RESUMO
INTRODUCTION: Integrating palliative care (PC) early in the illness course for patients with serious cancers improves their outcomes and is recommended by national organisations such as the American Society of Clinical Oncology. However, monthly visits with PC clinicians from the time of diagnosis can be challenging to implement due to the lack of specialty-trained PC clinicians and resources. Therefore, we developed a stepped care model to triage PC service based on patients' needs. METHODS AND ANALYSIS: We are conducting a non-blinded, randomised trial to evaluate the non-inferiority of a stepped PC model compared with an early integrated PC model for improving patients' quality of life (QOL) at 24 weeks (primary outcome). Patients assigned to early integrated PC meet with PC every 4 weeks throughout their illness. Patients assigned to stepped PC have PC visits only at clinically significant points in their illness (eg, cancer progression) unless their QOL decreases, at which time they are 'stepped up' and meet with PC every 4 weeks throughout the remainder of their illness. Secondary aims include assessing whether stepped PC is non-inferior to early integrated PC regarding patient-clinician communication about end of life care and length of stay on hospice as well as comparing resource utilisation. Patients are recruited from the Massachusetts General Hospital Cancer Center, Boston, Massachusetts; Duke Cancer Center, Durham, North Carolina and University of Pennsylvania Abramson Cancer Center, Philadelphia, Pennsylvania. The target sample size is 510 patients. ETHICS AND DISSEMINATION: The study is funded by the National Cancer Institute, approved by the Dana-Farber/Harvard Cancer Center Institutional Review Board and will be reported in accordance with the Consolidated Standards of Reporting Trials statement. We will disseminate results through professional society meetings, peer-reviewed publications and presentations to patient organisations. TRIAL REGISTRATION NUMBER: NCT03337399.
Assuntos
Enfermagem de Cuidados Paliativos na Terminalidade da Vida , Neoplasias Pulmonares , Assistência Terminal , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/terapia , Cuidados Paliativos/métodos , Qualidade de Vida , Ensaios Clínicos Controlados Aleatórios como Assunto , Assistência Terminal/métodosRESUMO
PURPOSE: Insured patients with cancer face high treatment-related, out-of-pocket (OOP) costs and often cannot access financial assistance. We conducted a randomized, controlled trial of Bridge, a patient-facing app designed to identify eligible financial resources for patients. We hypothesized that patients using Bridge would experience greater OOP cost reduction than controls. METHODS: We enrolled patients with cancer who had OOP expenses from January 2018 to March 2019. We randomly assigned patients 1:1 to intervention (Bridge) versus control (financial assistance educational websites). Primary and secondary outcomes were self-reported OOP costs and subjective financial distress 3 months postenrollment. In post hoc analyses, we analyzed application for and receipt of financial assistance at 3 months postenrollment. We used chi-square, Mann-Whitney tests, and logistic regression to compare study arms. RESULTS: We enrolled 200 patients. The median age was 57 years (IQR, 47.0-63.0). Most patients had private insurance (71%), and the median household income was $62,000 in US dollars (USD) (IQR, $36,000-$100,000 [USD]). Substantial missing data precluded assessment of primary and secondary outcomes. In post hoc analyses, patients in the Bridge arm were more likely than controls to both apply for and receive financial assistance. CONCLUSION: We were unable to test our primary outcome because of excessive missing follow-up survey data. In exploratory post hoc analyses, patients who received a financial assistance app were more likely to apply for and receive financial assistance. Ultimately, our study highlights challenges faced in identifying measurable outcomes and retaining participants in a randomized, controlled trial of a mobile app to alleviate financial toxicity.
Assuntos
Aplicativos Móveis , Neoplasias , Gastos em Saúde , Humanos , Renda , Pessoa de Meia-Idade , Neoplasias/terapia , Inquéritos e QuestionáriosRESUMO
Patients with advanced cancer and their families frequently encounter clinical and logistical challenges related to end-of-life care. Hospice provides interdisciplinary and holistic care to meet patients' biomedical, psychosocial, and spiritual needs in the last phases of life. Despite increasing general acceptance and use among patients with cancer, hospice remains underused. Underuse stems from ongoing misconceptions regarding hospice and its purpose, coupled with the rapid development of novel anticancer treatments, such as immunotherapies and targeted therapies, that have changed the landscape of possibilities. Furthermore, rapid evolutions in how end-of-life care is structured and reimbursed for will affect how oncology patients will intersect with hospice care. In this review, we explore the current and future challenges to greater integration of hospice care in the care of patients with advanced cancer and propose five recommendations as part of the path forward.
