RESUMO
AIM: Dermaseptins are one of the main families of antimicrobial peptides (AMPs) derived from the skin secretions of Hylidae frogs. Among them, dermaseptin S4 (DS4) is characterized by its broad-spectrum of activity against bacteria, protozoa, and fungi. In this study, the physicochemical properties of the native peptide DS4 (1-28) and two derivatives [DS4 (1-28)a and DS4 (1-26)a] isolated from the skin of the frog Phyllomedusa sauvagii were investigated and their antimicrobial properties against two marine pathogenic bacteria (V. harveyi and V. anguillarum) were examined. METHODS AND RESULTS: The results indicate that the peptide DS4 (1-26)a has high antibacterial activity against the tested strains and low hemolytic activity (< 30% lysis at the highest tested concentration of 100 µg mL -1) compared to the other two peptides tested. In addition, all three peptides affect the membrane and cell wall integrity of both pathogenic bacteria, causing leakage of cell contents, with DS4 (1-26)a having the most severe impact. These skills were corroborated by transmission electron microscopy and by the variation of cations in their binding sites due to the effects caused by the AMPs. CONCLUSIONS: These results suggest that DS4 and its derivatives, in particular the truncated and amidated peptide DS4 (1-26)a could be effective in the treatment of infections caused by these marine pathogenic bacteria. Future studies are required to validate the use of DS4 in vivo for the prevention of bacterial diseases in fish.
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Natural peptides isolated from animal venoms generally target cell surface receptors with high affinity and selectivity. On many occasions, some of these receptors are over-expressed in cancer cells. Herein, we identified Lqh-8/6 as a natural peptide analog of chlorotoxin, a proven and useful compound for the diagnosis and treatment of glioma. Lqh-8/6 and two other natural analogues were chemically synthesized for the first time and evaluated for their ability to label, detect and prevent glioma growth in vitro. We demonstrate that a biotinylated version of Lqh-8/6 allows both the labeling of glioma cell lines and the detection of glioma in brain sections of glioma allograft Fisher rats. Lqh-8/6 has intrinsic anti-invasive properties but is non-toxic to glioma cells. To confer anti-tumor properties to Lqh-8/6, we chemically coupled doxorubicin to the glioma-targeting peptide using click chemistry. To this end, we successfully chemically synthesized Lqh-8/6-azide and doxorubicin-alkyne without impairing the toxic nature of doxorubicin. The toxin-drug conjugate efficiently promotes the apoptosis of glioma cells in vitro. This example contributes to the concept that animal venom peptides constitute exquisite warheads for delivering toxic chemical conjugates, a parallel to the popular concept of antibody-drug conjugates for the treatment of cancer.
RESUMO
BACKGROUND: Flavonoids, which existed nearly in all fruits and vegetables, are considered as a class of plant-secondary metabolites with a polyphenolic structure and have properties with health-improving potential. Yet, not so many experimental focus on the benefits of flavonoid in vivo after external application. Here we assessed the impacts of naringin in vitro and in vivo in the human glioma U-87 cells implanted into athymic mice. METHODS: Tumor size and animal survival time were followed in naringin-treated mice bearing subcutaneous gliomas. To define the effects of naringin on angiogenesis, in vitro, tube formation and migration were assayed using endothelial HUVEC cell line. RESULTS: Low concentration of naringin remarkably inhibited tubulogenesis and reduced cell invasion. Moreover, naringin has been shown to have a toxicity effect on U-87 cells in a dose-dependent way. Furthermore, naringin administration (120 mg/kg/day) applies serious anti-cancer belongings on glioblastoma, as demonstrated by a slow cancer progression. CONCLUSIONS: Our study has provided the first evidence on the antitumor effect of naringin, which is somehow due to the inhibition of invasion and angiogenesis.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Flavanonas/uso terapêutico , Glioblastoma/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Animais , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Flavanonas/farmacologia , Glioblastoma/irrigação sanguínea , Glioblastoma/patologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Camundongos Nus , Neovascularização Patológica/patologia , Carga Tumoral/efeitos dos fármacosRESUMO
OBJECTIVE: We synthesized new tamoxifen derivatives as anticancer drug candidates and elaborated on convection-enhanced delivery (CED) as a strategy for delivery. METHODS: To overcome the issue of their poor solubility, these ferrocenyl-tamoxifen derivatives were esterified and encapsulated into different nanocarriers, that is lipid (LNC) and polymeric nanocapsules (PNL-NC). We describe the chemistry, the encapsulation and the physicochemical characterization of these formulations. KEY FINDINGS: Starting compounds [phthalimido-ferrocidiphenol and succinimido-ferrocidiphenol], esterified prodrugs and their nanocapsules formulations were characterized. These drug candidates displayed a strong in vitro activity against breast and glioblastoma cancer cells. The ester prodrugs were toxic for glioblastoma cells (IC50 = 9.2 × 10-2 µm and 6.7 × 10-2 µm, respectively). The IC50 values for breast cancer cells were higher for these compounds. The encapsulation of the esterified compounds in LNCs (≈50 nm) or PCL-NCs (≈300 nm) did not prevent their efficacy on glioblastoma cells. These anticancer effects were due to both blockade in the S-phase of the cell cycle and apoptosis. Moreover, the tamoxifen derivatives-loaded nanocapsules induced no toxicity for healthy astrocytes and showed no haemolytic properties. Loaded Lipid Nanocapsules (LNCs) presented interesting profiles for the optimal delivery of active compounds. CONCLUSIONS: Phthalimido- and Succinimido-esters represent an innovative approach to treat cancers with cerebral localizations such as glioblastoma or brain metastases from breast cancers.
