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1.
Cell Chem Biol ; 30(10): 1235-1247.e6, 2023 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-37531956

RESUMO

Targeting transcription replication conflicts, a major source of endogenous DNA double-stranded breaks and genomic instability could have important anticancer therapeutic implications. Proliferating cell nuclear antigen (PCNA) is critical to DNA replication and repair processes. Through a rational drug design approach, we identified a small molecule PCNA inhibitor, AOH1996, which selectively kills cancer cells. AOH1996 enhances the interaction between PCNA and the largest subunit of RNA polymerase II, RPB1, and dissociates PCNA from actively transcribed chromatin regions, while inducing DNA double-stranded breaks in a transcription-dependent manner. Attenuation of RPB1 interaction with PCNA, by a point mutation in RPB1's PCNA-binding region, confers resistance to AOH1996. Orally administrable and metabolically stable, AOH1996 suppresses tumor growth as a monotherapy or as a combination treatment but causes no discernable side effects. Inhibitors of transcription replication conflict resolution may provide a new and unique therapeutic avenue for exploiting this cancer-selective vulnerability.


Assuntos
Cromatina , Neoplasias , Humanos , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Neoplasias/tratamento farmacológico , DNA , Replicação do DNA
2.
Genes (Basel) ; 14(7)2023 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-37510250

RESUMO

This article reviews the currently used therapeutic strategies to target DNA replication stress for cancer treatment in the clinic, highlighting their effectiveness and limitations due to toxicity and drug resistance. Cancer cells experience enhanced spontaneous DNA damage due to compromised DNA replication machinery, elevated levels of reactive oxygen species, loss of tumor suppressor genes, and/or constitutive activation of oncogenes. Consequently, these cells are addicted to DNA damage response signaling pathways and repair machinery to maintain genome stability and support survival and proliferation. Chemotherapeutic drugs exploit this genetic instability by inducing additional DNA damage to overwhelm the repair system in cancer cells. However, the clinical use of DNA-damaging agents is limited by their toxicity and drug resistance often arises. To address these issues, the article discusses a potential strategy to target the cancer-associated isoform of proliferating cell nuclear antigen (caPCNA), which plays a central role in the DNA replication and damage response network. Small molecule and peptide agents that specifically target caPCNA can selectively target cancer cells without significant toxicity to normal cells or experimental animals.


Assuntos
Neoplasias , Animais , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Replicação do DNA , Dano ao DNA , Oncogenes
3.
Cells ; 10(11)2021 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-34831131

RESUMO

Peptides are increasingly being developed for use as therapeutics to treat many ailments, including cancer. Therapeutic peptides have the advantages of target specificity and low toxicity. The anticancer effects of a peptide can be the direct result of the peptide binding its intended target, or the peptide may be conjugated to a chemotherapy drug or radionuclide and used to target the agent to cancer cells. Peptides can be targeted to proteins on the cell surface, where the peptide-protein interaction can initiate internalization of the complex, or the peptide can be designed to directly cross the cell membrane. Peptides can induce cell death by numerous mechanisms including membrane disruption and subsequent necrosis, apoptosis, tumor angiogenesis inhibition, immune regulation, disruption of cell signaling pathways, cell cycle regulation, DNA repair pathways, or cell death pathways. Although using peptides as therapeutics has many advantages, peptides have the disadvantage of being easily degraded by proteases once administered and, depending on the mode of administration, often have difficulty being adsorbed into the blood stream. In this review, we discuss strategies recently developed to overcome these obstacles of peptide delivery and bioavailability. In addition, we present many examples of peptides developed to fight cancer.


