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RLIP acts as a transporter that responds to stress and provides protection, specifically against glutathione-electrophile conjugates and xenobiotic toxins. Its increased presence in malignant cells, especially in cancer, emphasizes its crucial anti-apoptotic function. This is achieved by selectively regulating the cellular levels of pro-apoptotic oxidized lipid byproducts. Suppressing the progression of tumors in human xenografts can be achieved by effectively inhibiting RLIP, a transporter in the mercapturic acid pathway, without involving chemotherapy. Utilizing ovarian cancer (OC) cell lines (MDAH2774, OVCAR4, and OVCAR8), we observed that agents targeting RLIP, such as RLIP antisense and RLIP antibodies, not only substantially impeded the viability of OC cells but also remarkably increased their sensitivity to carboplatin. To delve further into the cytotoxic synergy between RLIP antisense, RLIP antibodies, and carboplatin, we conducted investigations in both cell culture and xenografts of OC cells. The outcomes revealed that RLIP depletion via phosphorothioate antisense led to rapid and sustained remissions in established subcutaneous human ovary xenografts. Furthermore, RLIP inhibition by RLIP antibodies exhibited comparable efficacy to antisense and enhanced the effectiveness of carboplatin in MDAH2774 OC xenografts. These investigations underscore RLIP as a central carrier crucial for supporting the survival of cancer cells, positioning it as a suitable focus for cancer treatment.
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The incidence of malignant melanoma, a neoplasm of melanocytic cells, is increasing rapidly. The lymph nodes are often the first site of metastasis and can herald systemic dissemination, which is almost uniformly fatal. RLIP, a multi-specific ATP-dependent transporter that is over-expressed in several types of cancers, plays a central role in cancer cell resistance to radiation and chemotherapy. RLIP appears to be necessary for cancer cell survival because both in vitro cell culture and in vivo animal tumor studies show that the depletion or inhibition of RLIP causes selective toxicity to malignant cells. RLIP depletion/inhibition triggers apoptosis in cancer cells by inducing the accumulation of endogenously formed glutathione-conjugates. In our in vivo studies, we administered RLIP antibodies or antisense oligonucleotides to mice bearing subcutaneous xenografts of SKMEL2 and SKMEL5 melanoma cells and demonstrated that both treatments caused significant xenograft regression with no apparent toxic effects. Anti-RLIP antibodies and antisense, which respectively inhibit RLIP-mediated transport and deplete RLIP expression, showed similar tumor regressing activities, indicating that the inhibition of RLIP transport activity at the cell surface is sufficient to achieve anti-tumor activity. Furthermore, RLIP antisense treatment reduced levels of RLIP, pSTAT3, pJAK2, pSrc, Mcl-1 and Bcl2, as well as CDK4 and cyclin B1, and increased levels of Bax and phospho 5' AMP-activated protein kinase (pAMPK). These studies indicate that RLIP serves as a key effector in the survival of melanoma cells and is a valid target for cancer therapy. Overall, compounds that inhibit, deplete or downregulate RLIP will function as wide-spectrum agents to treat melanoma, independent of common signaling pathway mutations.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Proteínas Ativadoras de GTPase/genética , Janus Quinase 2/genética , Melanoma/genética , Fator de Transcrição STAT3/genética , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/imunologia , Animais , Anticorpos Anti-Idiotípicos/farmacologia , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Proteínas Ativadoras de GTPase/imunologia , Humanos , Melanoma/patologia , Camundongos , Proteínas de Neoplasias/genética , Transdução de Sinais/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Breast cancer (BC) remains one of the major causes of cancer deaths in women. Over half of all BCs carry genetic defects in the gene encoding p53, a powerful tumor suppressor. P53 is known as the 'guardian of the genome' because it is essential for regulating cell division and preventing tumor formation. Ral-interacting protein (RLIP) is a modular protein capable of participating in many cellular functions. Blocking this stress-responsive protein, which is overexpressed during malignancy, enables BC cells to overcome the deleterious effects of p53 loss more effectively. In the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas9) system, a single-guide RNA (sgRNA) recognizes a specific DNA sequence and directs the endonuclease Cas9 to make a double-strand break, which enables editing of targeted genes. Here, we harnessed CRISPR/Cas9 technology to target the RLIP gene in BC cells. We screened sgRNAs using a reporter system and lentivirally delivered them, along with Cas9, to BC cells for validation. We then assessed the survival, proliferation, and tumorigenicity of BC cells in