nutlin, and to the pan-Bcl-2 antagonist BH3-M6. Finally, in vivo, PI-1840 but not bortezomib suppresses the growth in nude mice of human breast tumor xenografts. These results warrant further evaluation of a noncovalent and rapidly reversible proteasome inhibitor as potential anticancer agents against solid tumors.
Assuntos
Acetamidas/farmacologia , Antineoplásicos/farmacologia , Oxidiazóis/farmacologia , Inibidores de Proteassoma/farmacologia , Animais , Western Blotting , Ácidos Borônicos/farmacologia , Bortezomib , Linhagem Celular Tumoral , Humanos , Camundongos , Pirazinas/farmacologiaRESUMO
KRAS mutations are prevalent in pancreatic and lung cancers, but not all mutant (mt) KRAS tumors are addicted to mt KRAS. Here, we discovered a 30-gene transcriptome signature "KDS30" that encodes a novel EGFR/ERBB2-driven signaling network and predicts mt KRAS, but not NRAS or HRAS, oncogene addiction. High KDS30 tumors from mt KRAS lung and pancreatic cancer patients are enriched in genes upregulated by EGFR, ERBB2, mt KRAS or MEK. EGFR/ERBB2 (neratinib) and MEK (cobimetinib) inhibitor combination inhibits tumor growth and prolongs mouse survival in high, but not low, KDS30 mt KRAS lung and pancreatic xenografts, and is synergistic only in high KDS30 mt KRAS patient-derived organoids. Furthermore, mt KRAS high KDS30 lung and pancreatic cancer patients live significantly shorter lives than those with low KDS30. Thus, KDS30 can identify lung and pancreatic cancer patients whose tumors are addicted to mt KRAS, and predicts EGFR/ERBB2 and MEK inhibitor combination response.
RESUMO
Currently, there are no clinically approved drugs that directly thwart mutant KRAS G12D, a major driver of human cancer. Here, we report on the discovery of a small molecule, KRB-456, that binds KRAS G12D and inhibits the growth of pancreatic cancer patient-derived tumors. Protein nuclear magnetic resonance studies revealed that KRB-456 binds the GDP-bound and GCP-bound conformation of KRAS G12D by forming interactions with a dynamic allosteric binding pocket within the switch-I/II region. Isothermal titration calorimetry demonstrated that KRB-456 binds potently to KRAS G12D with 1.5-, 2-, and 6-fold higher affinity than to KRAS G12V, KRAS wild-type, and KRAS G12C, respectively. KRB-456 potently inhibits the binding of KRAS G12D to the RAS-binding domain (RBD) of RAF1 as demonstrated by GST-RBD pulldown and AlphaScreen assays. Treatment of KRAS G12D-harboring human pancreatic cancer cells with KRB-456 suppresses the cellular levels of KRAS bound to GTP and inhibits the binding of KRAS to RAF1. Importantly, KRB-456 inhibits P-MEK, P-AKT, and P-S6 levels in vivo and inhibits the growth of subcutaneous and orthotopic xenografts derived from patients with pancreatic cancer whose tumors harbor KRAS G12D and KRAS G12V and who relapsed after chemotherapy and radiotherapy. These results warrant further development of KRB-456 for pancreatic cancer. SIGNIFICANCE: There are no clinically approved drugs directly abrogating mutant KRAS G12D. Here, we discovered a small molecule, KRB-456, that binds a dynamic allosteric binding pocket within the switch-I/II region of KRAS G12D. KRB-456 inhibits P-MEK, P-AKT, and P-S6 levels in vivo and inhibits the growth of subcutaneous and orthotopic xenografts derived from patients with pancreatic cancer. This discovery warrants further advanced preclinical and clinical studies in pancreatic cancer.