Assuntos
Cuidados Paliativos na Terminalidade da Vida , Hospitais para Doentes Terminais , Neoplasias , Assistência Terminal , Humanos , Estudos Interdisciplinares , Neoplasias/terapiaRESUMO
CONTEXT: Low utilization of palliative care services warrant testing of new solutions to educate and engage patients around the benefits of palliative care. OBJECTIVES: We sought out to develop and test a novel, mobile health solution to prepare patients for an upcoming outpatient palliative care appointment. METHODS: After developing a web-based tool called PCforMe (Palliative Care for Me), we conducted a randomized, active-controlled, trial of PCforMe. The primary outcome was the score on the System Usability Scale (SUS). Secondary outcomes were patient self-efficacy and change in knowledge. We compared PCforMe to three common online resources for patients seeking information about palliative care. RESULTS: A total of 80 patients were randomized. There were no significant demographic differences. Mean SUS score for PCforMe was 78.2, significantly above the normative average SUS score of 68 (P-value < 0.0001). Mean change in Perceived Efficacy in Patient-Physician Interactions score was -2.2 for PCforMe and -1.7 for control group (P-value = 0.72). Preparedness for an upcoming palliative care visit increased 50% in the intervention group and 13.3% in the control group. Difference in the number of patients with improved knowledge regarding palliative care approached significance (P = 0.06). Lastly, we found that the no-show rate was lower during Q1 2017 (during trial) and Q1 2016 (before trial), at 11.7% and 21%, respectively (P < 0.05). Comparing the full calendar year (CY) 2016 with 2017, we did not find a statistical difference (CY 2016 of 18.8% and 15% in CY 2017; P = 0.22). CONCLUSION: PCforMe is a usable mobile health tool to prepare patients for an upcoming palliative care appointment. Further research is needed to test effectiveness.
Assuntos
Pacientes Ambulatoriais , Cuidados Paliativos , Aceitação pelo Paciente de Cuidados de Saúde , Telemedicina , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Adulto JovemRESUMO
PURPOSE: We investigated the effects of two peptides from the ras-p21 protein, corresponding to residues 35-47 (PNC-7) and 96-110 (PNC-2), on two ras-transformed human cancer cell lines, HT1080 fibrosarcoma and MIAPaCa-2 pancreatic cancer cell lines. In prior studies, we found that both peptides block oncogenic, but not insulin-activated wild-type, ras-p21-induced oocyte maturation. When linked to a transporter penetratin peptide, these peptides induce reversion of ras-transformed rat pancreatic cancer cells (TUC-3) to the untransformed phenotype. METHODS: These peptides and a control peptide, linked to a penetratin peptide, were incubated with each cell lines. Cell counts were obtained over several weeks. The cause of cell death was determined by measuring caspase as an indicator of apoptosis and lactate dehydrogenase (LDH) as marker of necrosis. Since both peptides block the phosphorylation of jun-N-terminal kinase (JNK) in oocytes, we blotted cell lysates of the two cancer cell lines for the levels of phosphorylated JNK to determine if the peptides reduced these levels. RESULTS: We find that both peptides, but not control peptides linked to the penetratin sequence, induce phenotypic reversion of the HT-1080 cell line but cause tumor cell necrosis of the MIA-PaCa-2 cell line. On the other hand, neither peptide has any effect on the viability of an untransformed pancreatic acinar cell line, BMRPA1. We find that, while total JNK levels remain constant during peptide treatment, phosphorylated JNK levels decrease dramatically, consistent with the mechanisms of action of these peptides. CONCLUSION: We conclude that these peptides block tumor but not normal cell growth likely by blocking oncogenic ras-p21-induced phosphorylation of JNK, an essential step on the oncogenic ras-p21-protein pathway. These peptides are therefore promising as possible anti-tumor agents.