vitro and the growth of tumors in vivo after CRISPR-mediated knockdown of RLIP. Doxycycline-inducible expression of Cas9 in BC cells transduced with lentiviral vectors encoding the sgRNAs disrupted the RLIP gene, leading to inhibition of BC cell proliferation both in vitro and in vivo, with resected tumors showing reduced levels of the survival and proliferation markers Ki67, RLIP, pAkt, and survivin, the cell cycle protein CDK4, and the mesenchymal marker vimentin, as well as elevated levels of the differentiation protein E-cadherin and pro-apoptotic protein Bim. Inducible Cas9/sgRNA-transduced BC cells without doxycycline treatment did not exhibit altered cell survival or proliferation in vitro or in vivo. Our study provides proof-of-concept that the CRISPR/Cas9 system can be utilized to target RLIP in vitro and in vivo.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Neoplasias da Mama/terapia , Sistemas CRISPR-Cas/genética , Proteínas Ativadoras de GTPase/genética , Terapia Genética/métodos , Transportadores de Cassetes de Ligação de ATP/metabolismo , Apoptose/genética , Neoplasias da Mama/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Feminino , Proteínas Ativadoras de GTPase/metabolismo , Edição de Genes/métodos , Técnicas de Silenciamento de Genes , Humanos , Estudo de Prova de Conceito , RNA Guia de Cinetoplastídeos/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The overexpression and amplification of the protooncogene neu (ERBB2) play an important role in the development of aggressive breast cancer (BC) in humans. Ral-interacting protein (RLIP), a modular stress-response protein with pleiotropic functions, is overexpressed in several types of cancer, including BC. Here, we show that blocking RLIP attenuates the deleterious effects caused by the loss of the tumor suppressor p53 and inhibits the growth of human BC both in vitro and in vivo in MMTV-neu mice. In addition, we show that treatment with the diet-derived, RLIP-targeting chemotherapeutic 2'-hydroxyflavanone (2HF), alone or in combination with RLIP-specific antisense RNA or antibodies, significantly reduced the cumulative incidence and/or burden of mammary hyperplasia and carcinoma in MMTV-neu mice. 2HF treatment correlated with reduced tumor cell proliferation and increased apoptosis, and the average number of Ki67-positive (proliferating) cells was significantly lower in the tumors of 2HF-treated mice than in the tumors of control mice. Furthermore, targeting RLIP also resulted in the overexpression of E-cadherin and the infiltration of CD3+ T cells into mammary tumors. Taken together, these results underscore the translational potential of RLIP-targeting agents and provide a strong rationale to validate them in the clinic.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Anticarcinógenos/farmacologia , Neoplasias da Mama/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Neoplasias Mamárias Experimentais/prevenção & controle , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Flavanonas/farmacologia , Proteínas Ativadoras de GTPase/genética , Humanos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Glândulas Mamárias Animais/efeitos dos fármacos , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos Transgênicos , Terapia de Alvo Molecular , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptor ErbB-2/metabolismoRESUMO
TP53 (p53) is a tumor suppressor whose functions are lost or altered in most malignancies. p53 homozygous knockout (p53-/-) mice uniformly die of spontaneous malignancy, typically T-cell lymphoma. RALBP1 (RLIP76, Rlip) is a stress-protective, mercapturic acid pathway transporter protein that also functions as a Ral effector involved in clathrin-dependent endocytosis. In stark contrast to p53-/- mice, Rlip-/- mice are highly resistant to carcinogenesis. We report here that partial Rlip deficiency induced by weekly administration of an Rlip-specific phosphorothioate antisense oligonucleotide, R508, strongly inhibited spontaneous as well as benzo(a)pyrene-induced carcinogenesis in p53-/- mice. This treatment effectively prevented large-scale methylomic and transcriptomic abnormalities suggestive of inflammation found in cancer-bearing p53-/- mice. The remarkable efficiency with which Rlip deficiency suppresses spontaneous malignancy in p53-/- mice has not been observed with any previously reported pharmacologic or genetic intervention. These findings are supported by cross-breeding experiments demonstrating that hemizygous Rlip deficiency also reduces the spontaneous malignancy phenotype of p53+/- mice. Rlip is found on the cell surface, and antibodies directed against Rlip were found to inhibit growth and promote apoptosis of cell lines as effectively as Rlip siRNA. The work presented here investigates several features, including oxidative DNA damage of the Rlip-p53 association in malignant transformation, and offers a paradigm for the mechanisms of tumor suppression by p53 and the prospects of suppressing spontaneous malignancy in hereditary cancer syndromes such as Li-Fraumeni.