Assuntos
Neoplasias/tratamento farmacológico , Peptídeos/uso terapêutico , Peptídeos Penetradores de Células/farmacologia , Humanos , Modelos Biológicos , Nanopartículas/química , Peptídeos/farmacologia , Antígeno Nuclear de Célula em Proliferação/metabolismo
4.
Mol Ther Oncolytics ; 17: 250-256, 2020 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-32368614

RESUMO

Pancreatic ductal adenocarcinoma is a particularly difficult cancer to treat due to a lack of effective screening or treatment. Pancreatic cancer cells exhibit high proliferating cell nuclear antigen (PCNA) expression, which is associated with poor prognosis. PCNA, an important nuclear DNA replication and repair protein, regulates a myriad of proteins via the interdomain connector loop. Within this region, amino acids 126-133 are critical for PCNA interactions in cancer cells. Here, we investigate the ability of a decoy cell-penetrating peptide, R9-caPeptide, that mimics the interdomain connector loop region of PCNA to disrupt PCNA-protein interactions in pancreatic cancer cells. Our data suggest that R9-caPeptide causes dose-dependent toxicity in a panel of pancreatic cancer cell lines by inhibiting DNA replication fork progression and PCNA-regulated DNA repair, ultimately causing lethal DNA damage. Overall, these studies lay the foundation for novel therapeutic strategies that target PCNA in pancreatic cancer.

5.
Clin Cancer Res ; 24(23): 6053-6065, 2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-29967249

RESUMO

PURPOSE: Proliferating cell nuclear antigen (PCNA) plays an essential role in regulating DNA synthesis and repair and is indispensable to cancer cell growth and survival. We previously reported a novel cancer associated PCNA isoform (dubbed caPCNA), which was ubiquitously expressed in a broad range of cancer cells and tumor tissues, but not significantly in nonmalignant cells. We found the L126-Y133 region of caPCNA is structurally altered and more accessible to protein-protein interaction. A cell-permeable peptide harboring the L126-Y133 sequence blocked PCNA interaction in cancer cells and selectively kills cancer cells and xenograft tumors. On the basis of these findings, we sought small molecules targeting this peptide region as potential broad-spectrum anticancer agents. EXPERIMENTAL DESIGN: By computer modeling and medicinal chemistry targeting a surface pocket partly delineated by the L126-Y133 region of PCNA, we identified a potent PCNA inhibitor (AOH1160) and characterized its therapeutic properties and potential toxicity. RESULTS: AOH1160 selectively kills many types of cancer cells at below micromolar concentrations without causing significant toxicity to a broad range of nonmalignant cells. Mechanistically, AOH1160 interferes with DNA replication, blocks homologous recombination-mediated DNA repair, and causes cell-cycle arrest. It induces apoptosis in cancer cells and sensitizes them to cisplatin treatment. AOH1160 is orally available to animals and suppresses tumor growth in a dosage form compatible to clinical applications. Importantly, it does not cause significant toxicity at 2.5 times of an effective dose. CONCLUSIONS: These results demonstrated the favorable therapeutic properties and the potential of AOH1160 as a broad-spectrum therapeutic agent for cancer treatment.


Assuntos
Antineoplásicos/farmacologia , Biomarcadores Tumorais , Antígeno Nuclear de Célula em Proliferação/metabolismo , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quebras de DNA/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Desenvolvimento de Medicamentos , Humanos , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Antígeno Nuclear de Célula em Proliferação/química , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto
6.
J Chem Inf Model ; 57(12): 3011-3021, 2017 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-29095607

RESUMO

Proliferating cell nuclear antigen (PCNA) is a member of the family of sliding clamp proteins that serves as a clamp during DNA repair, DNA replication, cell cycle control, and multiple forms of chromatin modification. PCNA functions as a homotrimer and complexes with multiple proteins in order to carry out each of these varied functions. PCNA binds to different partner proteins in the same region of its structure, called the " interdomain connecting loop", but with different affinities. This interdomain connecting loop is an intrinsically disordered region that takes different conformations when binding to different partner proteins. In this work, we performed all-atom molecular dynamics simulations on PCNA trimer unbound to any partner protein, PCNA bound to peptides from different partner proteins, and PCNA bound to the full Fen 1 protein in two different conformations. Using this massive amount of simulation results, we analyzed whether PCNA in its free trimeric form samples conformations that are similar to those when it is bound to different partner proteins. We observed that PCNA samples many of these peptide-bound conformations even when not bound to the peptides and selects specific conformations when binding to partner proteins. We also identified PCNA-peptide interactions formed in the peptide bound simulation that play a crucial role in complex formation. The calculated binding energies correlate well with the measured binding affinities of various peptides to PCNA. Lastly, we studied the internal dynamics of PCNA and propose a mechanism through which PCNA recruits binding partners. This work highlights the functional role of intrinsically disordered regions in multifunctional proteins such as PCNA.