Assuntos
Antineoplásicos/química , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias da Mama/tratamento farmacológico , Portadores de Fármacos , Glioblastoma/tratamento farmacológico , Lipídeos/química , Nanocápsulas , Poliésteres/química , Tamoxifeno/farmacologia , Animais , Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Química Farmacêutica/métodos , Composição de Medicamentos , Liberação Controlada de Fármacos , Feminino , Glioblastoma/patologia , Humanos , Cinética , Masculino , Ratos Endogâmicos F344 , Solubilidade , Tamoxifeno/análogos & derivados , Tamoxifeno/síntese químicaRESUMO
Hospital wastewaters contain large amounts of pharmaceutical residues, which may eventually be discharged into the aquatic environment through wastewater treatment plants, raising the question of their impact on human and environmental health. This has prompted the launch of several monitoring studies into the most commonly administered compounds in urban wastewater. The aim of this study was, therefore, to explore the cytotoxic potential of wastewaters samples collected from seven hospitals in Tunisia. The physicochemical analyses showed a large fluctuation of certain parameters in the collected samples, such as chemical oxygen demand (ranged from 860 to 1720 mg L-1), biochemical oxygen demand (ranged from 385 to 747 mg L-1), total organic carbon (ranged from 256 to 562 g L-1), total suspended solids (ranged from 905 to 1450 mg L-1), conductivity (ranged from 3.31 to 7.14 µsm/cm), and turbidity (ranged from 100 to 480 NTU). The analysis using inductively coupled plasma mass spectrometry (ICP-MS) also showed that hospital wastewater contains high concentrations of Hg (ranged from 0.0024 to 0.019 mg L-1). This could be explained by the variation of the activity and the services in certain hospitals compared to others. All hospital wastewater samples induced the proliferation of human breast cancer cell line MDA-231, even at low concentrations (20 µL/assay). Moreover, the maximum induction reached at the concentration of 60 µL/assay in wastewater samples from hospitals located in Monastir, Sidi Bouzid, Mahdia, and Sfax with percentages of induction up to 42.33, 14, 7.61, and 5.42%, respectively. These observations could be due to the presence of endocrine disrupting compounds (EDCs) in these wastewaters. Given this, our results evidenced the potential risk of these hospital effluents to environmental and public health.