Assuntos
Proteínas Ativadoras de GTPase/deficiência , Neoplasias/genética , Neoplasias/prevenção & controle , Proteína Supressora de Tumor p53/genética , Animais , Apoptose , Feminino , Proteínas Ativadoras de GTPase/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias/metabolismo , Neoplasias/fisiopatologia , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/metabolismoRESUMO
Prostate cancer (CaP) is often androgen-sensitive malignancy and regresses upon inhibition of androgen signaling. However, CaP, nearly always develops androgen resistance and progresses to aggressive and lethal androgen-independent CaP, which lacks satisfactory therapy. For metastatic CaP, patients are often treated with Taxotere (docetaxel), a cytoskeleton-targeted chemotherapy drug, that provides transient palliative benefit but to which patients rapidly develop drug-resistance. Combination chemotherapy may be used instead, but is more toxic and adds little clinically relevant benefit over docetaxel. Therefore, novel strategies to enhance docetaxel efficacy are needed to effectively treat patients with metastatic CaP. The mercapturic acid pathway, which metabolizes genotoxic and pro-apoptotic toxins, is over-expressed in CaP and plays an important role in carcinogenesis, metastasis and therapy-resistance of CaP. Vicenin-2, a flavonoid derived from Tulsi (holy basil) as an active compound, inhibits the growth of CaP and increases the anti-tumor activity of docetaxel in-vitro and in-vivo. Taken together, the combination of vicenin-2 and docetaxel could be highly effective in the treatment of advanced and metastatic CaP due to their multi-targeting anti-tumor potential.
Assuntos
Acetilcisteína/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apigenina/metabolismo , Glucosídeos/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Docetaxel , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Masculino , Taxoides/farmacologia , Taxoides/uso terapêuticoRESUMO
Substantial evidence suggests that 7,12-dimethylbenzanthracene (DMBA)-induced mammary carcinogenesis in mice mimics human breast cancer (BC) in many respects. Therefore, it has been used extensively to evaluate preventive and therapeutic agents for human BC. Mammary carcinogenesis induced by DMBA administration in female SENsitive to CARcinogen (SENCAR) mice was characterized by histopathological analysis of the mammary glands and alterations to the phosphatidylinositol 3-kinase/protein kinase B/cyclin-dependent kinase 1 (PI3K/Akt/CDK1) pathway. We recently reported that 2'-hydroxyflavanone (2HF) is a promising diet-derived chemotherapeutic agent that suppresses BC growth in vitro and in vivo by targeting a 76 kDa ral-interacting protein (RLIP). The objective of the current study was to investigate the synergistic anticarcinogenic effects of RLIP inhibition/depletion and 2HF in an in vivo model of DMBA-induced mammary carcinogenesis in SENCAR mice. Mice were given 2HF (50 mg/kg, bw, orally on alternate days), RLIP antibody (Rab; 5 mg/kg, bw, ip weekly), RLIP antisense (RAS; 5 mg/kg, b.w., ip weekly), or a combination of 2HF + Rab + RAS. Animals were monitored daily, and 7 days after the first appearance of moribund behavior, tissues were harvested for morphological and immunohistological analysis. Western blot analyses were performed to determine the expression of anti- and proapoptotic proteins in the mammary glands. Our results reveal that 2HF, RAS, and Rab significantly prevented the carcinogenic effects of DMBA administration in the mammary glands and other organs. Further, mice treated with a combination of 2HF + RAS + Rab exhibited no carcinogenic effect of DMBA as compared to either or the single agent-treated mice. This study demonstrates for the first time the anticarcinogenic effects of 2HF and RLIP inhibition/depletion in vivo in a novel DMBA-induced model of BC in SENCAR mice and provides the rationale for further clinical investigation.