Assuntos
Neoplasias Pancreáticas , Proteínas Proto-Oncogênicas p21(ras) , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linhagem Celular Tumoral , Neoplasias Pancreáticas/tratamento farmacológico , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismoRESUMO
A critical hallmark of cancer cell survival is evasion of apoptosis. This is commonly due to overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-X(L), and Mcl-1, which bind to the BH3 α-helical domain of pro-apoptotic proteins such as Bax, Bak, Bad, and Bim, and inhibit their function. We designed a BH3 α-helical mimetic BH3-M6 that binds to Bcl-X(L) and Mcl-1 and prevents their binding to fluorescently labeled Bak- or Bim-BH3 peptides in vitro. Using several approaches, we demonstrate that BH3-M6 is a pan-Bcl-2 antagonist that inhibits the binding of Bcl-X(L), Bcl-2, and Mcl-1 to multi-domain Bax or Bak, or BH3-only Bim or Bad in cell-free systems and in intact human cancer cells, freeing up pro-apoptotic proteins to induce apoptosis. BH3-M6 disruption of these protein-protein interactions is associated with cytochrome c release from mitochondria, caspase-3 activation and PARP cleavage. Using caspase inhibitors and Bax and Bak siRNAs, we demonstrate that BH3-M6-induced apoptosis is caspase- and Bax-, but not Bak-dependent. Furthermore, BH3-M6 disrupts Bcl-X(L)/Bim, Bcl-2/Bim, and Mcl-1/Bim protein-protein interactions and frees up Bim to induce apoptosis in human cancer cells that depend for tumor survival on the neutralization of Bim with Bcl-X(L), Bcl-2, or Mcl-1. Finally, BH3-M6 sensitizes cells to apoptosis induced by the proteasome inhibitor CEP-1612.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/efeitos dos fármacos , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Proteínas de Membrana/metabolismo , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/farmacologia , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteína de Morte Celular Associada a bcl/metabolismo , Proteína bcl-X/metabolismo , Animais , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína 11 Semelhante a Bcl-2 , Caspase 3/genética , Caspase 3/metabolismo , Linhagem Celular Tumoral , Citocromos c/genética , Citocromos c/metabolismo , Dipeptídeos/farmacologia , Células HEK293 , Humanos , Proteínas de Membrana/genética , Mitocôndrias , Proteína de Sequência 1 de Leucemia de Células Mieloides , Ftalimidas/farmacologia , Inibidores de Proteases/farmacologia , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genética , Proteína de Morte Celular Associada a bcl/genética , Proteína bcl-X/genéticaRESUMO
PURPOSE: Among human cancers that harbor mutant (mt) KRas, some, but not all, are dependent on mt KRas. However, little is known about what drives KRas dependency. EXPERIMENTAL DESIGN: Global phosphoproteomics, screening of a chemical library of FDA drugs, and genome-wide CRISPR/Cas9 viability database analysis were used to identify vulnerabilities of KRas dependency. RESULTS: Global phosphoproteomics revealed that KRas dependency is driven by a cyclin-dependent kinase (CDK) network. CRISPR/Cas9 viability database analysis revealed that, in mt KRas-driven pancreatic cancer cells, knocking out the cell-cycle regulators CDK1 or CDK2 or the transcriptional regulators CDK7 or CDK9 was as effective as knocking out KRas. Furthermore, screening of a library of FDA drugs identified AT7519, a CDK1, 2, 7, and 9 inhibitor, as a potent inducer of apoptosis in mt KRas-dependent, but not in mt KRas-independent, human cancer cells. In vivo AT7519 inhibited the phosphorylation of CDK1, 2, 7, and 9 substrates and suppressed growth of xenografts from 5 patients with pancreatic cancer. AT7519 also abrogated mt KRas and mt p53 primary and metastatic pancreatic cancer in three-dimensional (3D) organoids from 2 patients, 3D cocultures from 8 patients, and mouse 3D organoids from pancreatic intraepithelial neoplasia, primary, and metastatic tumors. CONCLUSIONS: A link between CDK hyperactivation and mt KRas dependency was uncovered and pharmacologically exploited to abrogate mt KRas-driven pancreatic cancer in highly relevant models, warranting clinical investigations of AT7519 in patients with pancreatic cancer.