Assuntos
Antígeno Nuclear de Célula em Proliferação/metabolismo , Cristalografia por Raios X , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Peptídeos/química , Peptídeos/metabolismo , Antígeno Nuclear de Célula em Proliferação/química , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas
7.
PLoS One ; 11(12): e0169259, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28036377

RESUMO

We describe a gentle and rapid method to purify the intact multiprotein DNA replication complex using free flow electrophoresis (FFE). In particular, we applied FFE to purify the human cell DNA synthesome, which is a multiprotein complex that is fully competent to carry-out all phases of the DNA replication process in vitro using a plasmid containing the simian virus 40 (SV40) origin of DNA replication and the viral large tumor antigen (T-antigen) protein. The isolated native DNA synthesome can be of use in studying the mechanism by which mammalian DNA replication is carried-out and how anti-cancer drugs disrupt the DNA replication or repair process. Partially purified extracts from HeLa cells were fractionated in a native, liquid based separation by FFE. Dot blot analysis showed co-elution of many proteins identified as part of the DNA synthesome, including proliferating cell nuclear antigen (PCNA), DNA topoisomerase I (topo I), DNA polymerase δ (Pol δ), DNA polymerase ɛ (Pol ɛ), replication protein A (RPA) and replication factor C (RFC). Previously identified DNA synthesome proteins co-eluted with T-antigen dependent and SV40 origin-specific DNA polymerase activity at the same FFE fractions. Native gels show a multiprotein PCNA containing complex migrating with an apparent relative mobility in the megadalton range. When PCNA containing bands were excised from the native gel, mass spectrometric sequencing analysis identified 23 known DNA synthesome associated proteins or protein subunits.


Assuntos
DNA Polimerase Dirigida por DNA/análise , DNA Polimerase Dirigida por DNA/isolamento & purificação , Eletroforese/métodos , Complexos Multienzimáticos/análise , Complexos Multienzimáticos/isolamento & purificação , Antígenos Virais de Tumores/genética , Extratos Celulares/química , Linhagem Celular Tumoral , DNA Polimerase I/isolamento & purificação , DNA Polimerase II/isolamento & purificação , DNA Polimerase III/isolamento & purificação , Replicação do DNA , DNA Topoisomerases Tipo I/isolamento & purificação , Células HeLa , Humanos , Antígeno Nuclear de Célula em Proliferação/análise , Origem de Replicação/genética , Proteína de Replicação A/isolamento & purificação , Proteína de Replicação C/isolamento & purificação , Vírus 40 dos Símios/genética
8.
Cancer Biol Ther ; 17(3): 310-9, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26889573

RESUMO

Human DNA replication and repair is a highly coordinated process involving the specifically timed actions of numerous proteins and enzymes. Many of these proteins require interaction with proliferating cell nuclear antigen (PCNA) for activation within the process. The interdomain connector loop (IDCL) of PCNA provides a docking site for many of those proteins, suggesting that this region is critically important in the regulation of cellular function. Previous work in this laboratory has demonstrated that a peptide mimicking a specific region of the IDCL (caPeptide) has the ability to disrupt key protein-protein interactions between PCNA and its binding partners, thereby inhibiting DNA replication within the cells. In this study, we confirm the ability of the caPeptide to disrupt DNA replication function using both intact cell and in vitro DNA replication assays. Further, we were able to demonstrate that treatment with caPeptide results in a decrease of polymerase δ activity that correlates with the observed decrease in DNA replication. We have also successfully developed a surface plasmon resonance (SPR) assay to validate the disruption of the PCNA-pol δ interaction with caPeptide.