Assuntos
9,10-Dimetil-1,2-benzantraceno/toxicidade , Anticarcinógenos/farmacologia , Transformação Celular Neoplásica/patologia , Flavanonas/farmacologia , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Neoplasias Mamárias Experimentais/prevenção & controle , Animais , Proteína Quinase CDC2/metabolismo , Modelos Animais de Doenças , Feminino , Proteínas Ativadoras de GTPase/genética , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos SENCAR , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Breast cancer (BC) prevention and therapy in the context of life-style risk factors and biological drivers is a major focus of developmental therapeutics in oncology. Obesity, alcohol, chronic estrogen signaling and smoking have distinct BC precipitating and facilitating effects that may act alone or in combination. A spectrum of signaling events including enhanced oxidative stress and changes in estrogen-receptor (ER)-dependent and -independent signaling drive the progression of BC. Breast tumors modulate ERα/ERß ratio, upregulate proliferative pathways driven by ERα and HER2 with a parallel loss and/or downregulation of tumor suppressors such as TP53 and PTEN which together impact the efficacy of therapeutic strategies and frequently lead to emergence of drug resistance. Natural phytochemicals modulate oxidative stress, leptin, integrin, HER2, MAPK, ERK, Wnt/ß-catenin and NFκB signaling along with regulating ERα and ERß, thereby presenting unique opportunities for both primary and combinatorial interventions in BC. In this regard, this article focuses on critical analyses of the evidence from multiple studies on the efficacy of natural phytochemicals in BC. In addition, areas in which the combinations of such effective natural phytochemicals with approved and/or developing anticancer agents can be translationally beneficial are discussed to derive evidence-based inference for addressing challenges in BC control and therapy.
Assuntos
Biomarcadores Tumorais/antagonistas & inibidores , Neoplasias da Mama/prevenção & controle , Terapia de Alvo Molecular , Compostos Fitoquímicos/uso terapêutico , Pesquisa Translacional Biomédica , Neoplasias da Mama/metabolismo , Quimioterapia Combinada , Feminino , HumanosRESUMO
Consumption of citrus-fruits is associated with reduced incidence of breast cancer (BC), the most common cancer diagnosed in women across the globe. In this study, we investigated the anticancer potential of 2-Hydroxyflavanone (2HF) in BC. 2HF, a citrus-bioflavonoid, has demonstrated anticancer properties in various cancers, but its anticancer role in BC has not been well studied. We investigated the in vitro and in vivo growth inhibitory effects of 2HF in an array of BC lines and in xenograft mouse models of ER-positive and HER2-positive BC cells. Compared to control, 2HF treatment reduced cell viability and suppressed migratory and invasive potential of BC cells, while, no growth inhibitory effects were observed in non-tumorigenic breast epithelial cells. Further, 2HF inhibited the expression of RLIP76, a stress-defensive and anti-apoptotic protein, which is over-expressed in BC cells and simultaneously reduced proliferation of BC cells. Nude mice bearing MCF7 or SKBR3 BC cells xenografts treated with either 2HF or targeting RLIP76 by RLIP76-antisense or RLIP76-antibody treatment had significantly lower tumor-weight as compared to corresponding controls. In addition, Western-blotting and immunohistochemical analysis of tumor tissue from control and treatment group mice showed that 2HF decreased protein expression levels of RLIP76, and the decrease was similar to those seen following RLIP76-antisense treatment. Furthermore, 2HF decreased expression of Ki67, CD31, vimentin, inhibited phosphorylation of Akt and expression of survivin and Bcl2, and increased levels of Bax, E-cadherin, and cleaved-PARP. Therefore, our results indicate that 2HF may suppress BC growth in vitro and in vivo by targeting RLIP76, and may serve as a potential adjuvant treatment in BC patients.