Assuntos
Quinases Ciclina-Dependentes/fisiologia , Neoplasias Pancreáticas/etiologia , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Animais , Quinases Ciclina-Dependentes/metabolismo , Humanos , Camundongos , Fosforilação , ProteomaRESUMO
An obstacle to the development of chimeric antigen receptor (CAR) T cells is the limited understanding of CAR T-cell biology and the mechanisms behind their antitumor activity. We and others have shown that CARs with a CD28 costimulatory domain drive high T-cell activation, which leads to exhaustion and shortened persistence. This work led us to hypothesize that by incorporating null mutations of CD28 subdomains (YMNM, PRRP, or PYAP), we could optimize CAR T-cell costimulation and enhance function. In vivo, we found that mice given CAR T cells with only a PYAP CD28 endodomain had a significant survival advantage, with 100% of mice alive after 62 days compared with 50% for mice with an unmutated endodomain. We observed that mutant CAR T cells remained more sensitive to antigen after ex vivo antigen and PD-L1 stimulation, as demonstrated by increased cytokine production. The mutant CAR T cells also had a reduction of exhaustion-related transcription factors and genes such as Nfatc1, Nr42a, and Pdcd1 Our results demonstrated that CAR T cells with a mutant CD28 endodomain have better survival and function. This work allows for the development of enhanced CAR T-cell therapies by optimizing CAR T-cell costimulation.
Assuntos
Antígenos CD28/antagonistas & inibidores , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Citocinas/biossíntese , Feminino , Humanos , Imunoterapia Adotiva , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fatores de Transcrição NFATC/genética , Células NIH 3T3 , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Receptor de Morte Celular Programada 1/genética , Receptores de Antígenos Quiméricos/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Screening of the NCI Diversity Set-1 identified PI-083 (NSC-45382) a proteasome inhibitor selective for cancer over normal cells. Focused libraries of novel compounds based on PI-083 chloronaphthoquinone and sulfonamide moieties were synthesized to gain a better understanding of the structure-activity relationship responsible for chymotrypsin-like proteasome inhibitory activity. This led to the demonstration that the chloronaphthoquinone and the sulfonamide moieties are critical for inhibitory activity. The pyridyl group in PI-083 can be replaced with other heterocyclic groups without significant loss of activity. Molecular modeling studies were also performed to explore the detailed interactions of PI-083 and its derivatives with the beta5 and beta6 subunits of the 20S proteasome. The refined model showed an H-bond interaction between the Asp-114 and the sulfonamide moiety of the PI-083 in the beta6 subunit.
Assuntos
Naftoquinonas/química , Inibidores de Proteases/síntese química , Inibidores de Proteassoma , Antraciclinas/química , Sítios de Ligação , Simulação por Computador , Ligação de Hidrogênio , Naftoquinonas/síntese química , Naftoquinonas/farmacologia , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/químicaRESUMO
PURPOSE: Tipifarnib is a farnesyl transferase (FTase) inhibitor that has activity in metastatic breast cancer and enhances the efficacy of cytotoxic agents in preclinical models. We evaluated the biological effects of tipifarnib in primary breast cancers in vivo, whether adding tipifarnib to preoperative chemotherapy increased the pathologic complete response rate (pCR) at surgery, and determined whether biomarkers predictive of pCR could be identified. EXPERIMENTAL DESIGN: Forty-four patients with stage IIB-IIIC breast cancer received up to four cycles of neoadjuvant doxorubicin-cyclophosphamide (AC) every 2 weeks plus tipifarnib and filgrastim followed by surgery. Enzymatic assays measuring FTase activity and Western blotting for phospho (p)-signal transducer and activator of transcription 3 (STAT3), phospho-extracellular signal-regulated kinase, p-AKT, and p27 were done in 11 patients who agreed to optional tissue biopsies before therapy and 2 hours after the final dose of tipifarnib during the first cycle, and predictive biomarkers were evaluated by immunohistochemistry in 33 patients. The trial was powered to detect an improvement in breast pCR rate of 10% or less expected for AC alone to 25% for AC-tipifarnib (alpha = 0.05, beta = 0.10). RESULTS: Eleven patients had a breast pCR (25%; 95% confidence interval, 13-40%). FTase enzyme activity decreased in all patients (median, 91%; range, 24-100%) and p-STAT3 expression decreased in 7 of 9 (77%) patients. Low tumor Ki-67 expression (below the median of 60%) at baseline was significantly associated with resistance to therapy (P = 0.01). CONCLUSION: Tipifarnib inhibits FTase activity in human breast tumors in vivo, is associated with down-regulation of p-STAT3, and enhances the breast pCR rate, thus meriting further evaluation.