Assuntos
Materiais Biomiméticos/farmacologia , Neoplasias/tratamento farmacológico , Fragmentos de Peptídeos/farmacologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proliferação de Células/efeitos dos fármacos , DNA Polimerase I/metabolismo , DNA Polimerase III/metabolismo , Replicação do DNA/efeitos dos fármacos , Células HeLa , Humanos , Terapia de Alvo Molecular , Neoplasias/metabolismo , Neoplasias/patologia , Ressonância de Plasmônio de Superfície
9.
Cell Rep ; 10(8): 1288-96, 2015 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-25732820

RESUMO

Recent evidence supports the presence of an L-glutamyl methyltransferase(s) in eukaryotic cells, but this enzyme class has been defined only in certain prokaryotic species. Here, we characterize the human C6orf211 gene product as "acidic residue methyltransferase-1" (Armt1), an enzyme that specifically targets proliferating cell nuclear antigen (PCNA) in breast cancer cells, predominately methylating glutamate side chains. Armt1 homologs share structural similarities with the SAM-dependent methyltransferases, and negative regulation of activity by automethylation indicates a means for cellular control. Notably, shRNA-based knockdown of Armt1 expression in two breast cancer cell lines altered survival in response to genotoxic stress. Increased sensitivity to UV, adriamycin, and MMS was observed in SK-Br-3 cells, while in contrast, increased resistance to these agents was observed in MCF7 cells. Together, these results lay the foundation for defining the mechanism by which this post-translational modification operates in the DNA damage response (DDR).


Assuntos
Reparo do DNA , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteína O-Metiltransferase/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Dano ao DNA , Humanos , Células MCF-7 , Metilação , Dados de Sequência Molecular , Antígeno Nuclear de Célula em Proliferação/química , Proteína O-Metiltransferase/antagonistas & inibidores , Proteína O-Metiltransferase/genética , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Alinhamento de Sequência , Especificidade por Substrato
10.
Mol Pharmacol ; 87(6): 996-1005, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25814515

RESUMO

COH29 [N-(4-(3,4-dihydroxyphenyl)-5-phenylthiazol-2-yl)-3,4-dihydroxybenzamide], a novel antimetabolite drug developed at City of Hope Cancer Center, has anticancer activity that stems primarily from the inhibition of human ribonucleotide reductase (RNR). This key enzyme in deoxyribonucleotide biosynthesis is the target of established clinical agents such as hydroxyurea and gemcitabine because of its critical role in DNA replication and repair. Herein we report that BRCA-1-defective human breast cancer cells are more sensitive than wild-type BRCA-1 counterparts to COH29 in vitro and in vivo. Microarray gene expression profiling showed that COH29 reduces the expression of DNA repair pathway genes, suggesting that COH29 interferes with these pathways. It is well established that BRCA1 plays a role in DNA damage repair, especially homologous recombination (HR) repair, to maintain genome integrity. In BRCA1-defective HCC1937 breast cancer cells, COH29 induced more double-strand breaks (DSBs) and DNA-damage response than in HCC1937 + BRCA1 cells. By EJ5- and DR-green fluorescent protein (GFP) reporter assay, we found that COH29 could inhibit nonhomologous end joining (NHEJ) efficiency and that no HR activity was detected in HCC1937 cells, suggesting that repression of the NHEJ repair pathway may be involved in COH29-induced DSBs in BRCA1-deficient HCC1937 cells. Furthermore, we observed an accumulation of nuclear Rad51 foci in COH29-treated HCC1937 + BRCA1 cells, suggesting that BRCA1 plays a crucial role in repairing and recovering drug-induced DNA damage by recruiting Rad51 to damage sites. In summary, we describe here additional biologic effects of the RNR inhibitor COH29 that potentially strengthen its use as an anticancer agent.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Benzamidas/farmacologia , Reparo do DNA/efeitos dos fármacos , Ribonucleotídeo Redutases/antagonistas & inibidores , Tiazóis/farmacologia , Animais , Antimetabólitos Antineoplásicos/uso terapêutico , Proteína BRCA1/genética , Benzamidas/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Feminino , Xenoenxertos , Humanos , Camundongos Endogâmicos NOD , Testes de Mutagenicidade , Transplante de Neoplasias , Tiazóis/uso terapêutico , Peixe-Zebra
11.
EBioMedicine ; 2(12): 1923-31, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26844271