Assuntos
Antineoplásicos/farmacocinética , Transformação Celular Neoplásica/efeitos dos fármacos , Proteína Oncogênica p21(ras)/fisiologia , Fragmentos de Peptídeos/farmacologia , Antineoplásicos/química , Caspases/biossíntese , Técnicas de Cultura de Células , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/biossíntese , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Necrose , Proteína Oncogênica p21(ras)/química , Fragmentos de Peptídeos/química , FosforilaçãoRESUMO
In a study of interactions between the raf-MEK-MAPK (ERK) and JNK-jun pathways, we found previously that JNK can induce phosphorylation of raf but not vice versa. In this study, we investigate the nature of the JNK-induced phosphorylation of raf. In in vitro experiments in which immunobead-bound raf is phosphorylated by activated JNK, we find strong phosphorylation signals at raf-Ser259 and Ser338. The Ser259 phosphorylation is surprising since it is associated with inhibition of migration of raf to the cell membrane where it can interact with ras-p21. We also find that in oocytes induced to mature with oncogenic ras-p21, which induces high levels of phosphorylated JNK and MAPK, the same pattern of phosphorylation of raf occurs. In contrast, in oocytes induced to mature with insulin, which requires activation of wild-type ras-p21, phosphorylation of raf-Ser338 but not raf-Ser259 occurs. In oncogenic ras-transformed human pancreatic cancer MIA-PaCa-2 cells, phosphorylation of both raf serines occurs. Treatment of these cells with the ras peptide, PNC-2 attached to a penetrating sequence that blocks JNK and MAPK phosphorylation and induces tumor cell necrosis, results in a marked decrease in phosphorylation of raf-Ser259, but not that of raf-Ser338. These results suggest that oncogenic ras-p21 induces phosphorylation of both raf-Ser259 and Ser338 and that raf-Ser 259 phosphorylation may be effected by activated JNK.
Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteína Oncogênica p21(ras)/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Animais , Especificidade de Anticorpos , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Ativação Enzimática , Humanos , Modelos Biológicos , Neoplasias Pancreáticas/metabolismo , Fosfopeptídeos/metabolismo , Fosforilação , XenopusRESUMO
We have previously found that oncogenic ras-p21 and insulin, which activates wild-type ras-21 protein, both induce Xenopus laevis oocyte maturation that is dependent on activation of raf. However, oncogenic ras-p21 utilizes raf-dependent activation of the two classic raf targets, MEK and MAP kinase (MAPK or ERK) while insulin-activated wild-type ras-p21 does not depend on activation of these two kinases. Utilizing a microarray containing the entire Xenopus genome, we discovered two dual specificity kinases, T-Cell Origin Protein Kinase (TOPK), known to bind to raf and the nuclear kinase, DYRK1A, that are expressed at much higher levels in insulin-matured oocytes. Using SiRNA's directed against expression of both of these proteins, we now show that each inhibits insulin-but not oncogenic ras-p21-induced oocyte maturation. Control siRNA's have no effect on either agent in induction of maturation. We find that each SiRNA "knocks down" expression of its target protein while not affecting expression of the other protein. These results suggest that both proteins are required for maturation induced by wild-type, but not oncogenic, ras-p21. They also suggest that oncogenic and wild-type ras-p21 utilize pathways that become divergent downstream of raf. On the basis of these findings, we propose a model for two signal transduction pathways by oncogenic and activated wild-type ras-p21 showing points of overlap and divergence.
Assuntos
Oócitos/enzimologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas de Xenopus/fisiologia , Animais , Insulina/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteína Oncogênica p21(ras)/metabolismo , Oócitos/crescimento & desenvolvimento , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Interferência de RNA , Transdução de Sinais , Especificidade por Substrato , Proteínas de Xenopus/antagonistas & inibidores , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
Among the members of the cytochrome P450 superfamily, P450 2E1 is most often associated with the production of reactive oxygen species and subsequent cellular toxicity. We sought to identify a structural basis for this distinguishing feature of P450 2E1 by examining its carbon monoxide binding kinetics as a probe of conformation/dynamics. We employed liver microsomes from wild-type and P450 2E1 knockout mice in order to characterize this P450 in a natural membrane environment. The CO binding kinetics of the P450s of wild-type microsomes had a rapid component that was absent in the knockout microsomes. Data analysis using the maximum entropy method (MEM) correspondingly identified two distinct kinetic components in the wild-type microsomes and only one component in the knockout microsomes. The rapid kinetic component in wild-type microsomes was attributed to endogenous P450 2E1, while the slower component was derived from the remaining P450s. In addition, rapid binding kinetics and a single component were also observed for human P450 2E1 in a baculovirus expression system, in the absence of other P450s. Binding kinetics of both mouse and human P450 2E1 were slowed in the presence of ethanol, a modulator of this P450. The unusually rapid CO binding kinetics of P450 2E1 indicate that it is more dynamically mobile than other P450s and thus able to more readily interconvert among alternate conformations. This suggests that conformational switching during the catalytic cycle may promote substrate release from a short-lived binding site, allowing activated oxygen to attack other targets with toxic consequences.