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos Fitogênicos/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Flavanonas/administração & dosagem , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Animais , Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Flavanonas/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Refractory and relapsed neuroblastoma (NB) present with significant challenges in clinical management. Though primary NBs largely with wild-type p53 respond well to interventions, dysfunctional signaling in the p53 pathways in a MYCN oncogene driven background is found in a number of children with NB. The p53-mutant NB is largely unresponsive to available therapies and p53-independent targeted therapeutics represents a vital need in pediatric oncology. We analyzed the findings on mercapturic acid pathway (MAP) transporter RLIP76, which has broad and critical effects on multiple pathways as essential for carcinogenesis, oxidative stress and drug-resistance, is over-expressed in NB. RLIP76 inhibition by antibodies or depletion by antisense causes apoptosis and sensitization to chemo-radiotherapy in many cancers. In addition, recent studies indicate that the interactions between p53, MYCN, and WNT regulate apoptosis resistance and protein ubiquitination. RLIP76 and p53 interact with each other and colocalize in NB cells. Targeted depletion/inhibition of RLIP76 causes apoptosis and tumor regression in NB irrespective of p53 status. In the present review, we discuss the mechanisms and the role of RLIP76 in oxidative stress, drug-resistance and clathrin-dependent endocytosis (CDE), and analyze the molecular basis for the role of RLIP76 targeted approaches in the context principal drivers of NB pathogenesis, progression and drug-resistance. The evidence from RLIP76 studies in other cancers, when taken in the context of our recent RLIP76 focused mechanistic studies in NB, provides strong basis for further characterization and development of RLIP76 targeted therapies for NB.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Neuroblastoma/tratamento farmacológico , Animais , Anticorpos/uso terapêutico , Apoptose , Transporte Biológico , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Criança , Clatrina/metabolismo , Resistencia a Medicamentos Antineoplásicos , Endocitose , Glutationa , Humanos , Terapia de Alvo Molecular , Mutação , Recidiva Local de Neoplasia , Neuroblastoma/patologia , Estresse Oxidativo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Mitochondrial oxidative phosphorylation produces most of the energy in aerobic cells by coupling respiration to the production of ATP. Mitochondrial uncouplers, which reduce the proton gradient across the mitochondrial inner membrane, create a futile cycle of nutrient oxidation without generating ATP. Regulation of mitochondrial dysfunction and associated cellular bioenergetics has been recently identified as a promising target for anticancer therapy. Here, we show that SR4 is a novel mitochondrial uncoupler that causes dose-dependent increase in mitochondrial respiration and dissipation of mitochondrial membrane potential in HepG2 hepatocarcinoma cells. These effects were reversed by the recoupling agent 6-ketocholestanol but not cyclosporin A and were nonexistent in mitochondrial DNA-depleted HepG2 cells. In isolated mouse liver mitochondria, SR4 similarly increased oxygen consumption independent of adenine nucleotide translocase and uncoupling proteins, decreased mitochondrial membrane potential, and promoted swelling of valinomycin-treated mitochondria in potassium acetate medium. Mitochondrial uncoupling in HepG2 cells by SR4 results in the reduction of cellular ATP production, increased ROS production, activation of the energy-sensing enzyme AMPK, and inhibition of acetyl-CoA carboxylase and mammalian target of rapamycin signaling pathways, leading to cell cycle arrest and apoptosis. Global analysis of SR4-associated differential gene expression confirms these observations, including significant induction of apoptotic genes and down-regulation of cell cycle, mitochondrial, and oxidative phosphorylation pathway transcripts at 24 h post-treatment. Collectively, our studies demonstrate that the previously reported indirect activation of AMPK and in vitro anticancer properties of SR4 as well as its beneficial effects in both animal xenograft and obese mice models could be a direct consequence of its mitochondrial uncoupling activity.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Mitocôndrias Hepáticas/metabolismo , Proteínas de Neoplasias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Desacopladores/farmacologia , Proteínas Quinases Ativadas por AMP/genética , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Mitocôndrias Hepáticas/genética , Mitocôndrias Hepáticas/patologia , Proteínas de Neoplasias/genética , Consumo de Oxigênio/efeitos dos fármacos , Consumo de Oxigênio/genética , Serina-Treonina Quinases TOR/genéticaRESUMO