Assuntos
Proliferação de Células/efeitos dos fármacos , Disruptores Endócrinos/toxicidade , Monitoramento Ambiental/métodos , Hospitais , Águas Residuárias/química , Poluentes Químicos da Água/toxicidade , Análise da Demanda Biológica de Oxigênio , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Disruptores Endócrinos/análise , Humanos , Tunísia , Poluentes Químicos da Água/análiseRESUMO
A previous report has shown that a chimera between a platinum complexing agent (1) and the cell penetrating peptide maurocalcin, synthesized with D-amino acids, (DMCa), termed Pt-1-DMCa, is a highly successful anticancer compound that works by targeting the intracellular redox system in glioblastoma (GBM) cells. However, the detailed cellular mechanism whereby the conjugate specifically kills tumor cells remains unclear. Herein, we show that Pt-1-DMCa induces apoptosis in Human U87 GBM cells through reactive oxygen species (ROS)-dependent modulation of the PI3K/AKT/FoxO3a signalling pathway. First, we found that Pt-1-DMCa treatment of these cells induces inhibition of AKT and nuclear accumulation of FoxO3a thereby facilitating transcription of the target genes Bim and PTEN. Modulation of the AKT/FoxO3a/Bim signaling pathway by RNA interference confirms that these signaling events are critical for Pt-1-DMCa-induced apoptosis of U87 GBM cells. Furthermore, we reveal that FoxO3a-mediated up-regulation of PTEN exerts an additional inhibitory effect on the AKT survival pathway. Thus, our results demonstrate that the conjugate can induce ROS-dependent FoxO3a-mediated apoptosis in U87 cells through PTEN-mediated inhibition of the PI3K/AKT survival axis. Our results help elucidate the molecular mechanisms underlying Pt-1-DMCa-induced cell death in U87 GBM cells and support a theoretical basis for future applications of the MCa peptide.
Assuntos
Apoptose/efeitos dos fármacos , Proteína 11 Semelhante a Bcl-2/metabolismo , Proteína Forkhead Box O3/metabolismo , Glioblastoma/patologia , PTEN Fosfo-Hidrolase/metabolismo , Platina/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linhagem Celular Tumoral , Humanos , Platina/química , Venenos de Escorpião/química , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Naringin (4',5,7-trihydroxyflavanone 7-rhamnoglucoside), a natural flavonoid, has pharmacological properties. In the present study, we investigated the anti-metastatic activity of naringin and its molecular mechanism(s) of action in human glioblastoma cells. Naringin exhibits inhibitory effects on the invasion and adhesion of U87 cells in a concentration-dependent manner by Matrigel Transwell and cell adhesion assays. Naringin also inhibited the migration of U87 cells in a concentration-dependent manner by wound-healing assay. Additional experiments showed that naringin treatment reduced the enzymatic activities and protein levels of matrix metalloproteinase (MMP)-2 and MMP-9 using a gelatin zymography assay and western blot analyses. Furthermore, naringin was able to reduce the protein phosphorylation of extracellular signal-regulated kinase ERK, p38 mitogen-activated protein kinase and c-Jun N-terminal kinase by western blotting. Collectively, our data showed that naringin attenuated the MAPK signaling pathways including ERK, JNK and p38 and resulted in the downregulation of the expression and enzymatic activities of MMP-2, MMP-9, contributing to the inhibition of metastasis in U87 cells. These findings proved that naringin may offer further application as an antimetastatic agent.
Assuntos
Flavanonas/farmacologia , Glioblastoma/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Inibidores de Metaloproteinases de Matriz/farmacologia , Metástase Neoplásica/prevenção & controle , Movimento Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Glioblastoma/tratamento farmacológico , Glioblastoma/enzimologia , Glioblastoma/metabolismo , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Metástase Neoplásica/tratamento farmacológico , Proteínas Proto-Oncogênicas c-jun/metabolismo , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Nephrotoxicity is a common complication of cisplatin chemotherapy and thus limits the use of cisplatin in clinic. Naringin, a natural flavonoid, plays important roles in inflammation and apoptosis in some inflammatory diseases; however, its roles in cisplatin-induced nephrotoxicity remain unclear. In this study, we first assessed the involvement of ROS overproduction and inflammation in cisplatin-induced nephrotoxicity in aged rats, and then we investigated the changes of renal function, histological injury, inflammatory response, and apoptosis in renal tissues after treatment with naringin (20, 50 or 100 mg/kg body weight). Cisplatin resulted in an increase of renal markers, lipid peroxidation, protein and DNA oxidation, and ROS formation. Renal tumor necrosis factor-α (TNF-α) and nitrite levels were also elevated. Expressions of nuclear factor-kappa B (NF-κB), inductible nitric oxide synthase (iNOS), caspase-3 and p53 were up-regulated in renal tissues of Cis-treated rats compared with the normal control group. Histopathological changes were also observed in cisplatin group. Adminstration of naringin at different doses (25, 50 and 100 mg/kg) was able to protect against the deterioration in kidney function, abrogate the decline in antioxidant enzyme activities and suppressed the increase in TBARS, nitrite and TNF-α concentrations. Moreover, naringin inhibited NF-κB and iNOS pathways, caspase-3 and p53 activation and improved the histological changes induced by cisplatin. In conclusion, our studies suggest that oxidative stress and inflammation might play important roles in the development of cisplatin-induced nephrotoxicity and naringin might become an effective therapeutic strategy for this disease.