RESUMO

Dysregulated expression of MYC family genes is a hallmark of many malignancies. Unfortunately, these proteins are not amenable to blockade by small molecules or protein-based therapeutic agents. Therefore, we must find alternative approaches to target MYC-driven cancers. Amplification of MYCN, a MYC family member, predicts high-risk neuroblastoma (NB) disease. We have shown that R9-caPep blocks the interaction of PCNA with its binding partners and selectively kills human NB cells, especially those with MYCN amplification, and we now show the mechanism. We found elevated levels of DNA replication stress in MYCN-amplified NB cells. R9-caPep exacerbated DNA replication stress in MYCN-amplified NB cells and NB cells with an augmented level of MYC by interfering with DNA replication fork extension, leading to Chk1 dependence and susceptibility to Chk1 inhibition. We describe how these effects may be exploited for treating NB.


Assuntos
Peptídeos Penetradores de Células/metabolismo , Amplificação de Genes , Neuroblastoma/genética , Neuroblastoma/metabolismo , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/farmacologia , Quinase 1 do Ponto de Checagem , Replicação do DNA/efeitos dos fármacos , Sinergismo Farmacológico , Perfilação da Expressão Gênica , Humanos , Modelos Moleculares , Proteína Proto-Oncogênica N-Myc , Antígeno Nuclear de Célula em Proliferação/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Transdução de Sinais , Estresse Fisiológico
12.
Mol Pharmacol ; 87(2): 263-76, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25480843

RESUMO

Proliferating cell nuclear antigen (PCNA) is a highly conserved protein necessary for proper component loading during the DNA replication and repair process. Proteins make a connection within the interdomain connector loop of PCNA, and much of the regulation is a result of the inherent competition for this docking site. If this target region of PCNA is modified, the DNA replication and repair process in cancer cells is potentially altered. Exploitation of this cancer-associated region has implications for targeted breast cancer therapy. In the present communication, we characterize a novel peptide (caPeptide) that has been synthesized to mimic the sequence identified as critical to the cancer-associated isoform of PCNA. This peptide is delivered into cells using a nine-arginine linking mechanism, and the resulting peptide (R9-cc-caPeptide) exhibits cytotoxicity in a triple-negative breast cancer cell line, MDA-MB-436, while having less of an effect on the normal counterparts (MCF10A and primary breast epithelial cells). The novel peptide was then evaluated for cytotoxicity using various in vivo techniques, including ATP activity assays, flow cytometry, and clonogenetic assays. This cytotoxicity has been observed in other breast cancer cell lines (MCF7 and HCC1937) and other forms of cancer (pancreatic and lymphoma). R9-cc-caPeptide has also been shown to block the association of PCNA with chromatin. Alanine scanning of the peptide sequence, combined with preliminary in silico modeling, gives insight to the disruptive ability and the molecular mechanism of action of the therapeutic peptide in vivo.


Assuntos
Neoplasias da Mama/metabolismo , Citotoxinas/metabolismo , Mimetismo Molecular/fisiologia , Fragmentos de Peptídeos/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Animais , Neoplasias da Mama/genética , Citotoxinas/genética , Feminino , Humanos , Células MCF-7 , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Fragmentos de Peptídeos/genética , Antígeno Nuclear de Célula em Proliferação/genética , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Coelhos , Distribuição Aleatória
13.
Cancer Chemother Pharmacol ; 74(5): 981-93, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25190177