R-Ras small GTPase enhances cell spreading and motility via RalBP1/RLIP76, an R-Ras effector that links GTP-R-Ras to activation of Arf6 and Rac1 GTPases. Here, we report that RLIP76 performs these functions by binding cytohesin-2/ARNO, an Arf GTPase guanine exchange factor, and connecting it to R-Ras at recycling endosomes. RLIP76 formed a complex with R-Ras and ARNO by binding ARNO via its N-terminus (residues 1-180) and R-Ras via residues 180-192. This complex was present in Rab11-positive recycling endosomes and the presence of ARNO in recycling endosomes required RLIP76, and was not supported by RLIP76(Δ1-180) or RLIP76(Δ180-192). Spreading and migration required RLIP76(1-180), and RLIP76(Δ1-180) blocked ARNO recruitment to recycling endosomes, and spreading. Arf6 activation with an ArfGAP inhibitor overcame the spreading defects in RLIP76-depleted cells or cells expressing RLIP76(Δ1-180). Similarly, RLIP76(Δ1-180) or RLIP76(Δ180-192) suppressed Arf6 activation. Together these results demonstrate that RLIP76 acts as a scaffold at recycling endosomes by binding activated R-Ras, recruiting ARNO to activate Arf6, thereby contributing to cell spreading and migration.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteínas ras/metabolismo , Fator 6 de Ribosilação do ADP , Animais , Sequência de Bases , Movimento Celular/fisiologia , Endossomos/metabolismo , Feminino , Fibroblastos/metabolismo , Proteínas Ativadoras de GTPase/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Transdução de Sinais , Proteínas ras/genéticaRESUMO
4-Hydroxy-2-trans-nonenal (4HNE), one of the major end products of lipid peroxidation (LPO), has been shown to induce apoptosis in a variety of cell lines. It appears to modulate signaling processes in more than one way because it has been suggested to have a role in signaling for differentiation and proliferation. It has been known that glutathione S-transferases (GSTs) can reduce lipid hydroperoxides through their Se-independent glutathione-peroxidase activity and that these enzymes can also detoxify LPO end-products such as 4HNE. Available evidence from earlier studies together with results of recent studies in our laboratories strongly suggests that LPO products, particularly hydroperoxides and 4HNE, are involved in the mechanisms of stress-mediated signaling and that it can be modulated by the alpha-class GSTs through the regulation of the intracellular concentrations of 4HNE. We demonstrate that 4HNE induced apoptosis in various cell lines is accompanied with c-Jun-N-terminal kinase (JNK) and caspase-3 activation. Cells exposed to mild, transient heat or oxidative stress acquire the capacity to exclude intracellular 4HNE at a faster rate by inducing GSTA4-4 which conjugates 4HNE to glutathione (GSH), and RLIP76 which mediates the ATP-dependent transport of the GSH-conjugate of 4HNE (GS-HNE). The balance between formation and exclusion promotes different cellular processes - higher concentrations of 4HNE promote apoptosis; whereas, lower concentrations promote proliferation. In this article, we provide a brief summary of the cellular effects of 4HNE, followed by a review of its GST-catalyzed detoxification, with an emphasis on the structural attributes that play an important role in the interactions with alpha-class GSTA4-4. Taken together, 4HNE is a key signaling molecule and that GSTs being determinants of its intracellular concentrations, can regulate stress-mediated signaling, are reviewed in this article.
Assuntos
Aldeídos/metabolismo , Antioxidantes/metabolismo , Glutationa Transferase/metabolismo , Isoenzimas/metabolismo , Estresse Oxidativo/fisiologia , Transdução de Sinais/fisiologia , Animais , Apoptose/fisiologia , Proliferação de Células/fisiologia , Humanos , Peroxidação de Lipídeos/fisiologiaRESUMO
Despite recent improvements in chemotherapeutic approaches to treating kidney cancer, this malignancy remains deadly if not found and removed at an early stage of the disease. Kidney cancer is highly drug-resistant, which may at least partially result from high expression of transporter proteins in the cell membranes of kidney cells. Although these transporter proteins can contribute to drug-resistance, targeting proteins from the ATP-binding cassette transporter family has not been effective in reversing drug-resistance in kidney cancer. Recent studies have identified RLIP76 as a key stress-defense protein that protects normal cells from damage caused by stress conditions, including heat, ultra-violet light, X-irradiation, and oxidant/electrophilic toxic chemicals, and