Assuntos
Anti-Inflamatórios/uso terapêutico , Antineoplásicos/efeitos adversos , Antioxidantes/uso terapêutico , Cisplatino/efeitos adversos , Flavanonas/uso terapêutico , Nefropatias/induzido quimicamente , Rim/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Rim/imunologia , Rim/patologia , Nefropatias/tratamento farmacológico , Nefropatias/imunologia , Nefropatias/patologia , NF-kappa B/análise , NF-kappa B/genética , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/genéticaRESUMO
Gliomas are the most common and malignant primary brain tumors. They are associated with a poor prognosis despite the availability of multiple therapeutic options. Naringin, a common dietary flavonoid abundantly present in fruits and vegetables, is believed to possess strong anti-proliferative and anti-cancer properties. However, there are no reports describing its effects on the invasion and migration of glioblastoma cell lines. Our results showed that the treatment of U251 glioma cell lines with different concentrations of naringin inhibited the invasion and migration of these cells. In addition, we revealed a decrease in the levels of matrix metalloproteinases (MMP-2) and (MMP-9) expression as well as proteinase activity in U251 glioma cells. In contrast, the expression of tissue inhibitor of metalloproteinases (TIMP-1) and (TIMP-2) was increased. Furthermore, naringin treatment decreased significantly the phosphorylated level of p38. Combined treatment with a p38 inhibitor (SB203580) resulted in the synergistic reduction of MMP-2 and MMP-9 expressions correlated with an increase of TIMP-1 and TIMP-2 expressions and the anti-invasive properties. However, p38 chemical activator (anisomycin) could block these effects produced by naringin, suggesting a direct downregulation of the p38 signaling pathway. These data suggest that naringin may have therapeutic potential for controlling invasiveness of malignant gliomas by inhibiting of p38 signal transduction pathways.
Assuntos
Movimento Celular/efeitos dos fármacos , Flavanonas/farmacologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Imidazóis/farmacologia , Immunoblotting , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Invasividade Neoplásica , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Glioblastoma multiforme (GBM) is a highly malignant and aggressive primary brain tumor. In spite of an arsenal of therapeutic interventions, the prognosis of glioblastoma remains very poor. Cisplatin-based therapy is one of the most important chemotherapy treatments for GBM, although its efficacy is limited by drug resistance and undesirable side effects. In the present study, we designed a chimera molecule containing the platinum binding moiety MBL-III-7 (1) attached N-terminal to the sequence of d-maurocalcine (D-MCa), a protease-resistant and highly efficient cell-penetrating peptide (CPP) derived from the Tunisian chactid scorpion toxin, L-MCa. The concept behind this design is that MCa, through its cell retention properties, should reduce cell expulsion of the platinum complex and increase its efficiency. The anti-cancer properties of the synthesized platinum analogue Pt-MBL-III_7-D_MCa (Pt-1-DMCa) were assessed in human glioblastoma cells (U87) by assaying cell viability and apoptosis. The new molecule exhibited enhanced anti-cancer efficacy compared to cisplatin, especially at low doses. By inducing intracellular oxidative stress, Pt-1-DMCa potentiated platinum-induced DNA damage and led to enhanced p53 phosphorylation, followed by increased activation of both mitochondrial and death receptor pathways. Decreased phosphorylated AKT and ERK levels were associated with the apoptosis induced by the novel synthesized cisplatin analogue. Our results suggested that a chimera between platinum and a maurocalcine-derived CPP is a highly successful anti-cancer compound that works by targeting the intracellular redox system. Pt-1-DMCa is an interesting candidate for a preclinical assessment of platinum-based therapy in GBM treatments and possibly other cancer types.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Compostos Organoplatínicos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Venenos de Escorpião/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Glioblastoma/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Supressora de Tumor p53RESUMO