Assuntos
Citocromo P-450 CYP2E1/química , Membranas Intracelulares/enzimologia , Animais , Baculoviridae/enzimologia , Baculoviridae/genética , Sítios de Ligação/genética , Monóxido de Carbono/química , Monóxido de Carbono/metabolismo , Citocromo P-450 CYP2E1/deficiência , Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP2E1/metabolismo , Vetores Genéticos , Humanos , Membranas Intracelulares/metabolismo , Isoenzimas/química , Isoenzimas/deficiência , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Ligação Proteica/genética , Conformação Proteica , Especificidade por Substrato/genéticaRESUMO
CONTEXT: Measuring What Matters (MWM) for palliative care has prioritized data collection efforts for evaluating quality in clinical practice. How these measures can be implemented across diverse clinical settings using point-of-care data collection on quality is unknown. OBJECTIVES: To evaluate the implementation of MWM measures by exploring documentation of quality measure adherence across six diverse clinical settings inherent to palliative care practice. METHODS: We deployed a point-of-care quality data collection system, the Quality Data Collection Tool, across five organizations within the Palliative Care Research Cooperative Group. Quality measures were recorded by clinicians or assistants near care delivery. RESULTS: During the study period, 1989 first visits were included for analysis. Our population was mostly white, female, and with moderate performance status. About half of consultations were seen on hospital general floors. We observed a wide range of adherence. The lowest adherence involved comprehensive assessments during the first visit in hospitalized patients in the intensive care unit (2.71%); the highest adherence across all settings, with an implementation of >95%, involved documentation of management of moderate/severe pain. We observed differences in adherence across clinical settings especially with MWM Measure #2 (Screening for Physical Symptoms, range 45.7%-81.8%); MWM Measure #5 (Discussion of Emotional Needs, range 46.1%-96.1%); and MWM Measure #6 (Documentation of Spiritual/Religious Concerns, range 0-69.6%). CONCLUSION: Variations in clinician documentation of adherence to MWM quality measures are seen across clinical settings. Additional studies are needed to better understand benchmarks and acceptable ranges for adherence tailored to various clinical settings.
Assuntos
Cuidados Paliativos na Terminalidade da Vida/normas , Cuidados Paliativos/normas , Sistemas Automatizados de Assistência Junto ao Leito , Garantia da Qualidade dos Cuidados de Saúde , Estudos Transversais , Coleta de Dados , Feminino , Implementação de Plano de Saúde , Cuidados Paliativos na Terminalidade da Vida/métodos , Humanos , Masculino , Estudos ProspectivosRESUMO
CONTEXT: Measuring What Matters (MWM) prioritizes quality measures in palliative care practice. Hematologic malignancy patients are less likely to access palliative care, yet little is known about their unique needs. Differences in MWM adherence may highlight opportunities to improve palliative care in hematology. OBJECTIVES: To assess adherence to MWM measures by palliative care clinicians caring for patients with hematologic malignancies, compared to those with solid tumors. METHODS: We used the Quality Data Collection Tool to assess completion of MWM measures across nine sites. RESULTS: We included data from 678 patients' first visits and various care settings; 64 (9.4%) had a hematologic malignancy, whereas 614 (90.6%) had a solid tumor. Hematology patients were more likely to be seen in a hospital (52 or 81.3% vs. 420 or 68%), whereas solid tumor patients were more frequently seen at home or in clinics (160 or 26% vs. 7 or 10.9%). Of the nine MWM measures we assessed, high adherence (>90%) was seen regardless of tumor type in measures #3 (Pain Treatment), #7 (Spiritual Concerns), #8 (Treatment Preferences), and #9 (Care Consistent With Preferences). Clinicians seeing hematology patients were significantly less likely to meet measures #2 (Screening for Physical Symptoms; 57.8% vs. 84.2%, P < 0.001), and #5 (Discussion of Emotional Needs; 56.3% vs. 70.0%, P = 0.03). CONCLUSION: MWM adherence regarding symptom assessment and meeting emotional needs was lower for patients with hematologic malignancies compared to those with solid tumors. This finding suggests two key areas for quality improvement initiatives in palliative care for patients with hematologic malignancies.