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias da Mama/tratamento farmacológico , Ciclofosfamida/uso terapêutico , Doxorrubicina/uso terapêutico , Farnesiltranstransferase/antagonistas & inibidores , Quinolonas/uso terapêutico , Adulto , Idoso , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Ciclofosfamida/administração & dosagem , Ciclofosfamida/efeitos adversos , Doxorrubicina/administração & dosagem , Doxorrubicina/efeitos adversos , Feminino , Humanos , Antígeno Ki-67/metabolismo , Pessoa de Meia-Idade , Terapia Neoadjuvante , Quinolonas/administração & dosagem , Quinolonas/efeitos adversos , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
PURPOSE: Mutant KRAS is a major driver of pancreatic oncogenesis and therapy resistance, yet KRAS inhibitors are lacking in the clinic. KRAS requires farnesylation for membrane localization and cancer-causing activity prompting the development of farnesyltransferase inhibitors (FTIs) as anticancer agents. However, KRAS becomes geranylgeranylated and active when cancer cells are treated with FTIs. To overcome this geranylgeranylation-dependent resistance to FTIs, we designed FGTI-2734, a RAS C-terminal mimetic dual FT and geranylgeranyltransferase-1 inhibitor (GGTI). EXPERIMENTAL DESIGN: Immunofluorescence, cellular fractionation, and gel shift assays were used to assess RAS membrane association, Western blotting to evaluate FGTI-2734 effects on signaling, and mouse models to demonstrate its antitumor activity. RESULTS: FGTI-2734, but not the selective FTI-2148 and GGTI-2418, inhibited membrane localization of KRAS in pancreatic, lung, and colon human cancer cells. FGTI-2734 induced apoptosis and inhibited the growth in mice of mutant KRAS-dependent but not mutant KRAS-independent human tumors. Importantly, FGTI-2734 inhibited the growth of xenografts derived from four patients with pancreatic cancer with mutant KRAS (2 G12D and 2 G12V) tumors. FGTI-2734 was also highly effective at inhibiting, in three-dimensional cocultures with resistance promoting pancreatic stellate cells, the viability of primary and metastatic mutant KRAS tumor cells derived from eight patients with pancreatic cancer. Finally, FGTI-2734 suppressed oncogenic pathways mediated by AKT, mTOR, and cMYC while upregulating p53 and inducing apoptosis in patient-derived xenografts in vivo. CONCLUSIONS: The development of this novel dual FGTI overcomes a major hurdle in KRAS resistance, thwarting growth of patient-derived mutant KRAS-driven xenografts from patients with pancreatic cancer, and as such it warrants further preclinical and clinical studies.
Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Farnesiltranstransferase/antagonistas & inibidores , Mutação , Neoplasias Pancreáticas/tratamento farmacológico , Proteínas Proto-Oncogênicas p21(ras)/genética , Alquil e Aril Transferases/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Farnesiltranstransferase/metabolismo , Humanos , Masculino , Camundongos , Camundongos SCID , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
An ongoing strategy for cancer treatment is selective induction of apoptosis in cancer over normal cells. N-thiolated beta-lactams were found to induce DNA damage, growth arrest and apoptosis in cultured human cancer cells. However, whether these compounds have a similar effect in vivo has not been studied. We report here that treatment with the beta-lactam L-1 caused a significant inhibition of tumor growth in a breast cancer xenograft mouse model, associated with induction of DNA damage and apoptosis in vivo. These results suggest that the synthetic antibiotic N-thiolated beta-lactams hold great potential to be developed as novel anti-cancer drugs.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Monobactamas/uso terapêutico , Animais , Antibacterianos/farmacologia , Antígenos de Diferenciação/metabolismo , Apoptose , Dano ao DNA , Feminino , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Células Jurkat , Camundongos , Camundongos Nus , Monobactamas/farmacologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Bif-1, a member of the endophilin B protein family, interacts with Bax and promotes interleukin-3 withdrawal-induced Bax conformational change and apoptosis when overexpressed in FL5.12 cells. Here, we provide evidence that Bif-1 plays a regulatory role in apoptotic activation of not only Bax but also Bak and appears to be involved in suppression of tumorigenesis. Inhibition of endogenous Bif-1 expression in HeLa cells by RNA interference abrogated the conformational change of Bax and Bak, cytochrome c release, and caspase 3 activation induced