Doxorubicin (Dox) has demonstrated potent activity in treating malignant lymphomas but its therapeutic efficacy is hampered by induction of cardiotoxicity. This side effect is related to the ability of the drug to generate reactive oxygen species in cells. Previously, we demonstrated that coupling Dox to penetratin (Pen), a cell penetrating peptide, represent a valuable strategy to overcome drug resistance in CHO cells. In the present study, we evaluated the consequences of the conjugation of Dox to Pen in term of apoptosis induction. When tested on CHO cells, Dox-Pen generated a typical apoptotic phenotype but at lower dose that needed for unconjugated Dox. Cell death induction was associated with chromatin condensation, caspase activation, Bax oligomerisation and release of cytochrome c. By using reactive oxygen species and c-jun NH2-terminal kinase (JNK) inhibitors, we prevented Dox- and Dox-Pen-induced CHO cell death. The chimeric soluble DR5 receptor that inhibits TRAIL induced cell death does not prevent Dox or Dox-Pen-induced cytotoxicity. These observations indicate that conjugation of Dox to cell penetrating peptide does not impair the ability of the drug to trigger cell death through activation of the intrinsic pathway involving c-Jun NH2-terminal kinase but could exhibit less toxic side effects and could warrant its use in clinic.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Apoptose , Proteínas de Transporte/farmacologia , Doxorrubicina/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Animais , Antibióticos Antineoplásicos/metabolismo , Células CHO , Proteínas de Transporte/metabolismo , Peptídeos Penetradores de Células , Cricetinae , Cricetulus , Doxorrubicina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismoRESUMO
One of the major obstacles which are opposed to the success of anticancer treatment is the cell resistance that generally develops after administration of commonly used drugs. In this study, we try to overcome the tumour cell resistance of doxorubicin (Dox) by developing a cell-penetrating peptide (CPP)-anticancer drug conjugate in aim to enhance its intracellular delivery and that its therapeutic effects. For this purpose, two cell-penetrating peptides, penetratin (pene) and tat, derived from the HIV-1 TAT protein, were chemically conjugated to Dox. The cytotoxicity, intracellular distribution and uptake were accessed in CHO cells (Chinese Hamster Ovarian carcinoma cells), HUVEC (Human Umbilical Vein Endothelial Cells), differentiated NG108.15 neuronal cell and breast cancer cells MCF7drug-sensitive or MDA-MB 231 drug-resistant cell lines. The conjugates showed different cell killing activity and intracellular distribution pattern by comparison to Dox as assessed respectively by MTT-based colorimetric cellular cytotoxicity assay, confocal fluorescence microscopy and FACS analysis. After treatment with 3 microM with Dox-CPPs for 2h, pene increase the Dox cytotoxicity by 7.19-fold in CHO cells, by 11.53-fold in HUVEC cells and by 4.87-fold in MDA-MB 231 cells. However, cytotoxicity was decreased in NG108.15 cells and MCF7. Our CPPs-Dox conjugate proves the validity of CPPs for the cytoplasmic delivery of therapeutically useful molecules and also a valuable strategy to overcome drug resistance.
Assuntos
Antibióticos Antineoplásicos/metabolismo , Antibióticos Antineoplásicos/farmacologia , Proteínas de Transporte/metabolismo , Doxorrubicina/metabolismo , Doxorrubicina/farmacologia , Fragmentos de Peptídeos/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Animais , Antibióticos Antineoplásicos/química , Transporte Biológico , Células CHO , Proteínas de Transporte/química , Linhagem Celular Tumoral , Peptídeos Penetradores de Células , Cricetinae , Cricetulus , Doxorrubicina/química , Humanos , Fragmentos de Peptídeos/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/químicaRESUMO
Previous work from our laboratory has shown that coupling doxorubicin (Dox) to cell penetrating peptides (Dox-CPPs) is a good strategy to overcome Dox resistance in MDA-MB 231 breast cancer cells. We also reported that, in contrast to unconjugated Dox-induced cell death, the increase in apoptotic response does not involve the mitochondrial apoptotic pathway. In this study, we demonstrate that both Dox and Dox-CPPs can increase the density of the TRAIL receptors DR4 and DR5 at the plasma membrane and moderately sensitize MDA-MB 231 cells to exogeneously added recombinant TRAIL, as has already been shown for other chemotherapeutic drugs. Moreover, we show that Dox-CPPs, used alone, induce the clustering of TRAIL receptors into ceramide-enriched membrane lipid rafts, a property not shared by unconjugated Dox and that this process is due to the generation of ceramide during Dox-CPPs treatment. In addition, MDA-MB 231 cells were found to express TRAIL and we show that the increased apoptotic rate induced by Dox-CPPs is due to the sensitization of MDA-MB 231 cells to endogenous TRAIL. The capacity of Dox-CPPs to sensitize cancer cells to physiologic amounts of TRAIL suggests that, in addition to their efficiency in combination chemotherapy, these compounds might increase the response of tumor cells to cytotoxic lymphocyte-mediated killing via TRAIL.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Doxorrubicina/administração & dosagem , Portadores de Fármacos/administração & dosagem , Peptídeos/administração & dosagem , Ceramidas/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Microdomínios da Membrana/fisiologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