Assuntos
Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/terapia , Cuidados Paliativos , Estudos Transversais , Emoções , Feminino , Neoplasias Hematológicas/psicologia , Humanos , Masculino , Médicos , Estudos Prospectivos , Qualidade da Assistência à Saúde , Sistema de Registros , Resultado do TratamentoRESUMO
p53 is the most frequently altered gene in human cancer and therefore represents an ideal target for cancer therapy. Several amino terminal p53-derived synthetic peptides were tested for their antiproliferative effects on breast cancer cell lines MDA-MB-468 (mutant p53), MCF-7 (overexpressed wild-type p53), and MDA-MB-157 (null p53). p53(15)Ant peptide representing the majority of the mouse double minute clone 2 binding site on p53 (amino acids 12-26) fused to the Drosophila carrier protein Antennapedia was the most effective. p53(15)Ant peptide induced rapid, nonapoptotic cell death resembling necrosis in all breast cancer cells; however, minimal cytotoxicity was observed in the nonmalignant breast epithelial cells MCF-10-2A and MCF-10F. Bioinformatic/biophysical analysis utilizing hydrophobic moment and secondary structure predictions as well as circular dichroism spectroscopy revealed an alpha-helical hydrophobic peptide structure with membrane disruptive potential. Based on these findings, p53(15)Ant peptide may be a novel peptide cancer therapeutic because it induces necrotic cell death and not apoptosis, which is uncommon in traditional cancer therapy.
Assuntos
Neoplasias da Mama/patologia , Proteínas Nucleares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição , Proteína Supressora de Tumor p53/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Sequência de Aminoácidos , Animais , Proteína do Homeodomínio de Antennapedia , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Sítios de Ligação , Mama/citologia , Mama/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Caspases/efeitos dos fármacos , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Dicroísmo Circular , Inibidores de Cisteína Proteinase/farmacologia , Células Epiteliais/efeitos dos fármacos , Feminino , Proteínas de Homeodomínio/genética , Humanos , L-Lactato Desidrogenase/efeitos dos fármacos , L-Lactato Desidrogenase/metabolismo , Camundongos , Dados de Sequência Molecular , Mutação , Necrose , Conformação Proteica , Proteínas Proto-Oncogênicas c-mdm2 , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genéticaRESUMO
The cytochrome P450 enzymes collectively metabolize a wide range of xenobiotic and endogenous compounds. The broad substrate specificity of this superfamily derives from the multiplicity of P450s whose unique substrate specificity profiles reflect underlying differences in primary sequence. Experimental structures of P450s, where available, have provided great insight into the basis of substrate recognition. However, for those mammalian P450s whose structures have not been determined, homology modeling has become an increasingly important tool for understanding substrate specificity and mechanism. P450 modeling is often a challenging task, owing to the rather low sequence identity between target and template proteins. Although mammalian P450 models have previously been based on bacterial P450 structures, the recent advent of mammalian P450 structures holds great potential for generating more accurate homology models. Consequently, the substrate recognition properties of several mammalian P450s have been rationalized using the predicted substrate binding site of recently developed models. This review summarizes the major concepts and current approaches of molecular modeling of P450s.
Assuntos
Sistema Enzimático do Citocromo P-450/química , Algoritmos , Animais , Sítios de Ligação , Biologia Computacional/métodos , Simulação por Computador , Sequência Conservada , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Modelos Moleculares , Modelos Teóricos , Conformação Molecular , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Software , Especificidade por SubstratoRESUMO
1. Cytochrome P4501A2 (CYP1A2) activates a large number of procarcinogens to carcinogens. Phytochemicals such as flavones can inhibit CYP1A2 activity competitively, and hydroxylated derivatives of flavone (galangin) may be potent, selective inhibitors of CYP1A2 activity relative to CYP1A1 activity. Molecular modelling of the CYP1A2 interaction with hydroxylated derivatives of flavone suggests that a number of hydrophobic residues of the substrate-binding domain engage in hydrogen bonding with such inhibitors. 2. We have tested this model using site-directed mutagenesis of these residues in expression plasmids transfected into the human B-lymphoblastoid cell line, AHH-1 TK+/-. 3. Consistent with the molecular model's predicted placement in the active site, amino acid substitutions at the predicted residues abolished CYP1A2 enzymatic activity. 4. Transfected cell lines contained equal amounts of immunoreactive CYP1A2. 5. Our results support the molecular model's prediction of the critical amino acid residues present in the hydrophobic active site, residues that can hydrogen bond with CYP1A2 inhibitors and modify substrate binding and/or turnover.