RESUMO

BACKGROUND: An 8 amino acid peptide sequence derived from proliferating cell nuclear antigen (PCNA) has been shown to effectively kill several breast cancer and neuroblastoma cell lines when added exogenously to cell cultures. METHODS: In this study, the expression of the 8 amino acid peptide sequence (caPeptide) was placed under control of a tetracycline responsive promoter in MDA-MB-231 cells. RESULTS: Endogenous expression of the peptide resulted in an increase in genomic DNA damage. CaPeptide induction combined with treatment of sublethal doses of cisplatin resulted in a marked increase in death of the cisplatin-resistant MDA-MB-231 cell line. CaPeptide was found to interact with POLD3, one of the subunits of DNA polymerase delta necessary for binding to PCNA. CONCLUSION: These results suggest an important line of inquiry into the possible role that caPeptide might play in the reversal of cisplatin resistance in breast and other cancers. This is of particular interest in those cancers where cisplatin is the first line of chemotherapy and where the acquisition of resistance is a common malady.


Assuntos
Cisplatino/farmacologia , Dano ao DNA , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Oligopeptídeos/farmacologia , Sequência de Aminoácidos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , DNA Polimerase III/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Concentração Inibidora 50 , Dados de Sequência Molecular , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Subunidades Proteicas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo
14.
PLoS One ; 9(4): e94773, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24728180

RESUMO

Proliferating cell nuclear antigen (PCNA), through its interaction with various proteins involved in DNA synthesis, cell cycle regulation, and DNA repair, plays a central role in maintaining genome stability. We previously reported a novel cancer associated PCNA isoform (dubbed caPCNA), which was significantly expressed in a broad range of cancer cells and tumor tissues, but not in non-malignant cells. We found that the caPCNA-specific antigenic site lies between L126 and Y133, a region within the interconnector domain of PCNA that is known to be a major binding site for many of PCNA's interacting proteins. We hypothesized that therapeutic agents targeting protein-protein interactions mediated through this region may confer differential toxicity to normal and malignant cells. To test this hypothesis, we designed a cell permeable peptide containing the PCNA L126-Y133 sequence. Here, we report that this peptide selectively kills human neuroblastoma cells, especially those with MYCN gene amplification, with much less toxicity to non-malignant human cells. Mechanistically, the peptide is able to block PCNA interactions in cancer cells. It interferes with DNA synthesis and homologous recombination-mediated double-stranded DNA break repair, resulting in S-phase arrest, accumulation of DNA damage, and enhanced sensitivity to cisplatin. These results demonstrate conceptually the utility of this peptide for treating neuroblastomas, particularly, the unfavorable MYCN-amplified tumors.


Assuntos
Permeabilidade da Membrana Celular , Neuroblastoma/metabolismo , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Humanos , Neuroblastoma/patologia , Antígeno Nuclear de Célula em Proliferação/química , Ligação Proteica/efeitos dos fármacos , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
15.
PLoS One ; 9(4): e94443, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24732116

RESUMO

Glioblastoma (GBM) is the most common primary brain tumor, accounting for approximately 40% of all central nervous system malignancies. Despite standard treatment consisting of surgical resection, radiotherapy and/or chemotherapy, the prognosis for GBM is poor; with a median survival of 14.6 months. The cancer stem cell or cancer-initiating cell model has provided a new paradigm for understanding development and recurrence of GBM following treatment. Berbamine (BBM) is a natural compound derived from the Berberis amurensis plant, and along with its derivatives, has been shown to exhibit antitumor activity in several cancers. Here, we reported that a novel synthetic Berbamine derivative, BBMD3, inhibits cell viability and induces apoptosis of cancer stem-like cells (CSCs) in a time- and dose-dependent manner when the CSCs from four GBM patients (PBT003, PBT008, PBT022, and PBT030) were cultured. These CSCs grew in neurospheres and expressed CD133 and nestin as markers. Treatment with BBMD3 destroyed the neurosphere morphology, and led to the induction of apoptosis in the CSCs. Induction of apoptosis in these CSCs is dependent upon activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP). MicroRNA-4284 (miR-4284) was shown to be over-expressed about 4-fold in the CSCs following BBMD3 treatment. Furthermore, transfection of synthetic anti-sense oligonucleotide against human miR-4284 partially blocked the anticancer effects of BBMD3 on the GBM derived CSCs. BBMD3 also increased phosphorylation of the c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK), resulting in an increase expression of phosphorylated c-Jun and total c-Fos; the major components of transcriptional factor AP-1. The JNK-c-Jun/AP-1 signaling pathway plays an important role in the induction of apoptosis in response to UV irradiation and some drug treatments. Targeting glioblastoma stem-like cells with BBMD3 is therefore novel, and may have promise as an effective therapeutic strategy for treating GBM patients.