is crucial for protecting cancer cells from apoptosis. RLIP76 is the predominant glutathione-electrophile-conjugate (GS-E) transporter in cells, and inhibiting it with antibodies or through siRNA or antisense causes apoptosis in many cancer cell types. To date, blocking of RLIP76, either alone or in combination with chemotherapeutic drugs, as a therapeutic strategy for kidney cancer has not yet been evaluated in human clinical trials, although there is considerable potential for RLIP76 to be developed as a therapeutic agent for kidney cancer. In the present review, we discuss the mechanisms underlying apoptosis caused by RLIP76 depletion, the role of RLIP76 in clathrin-dependent endocytosis deficiency, and the feasibility of RLIP76-targeted therapy for kidney cancer.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias Renais/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Endocitose/fisiologia , Glutationa/metabolismo , Humanos , Neoplasias Renais/metabolismoRESUMO
Feeding a Western high-fat diet (HFD) to C57BL/6 mice induces obesity, associated with a chronic inflammatory state, lipid transport, and metabolic derangements, and organ system effects that particularly prominent in the kidneys. Here, we report that RLIP76 homozygous knock-out (RLIP76(-/-)) mice are highly resistant to obesity as well as these other features of metabolic syndrome caused by HFD. The normal increase in pro-inflammatory and fibrotic markers associated with HFD induced obesity in wild-type C57B mice was broadly and nearly completely abrogated in RLIP76(-/-) mice. This is a particularly striking finding because chemical markers of oxidative stress including lipid hydroperoxides and alkenals were significantly higher in RLIP76(-/-) mice. Whereas HFD caused marked suppression of AMPK in wild-type C57B mice, RLIP76(-/-) mice had baseline activation of AMP-activated protein kinase, which was not further affected by HFD. The baseline renal function was reduced in RLIP76(-/-) mice as compared with wild-type, but was unaffected by HFD, in marked contrast to severe renal impairment and glomerulopathy in the wild-type mice given HFD. Our findings confirm a fundamental role of RLIP76 in regulating the function of obesity-promoting pro-inflammatory cytokines, and provide a novel mechanism for targeted therapy of obesity and metabolic syndrome.
Assuntos
Citocinas/metabolismo , Gorduras na Dieta/efeitos adversos , Proteínas Ativadoras de GTPase/metabolismo , Síndrome Metabólica/metabolismo , Obesidade/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Biomarcadores/metabolismo , Citocinas/genética , Gorduras na Dieta/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Proteínas Ativadoras de GTPase/genética , Técnicas de Silenciamento de Genes , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/metabolismo , Síndrome Metabólica/induzido quimicamente , Síndrome Metabólica/genética , Camundongos , Camundongos Knockout , Obesidade/induzido quimicamente , Obesidade/genética , Estresse Oxidativo/genéticaRESUMO
Methylglyoxal (MGO) is a highly reactive dicarbonyl compound known to induce cellular injury and cytoxicity, including apoptosis in vascular cells. Vascular endothelial cell apoptosis has been implicated in the pathophysiology and progression of atherosclerosis. We investigated whether the advanced glycation end-product inhibitor LR-90 could prevent MGO-induced apoptosis in human umbilical vascular endothelial cells (HUVECs). HUVECs were pre-treated with LR-90 and then stimulated with MGO. Cell morphology, cytotoxicity and apoptosis were evaluated by light microscopy, MTT assay, and Annexin V-FITC and propidium iodide double staining, respectively. Levels of Bax, Bcl-2, cytochrome c, mitogen-activated protein kinases (MAPKs) and caspase activities were assessed by Western blotting. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. LR-90 dose-dependently prevented MGO-associated HUVEC cytotoxicity and apoptotic biochemical changes such as loss of MMP, increased Bax/Bcl-2 protein ratio, mitochondrial cytochrome c release and activation of caspase-3 and 9. Additionally, LR-90 blocked intracellular ROS formation and MAPK (p44/p42, p38, JNK) activation, though the latter seem to be not directly involved in MGO-induced HUVEC apoptosis. LR-90 prevents MGO-induced HUVEC apoptosis by inhibiting ROS and associated mitochondrial-dependent apoptotic signaling cascades, suggesting that LR-90 possess cytoprotective ability which could be beneficial in prevention of diabetic related-atherosclerosis.
Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Butiratos/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Aldeído Pirúvico/metabolismo , Caspases/metabolismo , Citocromos c/metabolismo , Ativação Enzimática , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Aldeído Pirúvico/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Nanoengineering has emerged as a progressive method in cancer treatment, offering precise and targeted delivery of therapeutic agents while concurrently reducing overall toxicity. This scholarly article delves into the innovative strategies and advancements in nanoengineering that bridge the gap between clinical practice and research in the field of cancer treatment. Various nanoengineered platforms such as nanoparticles, liposomes, and dendrimers are scrutinized for their capacity to encapsulate drugs, augment drug efficacy, and enhance pharmacokinetics. Moreover, the article investigates research breakthroughs that drive the progression and enhancement of nanoengineered remedies, encompassing the identification of biomarkers, establishment of preclinical models, and advancement of biomaterials, all of which are imperative for translating laboratory findings into practical medical interventions. Furthermore, the integration of nanotechnology with imaging modalities, which amplify cancer detection, treatment monitoring, and response assessment, is thoroughly examined. Finally, the obstacles and prospective directions in nanoengineering, including regulatory challenges and issues related to scalability, are examined. This underscores the significance of fostering collaboration among various entities in order to efficiently translate nanoengineered interventions into enhanced cancer therapies and patient management.
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The leading cause of cancer deaths worldwide is attributed to non-small cell lung cancer (NSCLC), necessitating a continual focus on improving the diagnosis and treatment of this disease. In this review, the latest breakthroughs and emerging trends in managing NSCLC are highlighted. Major advancements in diagnostic methods, including better imaging technologies and the utilization of molecular biomarkers, are discussed. These advancements have greatly enhanced early detection and personalized treatment plans. Significant improvements in patient outcomes have been achieved by new targeted therapies and immunotherapies, providing new hope for individuals with advanced NSCLC. This review discusses the persistent challenges in accessing advanced treatments and their associated costs despite recent progress. Promising research into new therapies, such as CAR-T cell therapy and oncolytic viruses, which could further revolutionize NSCLC treatment, is also highlighted. This review aims to inform and inspire continued efforts to improve outcomes for NSCLC patients globally, by offering a comprehensive overview of the current state of NSCLC treatment and future possibilities.
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The rise of drug resistance in cancer cells presents a formidable challenge in modern oncology, necessitating the exploration of innovative therapeutic strategies. This review investigates the latest advancements in overcoming drug resistance mechanisms employed by cancer cells, focusing on emerging therapeutic modalities. The intricate molecular insights into drug resistance, including genetic mutations, efflux pumps, altered signaling pathways, and microenvironmental influences, are discussed. Furthermore, the promising avenues offered by targeted therapies, combination treatments, immunotherapies, and precision medicine approaches are highlighted. Specifically, the synergistic effects of combining traditional cytotoxic agents with molecularly targeted inhibitors to circumvent resistance pathways are examined. Additionally, the evolving landscape of immunotherapeutic interventions, including immune checkpoint inhibitors and adoptive cell therapies, is explored in terms of bolstering anti-tumor immune responses and overcoming immune evasion mechanisms. Moreover, the significance of biomarker-driven strategies for predicting and monitoring treatment responses is underscored, thereby optimizing therapeutic outcomes. For insights into the future direction of cancer treatment paradigms, the current review focused on prevailing drug resistance challenges and improving patient outcomes, through an integrative analysis of these emerging therapeutic strategies.
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Gynecological cancers, including ovarian, cervical, endometrial, and vulvar cancers, present significant challenges in diagnosis and treatment globally. The tumor microenvironment (TME) plays a pivotal role in cancer progression and therapy response, necessitating a deeper understanding of its composition and dynamics. This review offers a comprehensive overview of the gynecological cancer tumor microenvironment, emphasizing its cellular complexity and therapeutic potential. The diverse cellular components of the TME, including cancer cells, immune cells, stromal cells, and extracellular matrix elements, are explored, elucidating their interplay in shaping tumor behavior and treatment outcomes. Across various stages of cancer progression, the TME exerts profound effects on tumor heterogeneity, immune modulation, angiogenesis, and metabolic reprogramming. The urgency for novel therapeutic strategies is underscored by understanding immune evasion mechanisms within the TME. Emerging approaches such as immunotherapy, stromal-targeting therapies, anti-angiogenic agents, and metabolic inhibitors are discussed, offering promising avenues for improving patient outcomes. Interdisciplinary collaborations and translational research are emphasized, aiming to advance precision oncology and enhance therapeutic efficacy in gynecological cancers.