Assuntos
Arginina/genética , Citocromo P-450 CYP1A2/metabolismo , Lisina/genética , Oxazinas/metabolismo , Substituição de Aminoácidos , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/genética , Sistema Enzimático do Citocromo P-450/metabolismo , DNA Complementar/metabolismo , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oxirredutases/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Tumorais CultivadasRESUMO
PURPOSE: From molecular modeling studies we found that two ras-p21 peptides, corresponding to p21 residues 35-47 (PNC-7) and 96-110 (PNC-2), selectively block oncogenic (Val 12-p21), but not insulin-activated wild-type, p21-induced oocyte maturation. Our purpose was to determine if these peptides block the growth of mammalian cancer cells but not their normal counterpart cells. METHODS: Since oncogenic ras has been implicated as a causative factor in over 90% of human pancreatic cancers, we have established a normal pancreatic acinar cell line (BMRPA1) and the corresponding ras-transformed pancreatic cancer cell line (TUC-3). We treated both cell lines with PNC-7 and PNC-2 and the unrelated negative control peptide, X13, attached to the penetratin sequence that allows membrane penetration and also transfected these cell lines with plasmids encoding all three peptides. RESULTS: Treatment of TUC-3 cells with each peptide resulted in their complete phenotypic reversion to the untransformed phenotype as revealed by the lack of tumor formation of these revertant cells implanted in the peritoneal cavities of nude mice. In contrast, treatment with X13-leader resulted in no inhibition of cell growth. Identical results were obtained when TUC-3 cells were transfected with plasmids expressing PNC-2, PNC-7 and X13. None of these peptides affected the normal growth of BMRPA1 cells. CONCLUSIONS: PNC-2 and PNC-7 peptides induce phenotypic reversion of ras-induced pancreatic cancer cells and have no effect on normal pancreatic cell growth. Since the plasmid encoding PNC-2 without penetratin also had the same effect on the TUC-3 cell line, we conclude that the penetratin sequence has no effect on the activity of this peptide. Since X13 attached to penetratin had no effect on TUC-3 cells, the effect is specific for PNC-2 and PNC-7 and further confirms that the effect is not caused by the penetratin sequence. PNC-2- and PNC-7-penetratin may therefore be useful in the treatment of ras-induced pancreatic carcinomas.
Assuntos
Modelos Moleculares , Proteína Oncogênica p21(ras) , Neoplasias Pancreáticas/genética , Peptídeos/uso terapêutico , Células Tumorais Cultivadas/efeitos dos fármacos , Sequência de Bases , Humanos , Proteína Oncogênica p21(ras)/análogos & derivados , Proteína Oncogênica p21(ras)/antagonistas & inibidores , FenótipoRESUMO
ras-p21 protein binds to the son-of-sevenless (SOS) guanine nucleotide-exchange promoter that allows it to exchange GDP for GTP. Previously, we performed molecular dynamics calculations on oncogenic (Val 12-) and wild-type ras-p21 bound to SOS. By superimposing the average structures of these two complexes, we identified four domains (residues 631-641, 676-691, 718-729, and 994-1004) in SOS that change conformation and were candidates for being effector domains. These calculations were performed in the absence of three crystallographically undefined loops (i.e., residues 591-596, 654-675, and 742-751). We have now modeled these loops into the SOS structure and have re-performed the dynamics calculations. We find that all three loop domains undergo large changes in conformation that involve mostly changes in their positioning and not their individual conformations. We have also identified another potential effector domain (i.e., residues 980-989). Overall, our current results suggest that SOS interactions with oncogenic ras-p21 may enhance ras-p21 mitogenic signaling through prolonging its activation by maintaining its binding to GTP and by allowing its effector domains to interact with intracellular targets.