Assuntos
Apoptose/efeitos dos fármacos , Benzilisoquinolinas/farmacologia , Glioblastoma/patologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/patologia , Fator de Transcrição AP-1/metabolismo , Regulação para Cima/efeitos dos fármacos , Benzilisoquinolinas/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Glioblastoma/enzimologia , Glioblastoma/genética , Humanos , MicroRNAs/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Fosforilação/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Regulação para Cima/genética
16.
Gynecol Oncol ; 131(1): 198-206, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23851291

RESUMO

OBJECTIVE: The objective of this study is to determine whether an altered DNA replication process is responsible for some of genetic damage observed in ovarian cancer. METHODS: The replication fidelity of the DNA synthetic process was evaluated in both malignant and non-malignant human ovarian cells. The types of replication errors produced were identified. In addition, kinetic analyses of the efficiency of ovarian cancer DNA polymerases for misincorporating nucleotides were performed. RESULTS: We report for the first time that ovarian cancer cells harbor an error promoting DNA replication apparatus which contributes to the decrease in DNA synthetic fidelity exhibited by these cells. Our study also shows that the decrease in DNA replication fidelity was not a result of an increased DNA replication activity. In addition, it was observed that the higher rate of DNA replication errors does not result in significant differences in the type of DNA replication-errors made during the DNA replication process; just the relative abundance. A detailed kinetic analysis of the efficiency of misincorporating nucleotides demonstrated that the DNA polymerases within the ovarian cancer cells exhibited a significant propensity for creating purine-pyrimidine nucleotide mismatches relative to non-malignant ovarian cells, while being only slightly more efficient at incorrectly pairing a purine nucleotide with a purine nucleotide. CONCLUSIONS: All together, these data suggest that the systematic analysis of the DNA replication process in ovarian cancer could uncover information on some of the molecular mechanisms that drive the accumulation of genetic damage, and probably contribute to the pathogenesis of the disease.


Assuntos
Carcinoma/genética , Replicação do DNA , DNA de Neoplasias/biossíntese , DNA Polimerase Dirigida por DNA , Complexos Multienzimáticos , Mutação , Neoplasias Ovarianas/genética , Linhagem Celular Tumoral , DNA Polimerase Dirigida por DNA/metabolismo , Nucleotídeos de Desoxiadenina/metabolismo , Feminino , Humanos , Cinética , Óperon Lac/genética , Ovário/citologia
17.
Front Oncol ; 3: 93, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23638435

RESUMO

Children with solid tumors represent a unique population. Recent improvements in pediatric solid tumor survival rates have been confined to low- and moderate-risk cancers, whereas minimal to no notable improvement in survival have been observed in high-risk and advanced-stage childhood tumors. Treatments for patients with advanced disease are rarely curative, and responses to therapy are often followed by relapse, which highlights the large unmet need for novel therapies. Recent advances in cancer treatment have focused on personalized therapy, whereby patients are treated with agents that best target the molecular drivers of their disease. Thus, a better understanding of the pathways that drive cancer or drug resistance is of critical importance. One such example is the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway, which is activated in many solid cancer patients and represents a target for therapy. PI3K/Akt/mTOR pathway activation has also been observed in tumors resistant to agents targeting upstream receptor tyrosine kinases (RTKs). Agents that target this pathway have the potential to shut down survival pathways, and are being explored both in the setting of pathway-activating mutations and for their ability to restore sensitivity to upstream signaling targeted agents. Here, we examine the role of the PI3K/Akt/mTOR pathway in pediatric solid tumors, review the novel agents being explored to target this pathway, and explore the potential role of the inhibition of this pathway in the clinical development of these agents in children.