In our previous studies, we demonstrated that the deglycosylation of bleomycin-A2 (BLM-A2) does not affect the capacity of this drug to induce cell death by apoptosis in a caspase-independent manner in laryngeal cancer cells (HEp-2), but suppresses the ability of BLM-A2 to induce ROS formation. We have now investigated the consequence of BLM-A2 deglycosylation in terms of the involvement of apoptotic pathways in HEp-2 cells. Apoptosis induced by bleomycin-A2 and deglyco-BLM-A2 is associated with the release of cytochrome c and AIF. Only Bax was oligomerized with BLM-A2-induced HEp-2 cell death. BLM-A2 and deglyco-BLM-A2-induced apoptosis depended on JNK activation but was independent of death receptors expression. In contrast, both of these drugs would sensitize HEp-2 cells to death receptor ligand-induced cell death. These observations indicate that the deglycosylation of BLM does not impair the ability of the drug to trigger cell death through activation of the intrinsic pathway by the release of AIF responsible for mitochondrial permeability and chromatin condensation independent of caspases activation.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Fator de Indução de Apoptose/metabolismo , Apoptose , Bleomicina/análogos & derivados , Carcinoma/enzimologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Neoplasias Laríngeas/enzimologia , Antracenos/farmacologia , Fator de Indução de Apoptose/efeitos dos fármacos , Bleomicina/farmacologia , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Proteína Ligante Fas/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Leucemia/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismoRESUMO
Doxorubicin (Dox) is a commonly used drug to treat various types of cancers. Previously, we demonstrated that coupling Dox to cell-penetrating peptides (CPPs) represent a valuable strategy to overcome drug resistance in MDA-MB 231 breast cancer cells. In the present study, we evaluated the properties of these Dox conjugates (Dox-CPPs) in terms of apoptosis induction. Dox-CPPs were found to induce apoptotic death in MDA-MB 231 cells at a lower dose than that needed for unconjugated Dox. Cell death induction was associated with Bax oligomerisation, release of cytochrome c, caspase activation, chromatin condensation and internucleosomal degradation. However, whereas Bcl-2 overexpression was very potent in inhibiting apoptosis triggered by Dox, this anti-apoptotic protein was largely inefficient in preventing Dox-CPPs-induced apoptosis. These observations suggest that mitochondrial disruption is the main event in Dox-induced apoptotic signaling but that Dox-CPPs are probably able to trigger additional apoptotic pathways independent of mitochondrial events. Thus, the higher efficacy of Dox conjugated to CCPs in apoptosis induction might not be due exclusively to increased drug accumulation but also to the activation of multiple apoptotic pathways.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Permeabilidade da Membrana Celular , Doxorrubicina/farmacologia , Portadores de Fármacos , Peptídeos/metabolismo , Animais , Antibióticos Antineoplásicos/metabolismo , Neoplasias da Mama/metabolismo , Células CHO , Caspases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Cricetinae , Cricetulus , Citocromos c/metabolismo , Relação Dose-Resposta a Droga , Doxorrubicina/análogos & derivados , Doxorrubicina/metabolismo , Ativação Enzimática , Feminino , Humanos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Proteína X Associada a bcl-2/metabolismoRESUMO