by various intrinsic death signals. Similar results were obtained in Bif-1 knockout mouse embryonic fibroblasts. While Bif-1 did not directly interact with Bak, it heterodimerized with Bax on mitochondria in intact cells, and this interaction was enhanced by apoptosis induction and preceded the Bax conformational change. Moreover, suppression of Bif-1 expression was associated with an enhanced ability of HeLa cells to form colonies in soft agar and tumors in nude mice. Taken together, these findings support the notion that Bif-1 is an important component of the mitochondrial pathway for apoptosis as a novel Bax/Bak activator, and loss of this proapoptotic molecule may contribute to tumorigenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose/fisiologia , Mitocôndrias/fisiologia , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Caspases/metabolismo , Células Cultivadas , Citocromos c/metabolismo , Ativação Enzimática , Fibroblastos/fisiologia , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Neoplasias Experimentais/genética , Conformação Proteica , RNA Interferente Pequeno/metabolismoRESUMO
Mutant KRas is a significant driver of human oncogenesis and confers resistance to therapy, underscoring the need to develop approaches that disable mutant KRas-driven tumors. Because targeting KRas directly has proven difficult, identifying vulnerabilities specific for mutant KRas tumors is an important alternative approach. Here we show that glycogen synthase kinase 3 (GSK3) is required for the in vitro and in vivo growth and survival of human mutant KRas-dependent tumors but is dispensable for mutant KRas-independent tumors. Further, inhibiting phosphorylation of GSK3 substrates c-Myc on T58 and ß-catenin on S33/S37/T41 and their subsequent upregulation contribute to the antitumor activity of GSK3 inhibition. Importantly, GSK3 blockade inhibits the in vivo growth of G12D, G12V, and G12C mutant KRas primary and metastatic patient-derived xenografts from pancreatic cancer patients who progressed on chemo- and radiation therapies. This discovery opens new avenues to target mutant KRas-dependent cancers.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fatores de Transcrição/metabolismo , beta Catenina/metabolismo , Células A549 , Animais , Linhagem Celular Tumoral , Feminino , Genes ras , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Nus , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/terapia , Proteínas Proto-Oncogênicas p21(ras)/genética , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tumor growth and metastasis depend on the formation of blood vessels, angiogenesis, to supply the developing mass with nutrients, oxygen, and waste removal. The proteasome, a massive multisubunit catabolic body, exerts a regulatory influence on angiogenesis. Inhibition of the proteasome activity has been found to inhibit angiogenesis and induce apoptosis in human cancer cells with limited toxicity to normal cells. Therefore, the dual action of angiogenesis inhibition and cell death induction makes proteasome inhibition an attractive modality for chemotherapy. A variety of proteasome inhibitors have been studied including: antibiotics such as lactacystin, the green tea polyphenols, and the boronic acid Velcade (MLN-341). Most recently, certain classes of copper compounds have been found to act as potent proteasome inhibitors. The potential of particular organic compounds, such as 8-hydroxyquinoline, to spontaneously bind with tumor cellular copper and form proteasome inhibitors provides a new modality of anti-proteasome and anti-angiogenesis chemotherapy. This review examines angiogenesis, the proteasome, representative proteasome inhibitors, and the emerging role of copper. The formation of new blood vessels, or angiogenesis, is an important and necessary function in both embryonic development and wound repair. Therefore, the ability to regenerate or form new vessels for blood flow is essential. The control of angiogenic pathways is tightly regulated in normal differentiated adult cells, which generally do not stimulate blood vessel growth unless injury occurs. However, cancerous tissues stimulate angiogenesis that in turn leads to increased tumor formation and possible metastases. Many of the factors involved in angiogenesis are regulated by the proteasome, which recently has become a focus in anti-cancer therapies due to its involvement in cell cycle and apoptosis control. Here we discuss angiogenesis and its relation to the proteasome. Additionally, current modalities of anti-angiogenic treatment, mainly proteasome inhibitory strategies, are reviewed. Furthermore, proteasome inhibitors, both natural and synthetic, and their anti-angiogenic effects as well as future approaches to anti-angiogenic chemotherapies are also discussed.
Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Inibidores de Proteassoma , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Ácidos Borônicos/farmacologia , Bortezomib , Humanos , Neoplasias/irrigação sanguínea , Neoplasias/enzimologia , Neoplasias/prevenção & controle , Neovascularização Patológica/prevenção & controle , Inibidores de Proteases/farmacologia , Pirazinas/farmacologiaRESUMO
Green tea has been shown to lower plasma cholesterol, associated with up-regulation of the low-density lipoprotein receptor (LDLR) although the responsible molecular mechanism is unknown. Previously, we reported that ester bond-containing green tea polyphenols (GTPs), such as (-)-epigallocatechin-3-gallate [(-)-EGCG], potently inhibit the tumor cellular proteasome activity, which may contribute to the cancer-preventative effect of green tea. In the current study, we hypothesize that the proteasome is a heart disease-associated molecular target of GTPs. We have shown that ester bond-containing GTPs, including (-)-EGCG, potently inhibit the proteasomal activity in intact hepatocellular carcinoma HepG2 and cervical carcinoma HeLa cells, as evident by accumulation of ubiquitinated proteins and three natural proteasome targets (p27, IkappaB-alpha and Bax). (-)-EGCG selectively inhibits the chymotrypsin-like, but not trypsin-like, activity of the proteasome. Associated with proteasome inhibition by ester bond-containing GTPs, there was a significant, time- and concentration-dependent increase in levels of the cleaved, activated, but not the precursor, form of sterol regulatory element-binding protein 2 (SREBP-2), an essential factor for LDLR transcription. Subsequently, LDL receptor expression was increased dramatically in HepG2 and HeLa cells treated with (-)-EGCG. Our results suggest that ester bond-containing GTPs inhibit ubiquitin/proteasome-mediated degradation of the active SREBP-2, resulting in up-regulation of LDLR. This identified molecular mechanism may be related to the previously reported cholesterol-lowering and heart disease-preventative effects of green tea.
Assuntos
Catequina/análogos & derivados , Catequina/metabolismo , Cisteína Endopeptidases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Multienzimáticos/metabolismo , Receptores de LDL/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo , Camellia sinensis/metabolismo , Proteínas de Ligação a DNA/genética , Ésteres , Células HeLa , Humanos , Complexos Multienzimáticos/antagonistas & inibidores , Complexo de Endopeptidases do Proteassoma , Receptores de LDL/genética , Proteína de Ligação a Elemento Regulador de Esterol 2 , Fatores de Transcrição/genéticaRESUMO
It has been suggested that proteasome activity is essential for tumor cell proliferation and drug resistance development. We have previously shown that natural and synthetic ester bond-containing tea polyphenols are selective inhibitors of the chymotrypsin-like activity of the proteasome. The most abundant catechin in green tea is (-)-epigallocatechin-3-gallate [(-)-EGCG], which has been found by many laboratories to exhibit the most potent anticancer activity. We have reported that (-)-EGCG is also the most effective proteasome inhibitor among all the natural green tea catechins tested. Unfortunately, (-)-EGCG is very unstable in neutral and alkaline conditions. In an attempt to increase the stability and thus the efficacy, we synthesized several (-)-EGCG analogs with acetyl protected -OH groups as prodrugs. Here we report, for the first time, that these acetylated synthetic tea analogs are much more potent than natural (-)-EGCG in inhibiting the proteasome in cultured tumor cells. Consistently, these protected analogs showed much higher potency than (-)-EGCG to inhibit proliferation and transforming activity and to induce apoptosis in human leukemic, prostate, breast, and simian virus 40-transformed cells. Additionally, these protected analogs had greatly reduced effects on human normal and non-transformed cells. Therefore, these peracetate protected tea polyphenols are more efficacious than (-)-EGCG and possess great potential to be developed into novel anticancer drugs. Identification of the cytosolic metabolite(s) of peracetate-protected polyphenols in cultured tumor cells and examination of their in vivo tumor growth-inhibitory activity are currently underway in our laboratory.