Assuntos
Proteína Oncogênica p21(ras)/química , Proteínas Son Of Sevenless/química , Animais , Sítios de Ligação , Humanos , Proteína Oncogênica p21(ras)/metabolismo , Estrutura Terciária de Proteína/fisiologia , Transdução de Sinais/fisiologia , Proteínas Son Of Sevenless/metabolismoRESUMO
Oncogenic ras-p21 directly activates jun-N-terminal kinase (JNK) and its substrate, jun as a unique step on its mitogenic signal transduction pathway. This activation is blocked by the specific JNK-jun inhibitor, glutathione-S-transferase-pi (GST-pi). Four domains of GST-pi have been implicated in this regulatory function: 34-50, 99-121, 165-182, and 194-201. The 34-50 domain is unique in that it does not affect GST-pi binding to JNK-jun but blocks jun phosphorylation by JNK. We now find that it completely blocks oncogenic (Val 12-) ras-p21-induced oocyte maturation but has no effect on insulin-induced oocyte maturation. Because the latter process requires activation of wild-type ras-p21, this peptide appears to be specific for inhibiting only the oncogenic form of ras-p21, suggesting its use in treating ras-induced tumors.
Assuntos
Glutationa Transferase/metabolismo , Isoenzimas/metabolismo , Mitose/fisiologia , Proteína Oncogênica p21(ras)/antagonistas & inibidores , Peptídeos/metabolismo , Transdução de Sinais/fisiologia , Animais , Feminino , Glutationa S-Transferase pi , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Neoplasias/metabolismo , Oócitos/metabolismo , XenopusRESUMO
In the accompanying paper, we found, using molecular dynamics calculations, four domains of the ras-specific SOS guanine nucleotide exchange protein (residues 589-601, 654-675, 746-761, and 980-989) that differ markedly in conformation when SOS is complexed with either oncogenic (Val 12-) ras-p21 or wild-type ras-p21. Three of these domains contain three crystallographically undefined loops that we modeled in these calculations, and one is a newly identified non-loop domain containing SOS residues 980-989. We have now synthesized peptides corresponding to these four domains and find that all of them block Val 12-ras-p21-induced oocyte maturation. All of them also block insulin-induced oocyte maturation, but two of these peptides, corresponding to SOS residues 589-601 and 980-989, block oncogenic ras to a significantly greater extent. These results suggest that SOS contains domains, including the three loop domains, that are important for ras signaling and that several of these domains can activate different pathways specific to oncogenic or wild-type ras-p21.
Assuntos
Biologia Computacional , Fragmentos de Peptídeos/metabolismo , Transdução de Sinais/fisiologia , Proteínas Son Of Sevenless/metabolismo , Proteínas ras/metabolismo , Animais , Feminino , Oócitos/metabolismo , Fragmentos de Peptídeos/química , Estrutura Terciária de Proteína , Proteínas Son Of Sevenless/química , Xenopus , Proteínas ras/químicaRESUMO
Cytochrome P450 2E1 (CYP2E1) is a key enzyme in the metabolic activation of many low molecular weight toxicants and also an important contributor to oxidative stress. A noninvasive method to monitor CYP2E1 activity in vivo would be of great value for studying the role of CYP2E1 in chemical-induced toxicities and stress-related diseases. In this study, a mass spectrometry-based metabolomic approach was used to identify a metabolite biomarker of CYP2E1 through comparing the urine metabolomes of wild-type (WT), Cyp2e1-null, and CYP2E1-humanized mice. Metabolomic analysis with multivariate models of urine metabolites revealed a clear separation of Cyp2e1-null mice from WT and CYP2E1-humanized mice in the multivariate models of urine metabolomes. Subsequently, 2-piperidone was identified as a urinary metabolite that inversely correlated to the CYP2E1 activity in the three mouse lines. Backcrossing of WT and Cyp2e1-null mice, together with targeted analysis of 2-piperidone in mouse serum, confirmed the genotype dependency of 2-piperidone. The accumulation of 2-piperidone in the Cyp2e1-null mice was mainly caused by the changes in the biosynthesis and degradation of 2-piperidone because compared with the WT mice, the conversion of cadaverine to 2-piperidone was higher, whereas the metabolism of 2-piperidone to 6-hydroxy-2-piperidone was lower in the Cyp2e1-null mice. Overall, untargeted metabolomic analysis identified a correlation between 2-piperidone concentrations in urine and the expression and activity of CYP2E1, thus providing a noninvasive metabolite biomarker that can be potentially used in to monitor CYP2E1 activity.