PURPOSE: The aim of this study is to overcome tumour cell resistance that generally develops after administration of commonly used anti-cancer drugs, such as doxorubicin. METHODS: Recently, cell penetrating peptides have been used for their ability to deliver non-permeant compounds into cells. One such cell penetrating peptide, maurocalcine, has been isolated from the venom of a Tunisian scorpion. Herein, we report the effects of doxorubicin covalently coupled to an analogue of maurocalcine on drug-sensitive or drug-resistant cell lines MCF7 and MDA-MB 231. RESULTS: We demonstrated the in vitro anti-tumoral efficacy of the doxorubicin maurocalcine conjugate. On a doxorubicin-sensitive cancer cell line, the maurocalcine-conjugated form appears slightly less efficient than doxorubicin itself. On the contrary, on a doxorubicin-resistant cancer cell line, doxorubicin coupling allows to overcome the drug resistance. This strategy can be generalized to other cell penetrating peptides since Tat and penetratin show similar effects. CONCLUSION: We conclude that coupling anti-tumoral drugs to cell penetrating peptides represent a valuable strategy to overcome drug resistance.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Doxorrubicina/farmacologia , Portadores de Fármacos , Resistencia a Medicamentos Antineoplásicos , Venenos de Escorpião/metabolismo , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/metabolismo , Neoplasias da Mama/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Química Farmacêutica , Relação Dose-Resposta a Droga , Doxorrubicina/química , Doxorrubicina/metabolismo , Composição de Medicamentos , Feminino , Humanos , Venenos de Escorpião/química , Venenos de Escorpião/toxicidade , Fatores de TempoRESUMO
Maurocalcine is a 33-mer peptide initially isolated from the venom of a Tunisian scorpion. It has proved itself valuable as a pharmacological activator of the ryanodine receptor and has helped the understanding of the molecular basis underlying excitation-contraction coupling in skeletal muscles. Because of its positively charged nature, it is also an innovative vector for the cell penetration of various compounds. We report a novel maurocalcine analog with improved properties: (i) the complete loss of pharmacological activity, (ii) preservation of the potent ability to carry cargo molecules into cells, and (iii) coupling chemistries not affected by the presence of internal cysteine residues of maurocalcine. We did this by replacing the six internal cysteine residues of maurocalcine by isosteric 2-aminobutyric acid residues and by adding an additional N-terminal biotinylated lysine (for a proof of concept analog) or an N-terminal cysteine residue (for a chemically competent coupling analogue). Additional replacement of a glutamate residue by alanyl at position 12 further improves the potency of these analogues. Coupling to several cargo molecules or nanoparticles are presented to illustrate the cell penetration potency and usefulness of these pharmacologically inactive analogs.
Assuntos
Portadores de Fármacos/farmacologia , Venenos de Escorpião/farmacologia , Animais , Células CHO , Cricetinae , Cricetulus , Dissulfetos/química , Portadores de Fármacos/síntese química , Portadores de Fármacos/química , Humanos , Camundongos , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Venenos de Escorpião/síntese química , Venenos de Escorpião/genética , Escorpiões/genética , Escorpiões/metabolismoRESUMO
Maurocalcine (MCa), initially identified from a Tunisian scorpion venom, defines a new member of the family of cell penetrating peptides by its ability to efficiently cross the plasma membrane. The initiating mechanistic step required for the cell translocation of a cell penetrating peptide implicates its binding onto cell surface components such as membrane lipids and/or heparan sulfate proteoglycans. Here we characterized the interaction of wild-type MCa and MCa K20A, a mutant analogue with reduced cell-penetration efficiency, with heparin (HP) and heparan sulfates (HS) through surface plasma resonance. HP and HS bind both to MCa, indicating that heparan sulfate proteoglycans may represent an important entry route of the peptide. This is confirmed by the fact that (i) both compounds bind with reduced affinity to MCa K20A and (ii) the cell penetration of wild-type or mutant MCa coupled to fluorescent streptavidin is reduced by about 50% in mutant Chinese hamster ovary cell lines lacking either all glycosaminoglycans (GAGs) or just HS. Incubating MCa with soluble HS, HP, or chondroitin sulfates also inhibits the cell penetration of MCa-streptavidin complexes. Analyses of the cell distributions of MCa/streptavidin in several Chinese hamster ovary cell lines show that the distribution of the complex coincides with the endosomal marker Lyso-Tracker red and is not affected by the absence of GAGs. The distribution of MCa/streptavidin is not coincident with that of transferrin receptors nor affected by a dominant-negative dynamin 2 K44A mutant, an inhibitor of clathrin-mediated endocytosis. However, entry of the complex is greatly diminished by amiloride, indicating the importance of macropinocytosis in MCa/streptavidin entry. It is concluded that (i) interaction of MCa with GAGs quantitatively improves the cell penetration of MCa, and (ii) GAG-dependent and -independent MCa penetration rely similarly on the macropinocytosis pathway.