Assuntos
Antineoplásicos/síntese química , Apoptose/efeitos dos fármacos , Flavonoides/síntese química , Fenóis/síntese química , Inibidores de Proteassoma , Chá/química , Antineoplásicos/farmacologia , Apoptose/fisiologia , Catequina/análogos & derivados , Catequina/farmacologia , Flavonoides/farmacologia , Humanos , Peróxido de Hidrogênio/metabolismo , Células Jurkat , Fenóis/farmacologia , Polifenóis , Células Tumorais CultivadasRESUMO
Induction of tumor cell death by chemotherapeutic modalities often occurs in a cell cycle-dependent manner. It has also been observed that several regulatory proteins involved in tumor chemosensitivity and apoptosis are expressed periodically during the cell cycle progression. However, the nature of cancer cellular chemosensitivity and mechanisms of action of anticancer drugs in different phases of the cell cycle still remain unknown. In this chapter we describe two methods (serum deprivation and pharmacological drug treatment) to synchronize human tumor cells in specific phases of the cell cycle, followed by induction of cell death by anticancer drugs. We also describe three methods (flow cytometry, Western blot, and DNA fragmentation) to measure the cell cycle-associated cytotoxic effects of chemotherapeutic agents.
Assuntos
Antineoplásicos/farmacologia , Ciclo Celular , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Apoptose , Western Blotting , Ciclo Celular/efeitos dos fármacos , Meios de Cultura Livres de Soro/farmacologia , DNA/metabolismo , Fragmentação do DNA , Citometria de Fluxo , Fase G1 , HumanosRESUMO
Based on the evidence from epidemiological, animal, in vitro data and human clinical trials, it is evident that isoflavones are promising agents for breast cancer chemoprevention. It is also evident that the form of isoflavone used (purified vs soy products), dose of isoflavone used (low vs high), timing and duration of exposure of isoflavones appears to play a major role in determining agonistic or antagonistic effects. Collectively, these isoflavones have enough evidence to warrant use in a number of clinical trials to examine its efficacy as a potential chemopreventive agent for breast cancer. In this comprehensive review, we attempt to summarize the evidence demonstrating the potential use of isoflavones in breast cancer chemoprevention and the rationale to examine a combination of biochemical, morphological and molecular intermediate endpoint biomarkers of breast cancer risk to examine the efficacy of this promising agent.
Assuntos
Anticarcinógenos/farmacologia , Biomarcadores Tumorais , Neoplasias da Mama/prevenção & controle , Isoflavonas/farmacologia , Animais , Mama/metabolismo , Neoplasias da Mama/epidemiologia , Ensaios Clínicos como Assunto , Feminino , Humanos , Glândulas Mamárias Animais/metabolismo , Extratos Vegetais/uso terapêutico , Complexo de Endopeptidases do Proteassoma/metabolismo , Risco , Glycine max/metabolismo , Esteroides/metabolismoRESUMO
The discovery of natural and synthetic antibiotics is one of the most important medical breakthroughs in human history. Many diseases, such as bacterial meningitis, pneumonia, and septicemia, are now curable with the use of antibiotics. Antibiotics are efficacious, generally well tolerated in patients, and have a low toxicity level. It is for these reasons antibiotics remain an attractive target for drug discovery. Traditional beta-lactam antibiotics (e.g. penicillins, penems, cephalosporins) have a bicyclic ring structure that is conformationally rigid and functions to inhibit bacterial cell wall synthesis. In addition to the bactericidal action of antibiotics, it has been discovered that many antibiotics are capable of inhibiting tumor cell growth. There are currently many antitumor antibiotics approved for cancer therapy, which work to inhibit tumor cell growth by DNA intercalation. The use of beta-lactams as prodrugs has also met with success by aiding delivery of the chemotherapeutic directly to tumor sites. Recently, a novel class of N-thiolated monobactams, so termed because they possess a monocyclic ring instead of the bicyclic ring, has been found to induce apoptosis potently and specifically in many tumor cell lines but not in normal, non-transformed cell lines. Other beta-lactams, such as the polyaromatics, have been found to slow or inhibit tumor cell growth, and the 4-alkylidene beta-lactams are capable of inhibiting matrix metalloproteinases and leukocyte elactase activity. These data indicate that synthesis and evaluation of beta-lactams are a promising area for further development in anticancer research.