18.
Mol Pharmacol ; 81(6): 811-9, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22399488

RESUMO

Proliferating cell nuclear antigen (PCNA), a potential anticancer target, forms a homotrimer and is required for DNA replication and numerous other cellular processes. The purpose of this study was to identify novel small molecules that modulate PCNA activity to affect tumor cell proliferation. An in silico screen of a compound library against a crystal structure of PCNA and a subsequent structural similarity search of the ZINC chemical database were carried out to derive relevant docking partners. Nine compounds, termed PCNA inhibitors (PCNA-Is), were selected for further characterization. PCNA-I1 selectively bound to PCNA trimers with a dissociation constant (K(d)) of ~0.2 to 0.4 µM. PCNA-Is promoted the formation of SDS-refractory PCNA trimers. PCNA-I1 dose- and time-dependently reduced the chromatin-associated PCNA in cells. Consistent with its effects on PCNA trimer stabilization, PCNA-I1 inhibited the growth of tumor cells of various tissue types with an IC(50) of ~0.2 µM, whereas it affected the growth of nontransformed cells at significantly higher concentrations (IC(50), ~1.6 µM). Moreover, uptake of BrdU was dose-dependently reduced in cells treated with PCNA-I1. Mechanistically the PCNA-Is mimicked the effect of PCNA knockdown by siRNA, inducing cancer cell arrest at both the S and G(2)/M phases. Thus, we have identified a class of compounds that can directly bind to PCNA, stabilize PCNA trimers, reduce PCNA association with chromatin, and inhibit tumor cell growth by inducing a cell cycle arrest. They are valuable tools in studying PCNA function and may be useful for future PCNA-targeted cancer therapy.


Assuntos
Divisão Celular , Cromatina/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Animais , Ciclo Celular , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Feminino , Humanos , Concentração Inibidora 50 , Masculino , Camundongos , Antígeno Nuclear de Célula em Proliferação/efeitos dos fármacos
19.
Int J Mol Sci ; 13(1): 1126-1153, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22312308

RESUMO

Childhood cancer is the leading cause of death by disease among U.S. children between infancy and age 15. Despite successes in treating solid tumors such as Wilms tumor, disappointments in the outcomes of high-risk solid tumors like neuroblastoma have precipitated efforts towards the early and accurate detection of these malignancies. This review summarizes available solid tumor serum biomarkers with a special focus on mediastinal and abdominal cancers in children.


Assuntos
Biomarcadores Tumorais/sangue , Neoplasias/metabolismo , Antígeno Ca-125/sangue , Catecolaminas/sangue , Criança , Enzimas/sangue , Humanos , Neoplasias do Mediastino/metabolismo , Neoplasias do Mediastino/patologia , Glicoproteínas de Membrana/sangue , Neoplasias/patologia , Hormônios Peptídicos/sangue , alfa-Fetoproteínas/análise
20.
J Cell Biochem ; 112(3): 756-60, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21328449

RESUMO

Post-translational modifications (PTMs) of nuclear proteins play essential roles in the regulation of gene transcription and signal transduction pathways. Numerous studies have demonstrated a correlation between specific nuclear protein isoforms and cellular malignant process. This communication reviews the impact of major PTM events such as phosphorylation, acetylation, methylation, ubiquitination, and sumoylation on several important nuclear proteins including p53, histones, proliferating cellular nuclear antigen (PCNA), and retinoblastoma protein (Rb) in the process. In addition, the implications of the PTMs as cancer biomarkers and therapeutic targets are considered.


Assuntos
Neoplasias/diagnóstico , Neoplasias/terapia , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Histonas/metabolismo , Humanos , Neoplasias/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína do Retinoblastoma/metabolismo , Fator de Transcrição STAT3/metabolismo , Proteína Supressora de Tumor p53/metabolismo
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